general information - pmda

21352135

GENERAL INFORMATION

G1 Physics and Chemistry

Guideline for Residual Solventsand Models for the Residual

Solvents Test1. Guideline for Residual Solvents

Refer to the Guideline for Residual Solvents in Phar-maceuticals (PAB/ELD Notification No. 307; dated March30, 1998).

Since the acceptable limits of residual solvents recom-mended in the Guideline were estimated to keep the safety ofpatients, the levels of residual solvents in pharmaceuticalsmust not exceed the limits, except for in a special case. Phar-maceutical manufacturers should assure the quality of theirproducts by establishing their own specification limits ormanufacturing process control limits for residual solventspresent in their products in consideration of the limitsrecommended in the Guideline and the observed values intheir products, and by performing the test with the productsaccording to the Residual Solvents Test.

2. Residual Solvents TestGenerally, the test is performed by using gas chromato-

graphy <2.02> as directed in the Residual Solvents Test<2.46>. If only the class 3 solvents with low toxic potential toman are present in the products, Loss on Drying Test <2.41>can be applied in place of gas chromatography, in which casethe limit value of residual solvents is not more than 0.5z.

The test may also be performed according to the EPmethod (2.4.24 Identification and control of residual sol-vents) or the USP method (<467> Residual Solvents). Even inthis case, description should be in the JP style and the systemsuitability test should be performed according to the JP rule.

3. Models for the operating conditions and system suita-bility of gas chromatography for residual solvents test

The following are typical examples of the operating condi-tions of gas chromatography for residual solvents test, de-scribed in the EP and the USP, but these do not necessarilyimply that other suitable operating conditions can not beused. In the operating conditions, generally, items requiredfor the test such as detector, column, column temperature,injection port temperature, detector temperature, carriergas, flow rate, and time span of measurement should be spe-cified, and in the system suitability, items such as test for re-quired detectability, system performance, and system repeat-ability should be specified. The following are several modelsfor the operating conditions and the system suitability:

3.1. Models for operating conditions for a head-spacesample injection device (Models described in the EP and theUSP)

(i) Operating conditions (1) for the head-space sample

injection device�Equilibration temperature A constant temperature offor inside vial about 809CEquilibration time for 60 minutesinside vialTransfer-line temperature A constant temperature of

about 859CCarrier gas NitrogenPressurisation time 30 secondsInjection volume of sample 1.0 mL(ii) Operating conditions (2) for the head-space sample


about 1109CCarrier gas NitrogenPressurisation time 30 secondsInjection volume of sample 1.0 mL(iii) Operating conditions (3) for the head-space sample


about 1059CCarrier gas NitrogenPressurisation time 30 secondsInjection volume of sample 1.0 mL

3.2. Models for operating conditions and system suitabilityof gas chromatography

(i) Test conditions (1) (A model described under Proce-dure A in the EP and the USP)Operating conditions�

Detector: Hydrogen flame-ionization detector.Column: Coat the inside wall of a fused silica tube, 0.32

mm (or 0.53 mm) in inside diameter and 30 m in length, to1.8 mm (or 3 mm) thickness with 6z cyanopropylphenyl-methyl silicon polymer for gas chromatography. Use a guardcolumn if necessary.

Column temperature: Maintain at 409C for 20 minutes,then increase to 2409C at 109C per minute if necessary, andkeep at 2409C for 20 minutes.

Injection port temperature: A constant temperature ofabout 1409C.

Detector temperature: A constant temperature of about2509C.

Carrier gas: Helium.Flow rate: 35 cm/second.Split ratio: 1:5.

System suitability�System performance: When the procedure is run with the

standard solution under the above operating conditions, the

21362136 JP XVIPhysics and Chemistry / General Information

resolution between the peaks is not less than 1.0. (Note: Inthe case that the number of substances to be tested is two ormore.)

System repeatability: When the test is repeated 3 timeswith the standard solution under the above operating condi-tions, the relative standard deviation of the peak areas of thesubstance to be tested is not more than 15z.

(ii) Test conditions (2) (A model described under Proce-dure B in the EP and the USP)Operating conditions�


mm (or 0.53 mm) in inside diameter and 30 m in length, to0.25 mm thickness with polyethylene glycol 20M for gaschromatography. Use a guard column if necessary.

Column temperature: Maintain at 509C for 20 minutes,then increase to 1659C at 69C per minute if necessary, andkeep at 1659C for 20 minutes.



Carrier gas: Helium.Flow rate: 35 cm/second.Split ratio: 1:5.


standard solution under the above operating conditions, theresolution between the peaks is not less than 1.0. (Note: Inthe case that the number of substances to be tested is two ormore.)


(iii) Test conditions (3) (A model described in Method Iunder Other Analytical Procedures in the USP)Operating conditions�


mm in inside diameter and 30 m in length, to 5 mm thicknesswith 5z phenyl-methyl silicon polymer for gas chromatog-raphy. If necessary use a guard column prepared by coatingthe inside wall of a fused silica tube, 0.53 mm in inside diam-eter and 5 m in length, to 5 mm thickness with 5z phenyl-methyl silicon polymer for gas chromatography.

Column temperature: Maintain at 359C for 5 minutes,then increase to 1759C at 89C per minute, further increase to2609C at 359C per minute if necessary, and keep at 2609Cfor 16 minutes.



Carrier gas: Helium.Flow rate: 35 cm/second.Split ratio: Splitless.


standard solution under the above operating conditions, theresolution between the peaks is not less than 1.0. (Note: Inthe case that the number of substances to be tested is two ormore.)


Inductively Coupled Plasma AtomicEmission Spectrometry

Inductively coupled plasma (ICP) atomic emission spec-trometry is a method for qualitative and quantitative analy-sis for the element in the sample specimen, which is nebu-lized into argon plasma induced by radio frequency power,by measuring the wavelength and the intensity of the emis-sion spectra generated by the target element, atomized andexcited in the plasma. Argon gas plasma used in this methodis characteristic of higher temperature of 6000 ¿ 8000 K andelectronic density of 1015 cm－3.

When the high energy is externally given to an atom, theorbit transition of the most exterior electron is occurred andattained to the excited state. When the excited state of atomreturns to the basic state, the energy obtained by the excita-tion is radiated as a light. The emitted light has an intrinsicwave number n and wave length l, attributed to the individ-ual element. Assuming the Plank constant h and the lightvelocity c, the emitted light energy DE is expressed by thefollowing formula.

DE＝ hn＝ hc/l

Since there are many of combinations of the orbit transi-tion energy level of the most exterior electron and the emit-ted light energy, usually, there are plenty of spectral linesemitted from an element, summing up every strong andweak emitted lights. However, the number of emitted lightsof being in the ultra-violet and visible region and having suit-able detection sensitivity for qualitative and quantitativeanalysis are limited to a certain number. Since the atomicemission spectra give intrinsic wave number or wave lengthfor the respective element, many of elements contained in asample specimen can be identified by the spectroscopicanalysis of the emitted lights. Furthermore, the quantitativeanalysis for an element can also be done by measuring thespectral intensity of the emitted light.

The characteristics of ICP emission spectrometry are sum-marized as follows;

( i ) Microanalysis for plenty of elements is possible.( ii ) Analytical precision is high, since the stable plasma

generation can be kept.(iii) Linearity of the calibration curve is ensured in the

wide range of 4 ¿ 5 digit.(iv) Multi-element simultaneous analysis is possible.( v ) Chemical interferences are almost negligible.

The most serious problem in the ICP emission spectro-metry is that the spectral interferences are inevitable to thisanalysis, and disturb the analysis for the target element. Itresults from many of emitted spectral lines due to coexistingelements, because the atomization and excitation of elementsare done in very high temperature of plasma. Thus it is thekey technique to obtain an accurate analytical result how toreduce the spectral interferences and how to correct the in-

21372137JP XVI General Information / Physics and Chemistry

terfered spectra.The present method is superior to analyze inorganic im-

purities and/or coexisting elements in drug substances andpreparations specifically, and also to analyze metal residuesin crude drugs and preparations. The qualitative and quan-titative analysis of alkali, alkali earth, and heavy metals arepossible, and also of many other elements which are requiredfor suitable control for the safety assurance of drug medi-cines. Furthermore, since the simultaneous analysis for lotsof elements are possible, this method can be used for theprofile analysis of inorganic impurities such as metal ele-ments for the quality control of drug substances.

In this General Information, a spectroscopic detectionmethod of atomic emission spectra (AES) due to metal ele-ments introduced into inductively coupled plasma (ICP), isdescribed as ICP-AES. Separately, since ICP is not only agood excitation source, but also a good ionizing source,inductively coupled plasma-mass spectrometry (ICP-MS) isalso possible by using ICP as an ionizing source.

1. Apparatus1.1. Composition of the apparatus

The apparatus is composed of the excitation source unit,sample introduction unit, the emission unit, the spectropho-tometry unit, the detection unit, and the data processingunit.

The excitation source unit is composed of the electricpower source circuit, the control circuit to supply and tocontrol electric energy, and the gas supplying part. The sam-ple introduction unit is the part of introducing the sampleinto the emission unit and composed of a nebulizer and aspray chamber, etc.

The emission unit is a part where the target element in thesample is excited and emitted, and it is composed of thetorch and the radio frequency induction coil. The torch usu-ally consists of a threefold tube, in which the sample is intro-duced by the central tube. Argon is used for the gas to formplasma and also to carry the sample. There are two types ofobservation way of the light emitted, one is the radial view-ing, and the other the axial viewing.

The spectroscope unit is a part separating the emitted lightinto the individual spectral line and composed of convergentlens system and optical elements such as the diffraction grat-ing, etc. There are two types of spectroscopes, one is thewavelength scanning type of monochrometer, the other thefixed wavelength type of polychrometer. For measuringspectral lines in the vacuum UV region, in which the wave-length is not more than 190 nm, the evacuation of the in-terior of the spectroscope or the substitution of air for argonor nitrogen gas are necessary.

Detection unit is a part converting the incident light inten-sity to the corresponding strength of the electric signal andcomposed of the detector and the signal processing system.Regarding the detector, either the photomultiplier or thecharge transfer device (CTD) such as the charge coupleddevice, the charge injection device, and the photo diode ar-ray etc., is used.

Data processing unit is a part of processing the detectedsignal and the measurement result and of giving the calibra-tion curve etc., which are given by the displaying apparatusor the printer, etc.

1.2. Attachments1.2.1. Ultrasonic nebulizer

It is an instrument for atomizing the sample solution withan ultrasonic vibrator, dehydrating it by heating and cool-ing, and introducing it into the emission unit by the carriergas. By using this attachment, the sample introducingefficiency is remarkably increased.1.2.2. Hydride generation apparatus

It is an instrument that, after reducing the compoundssuch as arsenic, selenium, and antimony in the sample solu-tion to the volatile hydrides with sodium tetrahydroborateetc., the vapor/liquid separation is carried out and the gascomponents only introduced into the emission unit with thecarrier gas. In comparison with the usual nebulizer, thesample introducing efficiency is remarkably increased.

2. Pretreatment of sampleUsually, organic samples such as drug substances are

ignited or digested by the dry ashing method or the aciddigestion method, respectively. Then the sample solution isprepared by dissolving the residues with small amount ofnitric acid or hydrochloric acid. Separately, as for the diges-tion resistant samples such as crude drugs, the microwavedigestion apparatus is available to the digestion of thosesamples in tightly closed vessels.

Where the sample can be dissolved in water or an ap-propriate solvent, the sample solution can be simply pre-pared by dilution or dissolution, provided that a correctanalysis is assured previously, either by the dry ashingmethod or the acid digestion method.2.1. Dilution and/or dissolution method

Take a specified amount of sample in the respective mono-graph, then dilute by water or an appropriate solvent, or dis-solve in water or a specified solvent to prepare the sample so-lution.2.2. Dry ashing method

Applying the ``Residue on Ignition Test'', take a specifiedamount of sample in the crucible, moisten it with a smallamount of sulfuric acid, then heat gently to carbonize itthoroughly. After cooling, moisten the charred residue witha small amount of sulfuric acid, heat gently again, thenignite the residue to ash in the range of 400 ¿ 6009C.Dissolve the ash by adding a small amount of nitric acid orhydrochloric acid to prepare the sample solution.

Separately, applying the 3rd Method in the ``Heavy MetalsLimit Test'', ignite the sample to ash without moistening itwith sulfuric acid. In this case, since it is digested and incin-erated in the open system, be careful to the vaporization lossof low boiling point elements such as mercury.2.3. Acid digestion method

Take an amount of sample into the beaker or the flask asspecified in the monograph, add nitric acid, sulfuric acid, orthe mixture of these acids, then digest by heating. If it is nec-essary, an auxiliary oxidant such as hydrogen peroxide maybe available. After the digestion is completed, add a smallamount of nitric acid or hydrochloric acid to the residues,dissolve them by heating to prepare the sample solution.

Acid digestion can be done either in the open or in theclosed system. Regarding the closed system, the heat diges-tion at high temperature under high pressure conditionsmight be applicable by using the pressure tolerable digestionvessel of polytetrafluoroethylene fitted with the outer tube ofstainless steel. Where the heat digestion method is used in the


closed system, be careful enough to accidents such as theexpansion and the liquid leak.2.4. Microwave digestion method

Take an amount of sample into the pressure tolerableclosed vessel as specified in the monograph, add an ap-propriate amount of nitric acid, then digest under heatingconditions by using the microwave digestion apparatus.Since it is a digestion procedure under high temperature/high pressure conditions, the temperature and the pressure inthe closed vessel should be appropriately controlled, in con-sideration of amounts of the acid and the sample.

The pretreatment method should be selected appropriate-ly, corresponding to the attribute of the sample and the tar-get element. Where the ignition or the digestion is done inthe open system, be careful to the vaporization loss of thetarget element or the contamination from the operating en-vironment. Furthermore, where the residues obtained by theignition or the digestion are dissolved in acid solution andprepared to the sample solution, if necessary, the filtrationwith membrane filter (pore size: 1 mm) may be useful.

3. Procedure3.1. Performance test of the spectroscope3.1.1. Wavelength calibration

The wavelength calibration should be suitably performedaccording to the indicated methods and procedures of theapparatus, since there is specific calibration method of theindividual instrument. For example, some of calibrationmethods are available, such as a method of using standardsolution containing several elements, spectral lines of mer-cury lamp, and emission lines of argon gas, etc.3.1.2. Wavelength resolution

The wavelength resolution is usually specified by the halfwavelength (nm) of the spectral lines for some representativeelements. During the lower and the higher wavelength, fol-lowing spectral lines of As (193.696 nm), Mn (257.610 nm),Cu (324.754 nm), and Ba (455.403 nm) are usually selected.The specification of the half wavelength for the individualelement is responsible to the user, because the characteristicsof the instrument and the spectroscope are intrinsic anddifferent each other.

However, in the case of the simultaneous analysis type ofinstrument fitted with semiconductor detector, this specifica-tion is not always required.3.2. Preparation of sample solution and standard solution

Sample of drug substances, preparations, and crude drugsare pretreated by using anyone of the method described inthe above section (2. Pretreatment of sample) and preparedto the sample solution. Usually, the sample solution is pre-pared to as a dilute nitric acid solution, while hydrochloricacid is also available in place of nitric acid.

Where the reference standard solutions are specified in theJapanese Pharmacopoeia (JP) and/or the Japanese Industri-al Standard (JIS), the standard solutions are prepared bydiluting those official standards to the definite concentra-tions with the water for ICP analysis etc. When those refer-ence standard solutions are not specified both in JP and JIS,the reference substances certified and provided by the offi-cial organization or the academic association etc., are usedfor the preparation of the standard solutions. Where the ref-erence standard solutions or the reference substances are notgiven by the above mentioned official or semi-official or-ganizations, either the pure metals or the compounds con-

taining the target element of high purity (not less than99.99z) can be used for preparing the calibration standards,in which one or more target elements are included. In case ofpreparing multi-component standard solution, the reagentsolutions and the element combinations are required not toform precipitates, and not to give spectral interferences withthe analytical line of the target element.3.3. Optimization of the operating conditions

The operating conditions are usually as follows.After the plasma is attained to the stable generation by the

warm-up driving for 15 ¿ 30 minutes, the optimum operat-ing conditions are adjusted for the intended use. Radio fre-quency power output of 0.8 ¿ 1.4 kW, argon gas flow ratefor the cooling gas of 10 ¿ 18 L/min, for auxiliary gas of0 ¿ 2 L/min, and for carrier gas of 0.5 ¿ 2 L/min, sampleintroduction of 0.5 ¿ 2 mL/min are usual operating condi-tions. Where the radial viewing method is applied, the obser-vation height is adjusted to 10 ¿ 25 mm above the inductioncoil, and the axial viewing is applied, the optical axis isadjusted to obtain the greatest emission intensity. The in-tegration time is set appropriately between 1 and several de-cades of second, in consideration of the stability of the emis-sion intensity.

As the analytical line of the individual target element, therepresentative emission lines are shown in Table 1. However,where the concentration of the target element is markedlyhigh, it is recommended either to dilute the sample solutionor to select an appropriate another spectral line, based on theexpected concentration of the element. Furthermore, whenthe spectral interferences due to coexisting elements are oc-curred, another spectral line should be selected so as to befree from the interference.

Where this test method is specified in the respective mono-graph, the necessary operating conditions such as the ana-lytical line (nm), the radio frequency power unit (kW), argongas flow rate (L/min) etc., should be specified for the in-tended use. However, all the specified conditions except forthe analytical line are the referring ones, and the optimiza-tion of those conditions are expected to be found accordingto the individual instrument and the observation way etc.3.4. Water and reagents

Water and reagents for this test are as follows.(i) As for water, use water for ICP analysis described

below,Water for ICP analysis: The electric conductivity should

be below 1 mS･cm－1 (259C). Further it should be con-firmed that the contaminated impurities do not inter-fere with the emission of the target element.

(ii) The quality of reagents should be suitable, as it doesnot contain the interfering substances of the test.

(iii) As for argon gas, use that specified below,Argon gas: The purity should be not less than 99.99

volz, as specified in JIS K 1105. Either the liquefiedargon or pressurized argon gas can be used.

3.5. ProcedureAfter confirmation of the normal action of the instrumen-

tal parts usually switched on electrically, the electric sourceof the instrument body and the surrounding equipmentsshould be switched on. After setting argon gas flow rate tothe decided value, switch on the radio frequency source, andgenerate argon plasma. After confirming the stable plasma isgiven, the sample solution and the standard solution pre-pared as specified in the individual monograph, are intro-

2139

Table 1 The representative analytical line (nm) of variousmetal elements

Al 396.153As 193.759B 249.773Ba 455.404Be 313.042Cd 214.438Co 228.616Cr 205.552Cu 324.754

Fe 259.940Hg 184.950In 230.606Ir 224.268Li 670.784Mg 279.553Mn 257.610Mo 202.030Ni 221.647

Os 225.585Pb 220.351Pd 340.458Pt 214.423Rb 780.023Rh 233.477Ru 240.272Sb 206.833Se 196.090

Sn 189.980Sr 407.771Tl 276.787V 309.311W 207.911Zn 213.856


duced into the plasma, and the emission intensity is meas-ured at the indicated analytical line. Where the qualitativetest for the confirmation or the identification is performed,the measurement wavelength range of the emission spectrashould be sufficiently wide, in which all the specified analyti-cal lines are included.

Where the emission lines in vacuum UV region are meas-ured by using the vacuum type spectroscope, the air aroundthe optical axis between the emission part and the spectro-scope part should be substituted for argon or nitrogen gas.

4. System suitabilityWhen this test is applied to the limit test or the quantita-

tive test of metal elements etc., it is required to confirm pre-viously whether the performance ability of the apparatus isadequate or not by the system suitability test specifiedbelow. However, in the assay test, the test for 4.1. Confir-mation of detection and evaluation of linearity, is notrequired.4.1. Confirmation of detection and evaluation of linearity

In the limit test of metal elements etc., where the pretreat-ment method of the sample specimen is specified in the re-spective monograph, it can be speculated what is the concen-tration of the specified limit in the sample solution (mg/mL).Assuming that the concentration of the target element in thestandard solution is 10 ¿ 20 times of that of the sample so-lution, the standard solution of the target element can beprepared, as mentioned in the section 3.2. Next, diluting thisstandard solution to 1/10 times of the concentration, this isthe system suitability test solution.

Under the optimum conditions for an individual instru-ment, ICP emission spectra of the standard solution and thesystem suitability test solution of the target element aremeasured and the followings should be confirmed. Regard-ing the system suitability test solution, it should be con-firmed that the spectra of the target element are clearly ob-served at the specified wavelength, and the emission intensityis within a specified range (for example, 80 ¿ 120z) com-pared to the theoretical emission intensity, which is calcu-lated by multiplying the concentration of the standard solu-tion by the dilution ratio.4.2. System repeatability

As for the standard solution of the target elementdescribed in the section 4.1, when the emission intensitymeasurement is repeated 6 times under the optimum condi-tions for an individual instrument, the relative standard de-viation of the emission intensity for the target elementshould be below a certain specified value (for example, notmore than 3.0z).

In case of the assay test, one of the standard solutions forthe calibration curve, which are specified in the monograph,can be selected as a test solution of the system repeatability.

5. Interferences and their reduction or correctionInterferences accompanied to ICP-AES are a general ex-

pression of the negative effects caused by the coexisting sub-stances or the matrix containing the target element. Variousinterferences are classified to the spectral interference andthe non-spectral one, and in the latter there are interferencessuch as the physical interference and the ionization interfer-ence etc. Those interferences can be removed or reduced bythe application of a suitable reduction or correctionmethods.5.1. Physical interference

When there are differences of physical properties such asthe viscosity, the density, and the surface tension etc. be-tween the sample solution and the standard solution, theanalytical result might be affected by the differences, due tothe induction of the difference in the sample nebulizationefficiency. It is called the physical interference. In order toremove or reduce this kind of interference, dilute the samplesolution until the interference cannot occur, or conform theliquid characteristics of the sample solution to those of thestandard solution (Matrix matching method ). Separately, itis a useful correction method to adopt the internal standardmethod (Intensity ratio method) or the standard additionmethod as an assay method.5.2. Ionization interference

The ionization interference indicates the effect that themarked increase of electronic density in plasma may affectthe ionization ratio of the target element, where the coexist-ing element highly contained in the sample solution are easilyionized and release a great number of electrons in the plas-ma. The reduction or the correction method against the ioni-zation interference effect is essentially the same as thatdescribed in the section 5.1 Physical interference. Further-more, the ionization interference is moderately reduced bythe selection of the observation way and the adjustments ofthe observation height, the radio frequency power output,and the flow rate of carrier gas, etc.5.3. Spectral interference

The spectral interference indicates that analytical resultsare affected by the overlapping of various emission lines orcontinuous spectra to the analytical line of the target ele-ment. The causes and the correction methods to this interfer-ence are shown below.5.3.1. Interference by the coexisting elements

This interference occurs when the emission line of coexist-ing element in the sample solution come close to the analyti-cal line of the target element. The degree of the interferencedepends on the resolution ability of the spectroscope, thewavelength difference between two emission lines, and theintensity ratio. To avoid this interference another analyticalline can also be available. Where an appropriate analyticalline cannot be found, it is necessary to do the spectral inter-ference correction.

The inter-element correction is one of the correctionmethods for the spectral interference. If the effect of coexist-ing element on the analytical line of the target element aremeasured in advance as a function of the emission intensityor the concentration by using the known concentration oftwo-component or multi-component standard solution, the


degree of interference on the emission intensity of the ana-lytical line or the concentration will be speculated by measur-ing the emission intensity of the coexisting element togetherwith measurement of the target element. Separately, thematrix matching method or the mathematical spectral sepa-ration technique, by which the spectra of coexisting elementsuperimposed to the analytical line are separated, can also beused for the correction.5.3.2. Background interference

The background interference indicates the following effectthat analytical results are affected by the increase of back-ground due to the emission lines caused by the element high-ly contained in the sample. In this case, this effect will be re-moved by applying the following correction method. Basedon the background behavior before and after the analyticalline, estimate the background intensity at the position of theanalytical line, and obtain an intrinsic emission intensity ofthe target element by subtraction of this background inten-sity from the apparent emission intensity.

Furthermore where the pretreatment of organic sample isnot complete, the molecular band spectra (NO, OH, NH,CH, etc.) generated by elements of N, O, H, C in the samplesolution may happen to affect the intensity of the analyticalline. This kind of interference is moderately reduced by theselection of the observation way and the adjustments of theobservation height, the radio frequency power output, andthe flow rate of carrier gas, etc.

6. Qualitative and quantitative analysis6.1. Qualitative analysis6.1.1. Identification of inorganic impurities such as metalelements

In JP, the confirmation test for drug substances is usuallyspecified by applying the method of spectral analysis, bywhich the structural features of the target molecule can betotally found. Drug substances frequently contain specificelements such as N, S, P, halogens, and metals etc. in theirmolecule, which are required for their existence in the in-dividual specification. Where the existence is not confirmedby such as spectral analyses, it is required for their confirma-tion by using chemical reactions. Thus the present methodcan be applied to their confirmation test, except for nitrogen(N).

When a few of wavelengths and their relative intensity ofthe target element in the sample solution coincide with thoseof the standard solution, the existence of the target elementis confirmed. In place of the standard solution, the spectralibrary attached to the individual instrument or the wave-length table provided by the academic society, can also beused for the confirmation.6.1.2. Profile analysis

If the target elements such as metal catalysts, inorganicelements, and other high toxicity elements such as Pb andAs, which should be ordinarily controlled in view of the drugsafety, are decided to be controlled, the profile analysis ofthose inorganic impurities can be performed by the presentmethod as a part of the good manufacturing practice of drugsubstances.

Although the analytical line of the individual element canbe selected referring to Table 1, an appropriate anotheremission line can also be available, when the spectral inter-ferences are supposed to affect the analytical results. Thestandard solution of the individual element is adjusted to a

suitable concentration, in consideration of a permitted limitof the respective element, specified separately. However,when the multi-component standard solution is prepared, itshould be confirmed beforehand that any trouble such ascoprecipitation does not occur.

The confirmation test of the target element should be doneaccording to the section 6.1.1, and the impurity content ofeach element in the sample specimen is roughly estimated bythe ratio of emission intensity of the sample solution and thestandard solution at the analytical line (1 point calibrationmethod).6.2. Quantitative analysis

The quantitative evaluation of inorganic impurities in thesample specimen is usually done by measuring the emissionintensity during a certain time of integration.6.2.1. Calibration curve method

Regarding to the target element, not less than 4 kinds ofthe calibration standard solutions with different concentra-tion are prepared. By using these calibration standards, plotthe emission intensity at the analytical line versus the concen-tration to obtain the calibration curve. After measuring theemission intensity of the sample solution, the concentrationof the target element is determined by the calibration curve.6.2.2. Internal standard method

Regarding to the target element, not less than 4 kinds ofcalibration standard solutions with different concentration,in which a definite concentration of internal standard is con-tained, are prepared. Usually, yttrium (Y) is used as an inter-nal standard. By using these calibration standards, plot theratio of emission intensity of the target element and the in-ternal standard versus the concentration to obtain thecalibration curve. In the preparation of the sample solution,the internal standard element is added to give the same con-centration as the calibration standards. After measuring theratio of the emission intensity of the target element and theinternal standard, the concentration of the target element isdetermined by the calibration curve.

Applying this method to inorganic impurity analysis, itshould be confirmed that the internal standard element is notcontained in the sample. As for the internal standard ele-ment, it is expected that changes of the emission intensitydue to the operational conditions and the solution character-istics, are similar to those of the target element, and theselection of emission line is necessary so as not to give thespectral interference to the analytical line.6.2.3. Standard addition method

Take not less than 4 sample solutions of equivalentvolume, add the target element to these solutions to preparethe calibration standard solutions with different concentra-tions containing zero addition. After measuring the emissionintensity of these calibration standards, plot the emission in-tensity at the analytical line versus the concentration. Theconcentration of the target element is determined to be theconcentration, at which the regression line and the abscissacross each other.

This method can be applied only to the case of analysiswith no spectral interference, or where the background andthe spectral interference are exactly corrected, and the rela-tion between the emission intensity and the concentrationkeeps good linearity.

References1) Japanese Industrial Standard: General Rules for Atomic


Emission Spectrometry (JIS K 0116), Japanese StandardAssociation (2003).

2) US Pharmacopeia 31 (2008), <730> PLASMA SPEC-TROCHEMISTRY.

3) European Pharmacopoeia 6.0 (2008), 2.2.57 INDUC-TIVELY COUPLED PLASMA-ATOMIC EMISSIONSPECTROMETRY.

4) Data Book on Analytical Chemistry, Ed. by The JapanSociety for Analytical Chemistry, pp. 88 � 90 (2004),Maruzen Press.

5) European Medicines Agency: Guideline on the specifica-tion limits for residues of metal catalysts or metal rea-gents (2008).

Near Infrared SpectrometryNear infrared spectrometry (NIR) is one of spectroscopic

methods used to qualitatively and quantitatively evaluatesubstances from analysis of data obtained by determiningtheir absorption spectrum of light in the near-infrared range.

The near-infrared range lies between the visible light andinfrared light, typically of wavelengths (wave numbers) be-tween 750 and 2500 nm (13333 � 4000 cm－1). The absorptionof near-infrared light occurs due to harmonic overtonesfrom normal vibration or combination tones in the infraredrange (4000 to 400 cm－1), primarily absorption of O�H,N�H, C�H and S�H that involve hydrogen atoms, in par-ticular. For instance the asymmetrical stretching vibration ofN�H occurs in the vicinity of 3400 cm－1, but the absorptiondue to the first harmonic overtone occurs in the vicinity of6600 cm－1 (wavelength 1515 nm), which is near double 3400cm－1.

Absorption in the near-infrared range is far weaker thanabsorption due to normal vibration that occurs in the in-frared range. Furthermore, in comparison with visible light,near-infrared light has longer wavelength, which makes itpossible for the light to penetrate to a depth of several mminto solid specimens including fine particles. This method isoften utilized as a nondestructive analysis, as changes occur-ring with absorbed light spectrum (transmitted light orreflected light) in this process provide physical and chemicalinformation pertaining to specimens.

Conventional spectrometry, such as calibration curvemethod, is used as a method for analyzing near-infraredabsorption spectrum whenever applicable. Ordinarily,however, chemometrics methods are used for analysis.Chemometrics ordinarily involve quantification of chemicaldata, as well as numerical and statistical procedures forcomputerization of information. Chemometrics for near-infrared spectrometry includes various types of multivariateanalysis such as multiple regression analysis, to performqualitative or quantitative evaluation of active substances.

Near-infrared spectrometry is used as a rapid and nondes-tructive method of analysis that replaces conventional andestablished analysis methods for water determinations orsubstance verifications. It is necessary to perform a compari-son test to evaluate this method against an existing analysismethod, to verify that this method is equivalent to suchexisting analysis method, before using this analysis methodas a quality evaluation test method in routine tests.

Applications of near-infrared spectrometry in the phar-

maceutical field include qualitative or quantitative evalua-tion of ingredients, additives or water contents of activesubstances or preparations. Furthermore, near-infraredspectrometry can also be used for evaluation of physicalconditions of substances, such as crystal forms, crystallinity,particle diameters. It is also possible to perform spectrome-try on samples that are located in a remote location awayfrom equipment main units, without sampling, by usingoptical fibers. It can therefore be used as an effective meansto perform pharmaceutical manufacturing process controlonline.

1. EquipmentNear-infrared spectrophotometers can either be a dis-

tributed near-infrared spectrophotometer or a Fourier trans-form near-infrared spectrophotometer1). Interference filter-type near-infrared spectrophotometers that use interferencefilter in the spectrometry section are also available, however,this type of equipment is hardly used in the field of phar-maceutical quality control.1.1. Distributed near-infrared spectrophotometer

This equipment is comprised of light source section, sam-ple section, spectrometry section, photometry section, signalprocessing section, data processing section, display-record-output section. Halogen lamps, tungsten lamps, light emit-ting diodes and other such devices that can emit high inten-sity near-infrared light in a stable manner are used in thelight source section. The sample section is comprised of asample cell and a sample holder. Equipment that have anoptical fiber section that is comprised of optical fibers and acollimator are equipped with a function for transmittinglight to sample section, which is remotely located away fromthe spectrophotometer main unit. Quartz is ordinarily usedas material for optical fibers.

The spectrometry section is intended to extract light of re-quired wavelength, using dispersive devices and is comprisedof slits, mirrors and dispersive devices. Potential dispersivedevices include prisms, diffraction grating, acousto-opticaltunable filters (AOTF), or liquid crystal tunable filters(LCTF).

The photometry section is comprised of detectors and am-plifiers. Sensors include semiconductor detectors (silicon,lead sulfide, indium-gallium-arsenic, indium-antimony), aswell as photomultiplier tubes. Detecting methods that usesemiconductor detectors generally perform detections withsingle elements, but there are also occasions where array-type detectors that use multiple elements are used. Such de-tectors are capable of simultaneously detecting multiplewavelengths (wave numbers). The signal processing sectionseparates signals required for measurements from output sig-nals fed by amplifiers and then outputs such isolated signals.The data processing section performs data conversions andspectral analysis, etc. The display-record-output section out-puts data, analysis results and data processing results to aprinter.1.2. Fourier transform near-infrared spectrophotometer

The configuration of the equipment is fundamentallysame as that of the distributed-type equipment described inSection 1.1, except for the spectrometry section and the sig-nal processing section.

The spectrometry section is comprised of interferometers,sampling signal generators, detectors, amplifiers, A/D con-version devices, etc. Interferometers include Michelson inter-


ferometers, transept interferometers and polarization inter-ferometers. The signal processing section is equipped withfunctions that are required for spectrometer, as well as afunction for translating acquired interference waveform(interferogram) into absorption spectrum by Fourier trans-formation.

2. DeterminationThere are three types of measurement methods that are

used with near-infrared spectrometry: transmittancemethod, diffuse reflectance method and transmittancereflectance method. The selection of measurement methodsrelies on the shape of samples and applications. The trans-mittance method or diffuse reflectance method is used forsolid samples, including fine particles. The transmittancemethod or transmittance reflectance method is used for liq-uid samples.2.1. Transmittance method

The degree of decay for incident light intensity as the lightfrom a light source passes through a sample, is representedas transmittance rate T (z) or absorbance A with the trans-mittance method. A sample is placed in the light path be-tween a light source and a detector, the arrangement ofwhich is ordinarily same as that of the spectroscopic method.

T＝ 100tT＝ I/I0＝ 10－acl

I0: Incident light intensityI: Transmitted light intensitya: Absorptivityc: Solution concentrationl: Layer length (sample thickness)

A＝－log t＝ log (1/t )＝ log (I0/I )＝ acl

This method is applied for taking measurements of sam-ples that are liquids and solutions. Quartz glass cells andflow cells are used, with the layer length of 1 � 5 mm along.Furthermore, this method can also be applied for takingmeasurements of samples that are solids, including fine par-ticles. It is also known as diffuse transmittance method.Selecting appropriate layer length is critical for this method,since the transmitted light intensity varies depending ongrain sizes and surface conditions of samples.2.2. Diffuse reflectance method

The ratio of the reflection light intensity I, emitted fromthe sample in a wide reflectance range and a control reflec-tion light intensity Ir emitted from surface of a substance, isexpressed as reflectance R (z) with the diffuse reflectancemethod. The near-infrared light penetrates to a depth ofseveral mm into solid samples, including fine particles. Inthat process, transmission, refraction, reflection and disper-sion are repeated, and diffusion takes place, but a portion ofthe diffused light is emitted again from the surface of thesample and captured by a detector. The spectrum for thediffuse reflectance absorbance (Ar) can ordinarily be ob-tained by plotting logarithm of inverse numbers for reflec-tance (1/r) against wavelengths (wave numbers).

R＝ 100rr＝ I/Ir

I: Reflection light intensity of light, diffuse reflected offthe sample

Ir: Control reflection light intensity of light emitted fromsurface of reference substance

Ar＝ log (1/r)＝ log (Ir/I )

The intensity of diffuse reflectance spectrum can also beexpressed with the Kubelka-Munk (K-M) function. The K-Mfunction is derived, based on the existence of a sample withsufficient thickness, and expressed in terms of light scatter-ing coefficient, which is determined by absorptivity, grainsize, shape and fill condition (compression).

This method is applied to solid samples, including fineparticles, and requires a diffuse reflector.2.3. Transmittance reflectance method

The transmittance reflectance method is a combination ofthe transmittance method and reflectance method. A mirroris used to re-reflect a light that has passed through a samplein order to take a measurement of transmittance reflectancerate, T* (z). Light path must be twice the thickness of thesample. On the other hand, the light reflected off a mirrorand enters into a detector is used as the control light. Whenthis method is applied to suspended samples, however, ametal plate or a ceramic reflector with rough surface thatcauses diffuse reflectance is used instead of a mirror.

Transmittance reflectance absorbance (A*) is obtained bythe following formula with this method:

T *＝ 100t*t*＝ I/IT

I: Intensity of transmitted and reflected light, in caseswhere a sample is placed

lT: Intensity of reflected light, in cases where is no sample

A*＝ log (1/t*)

This is a method that is applied to solid samples, includingfine particles, as well as liquids and suspended samples. Thethickness of a sample must be adjusted when applying thismethod to a solid sample. Ordinarily adjustment is made bysetting absorbance to 0.1 � 2 (transmittance of 79 � 1z),which provides the best linearity and S/N ratio of detector.A cell with appropriate layer length, according to the grainsize of the fine particle, must be selected when applying themethod to a fine particle sample.

3. Factors that affect spectrumFollowing items must be considered as factors that can af-

fect spectrum when applying near-infrared spectrometry,particularly when conducting quantitative analysis.

(i) Sample temperature: A significant change (wave-length shift, for example) can occur when the temperaturevaries by a several degree (9C). Care must be taken, particu-larly when the sample is a solution or contains water.

(ii) Water or residual solvent: Water or residual solventcontents of a sample, as well as water (humidity) in the en-vironment wherein measurements are taken, can potentiallysignificantly affect absorption band of the near-infraredrange.

(iii) Sample thickness: The thickness of a sample is afactor for spectral changes and therefore needs to be con-trolled at a certain thickness. A sample may be considered tobe of adequate thickness for the diffuse reflectance method,however, if the thickness is less than a certain amount, forexample, the sample may have to be placed on a supportplate with high reflectance to take measurements by thetransmittance reflectance method.

(iv) Fill condition of sample: The condition of samplefill can potentially affect spectrum, when taking measure-


ments of samples that are solids or fine particles. Care mustbe taken when filling samples in a cell, to ensure that a cer-tain amount is filled through a specific procedure.

(v) Optical characteristics of samples: When a sampleis physically, chemically or optically uneven, relatively largebeam size must be used, multiple samples must be used,measurements must be taken at multiple points on the samesample, or a sample must be pulverized to ensure averagingof the sample. Grain size, fill condition, as well as roughnessof surface can also affect fine particle samples.

(vi) Crystal forms: Variations in crystal structures(crystal forms) can also affect spectrum. In cases where mul-tiple crystal forms exist, it is necessary to have considerationfor characteristics of samples to be considered and care mustbe taken to ensure that even standard samples for calibrationcurve method have diversified distributions similar to that ofsamples that are subject to analysis.

(vii) Temporal changes in characteristics of samples:Samples can potentially undergo chemical, physical oroptical property changes, due to passing of time or storageafter sampling, and such changes affect spectrum in a subtlemanner. For instance even with identical samples, if elapsedtimes differ, then their characteristics of near-infrared spec-trum can vary significantly. In creating calibration curves,therefore, measurements must be taken offline in a laborato-ry or online in manufacturing process (or inline) and samplesfor calibration curves must be prepared with adequate con-siderations for the passing of time before measurements aretaken.

4. Control of equipment performance2,3)

4.1. Accuracy of wavelengths (wave numbers)The accuracy of wavelengths (wave numbers) of an equip-

ment is derived from the deviation of substances for whichpeak absorption wavelengths (wave numbers) have beendefined, such as polystyrene, mixture of rare earth oxides(dysprosium, holmium and erbium; 1:1:1) or steam, fromthe figures indicated on the equipment. Tolerance figures inthe vicinity of 3 peaks are ordinarily set in the followingmanner:

1200± 1 nm (8300± 8 cm－1)1600± 1 nm (6250± 4 cm－1)2000± 1.5 nm (5000± 4 cm－1)Since the location of absorption peaks vary, depending on

the substance used as reference, absorption peaks of wave-lengths (wave numbers) that are closest to the above 3 peaksare selected for suitability evaluations. A mixture of rareearth oxides, for instance, would indicate characteristicabsorption peaks at 1261 nm, 1681 nm and 1971 nm.

Absorption peaks at 1155 nm, 1417 nm, 1649 nm, 2352nm (layer length: 1.0 mm) can be used, when taking meas-urements with transmittance method that involve the use ofdichloromethane as reference. The absorption peak of steamat 7306.7 cm－1 can be used with a Fourier transformation-type spectrophotometer, as its wave number resolutionability is high.

Other substances can also be used as reference, so long astheir adequacy for the purpose can be verified.4.2. Spectroscopic linearity

Appropriate standard plates, such as plate-shaped poly-mer impregnated with varying concentrations of carbon(carbon-doped polymer standards), can be used to evaluatespectroscopic linearity. In order to verify linearity, however,

standard plates with no less than 4 levels of concentrationwithin the reflectance of 10 � 90z must be used. Whenmeasurements are expected to be taken with absorbance ofno less than 1.0, it is necessary to add standard plates withreflectance of either 2z or 5z or both.

In order to plot absorbance (AOBS) of such standard platesat locations in the vicinity of wavelengths 1200 nm, 1600 nmand 2000 nm against absorbance (AREF) assigned to eachstandard plate, verifications must be made to ensure that thegradient of linearity obtained are ordinarily within the range1.0± 0.05 for each of these wavelengths and 0± 0.05 forordinate intercept.4.3. Spectrophotometric noise

The spectrophotometric noise of the equipment can bechecked using appropriate reflectance standard plates, suchas white-colored reflecting ceramic tiles or reflective ther-moplastic resin (such as polytetrafluoroethylene).4.3.1. High flux noise

Spectrophotometric noise is evaluated by using standardplates with high reflectance, such as reflectance of 99z.Standard plates are used to take measurements for both sam-ples and control samples. Generally, the average value ob-tained from calculation of mean square root (RMS ) of noisefor each 100 nm segments in the wavelength range of 1200 �2200 nm ordinarily must not be more than 0.3× 10－3 andindividual values must not exceed 0.8× 10－3.

RMS＝s1/N・S(Ai－ Am)2t1/2

N: Number of measurement points per segmentAi: Absorbance at each measurement point of segmentAm: Average absorbance for segment

4.3.2. Low flux noiseSpectrophotometric noise is evaluated by using standard

plates with low reflectance, such as reflectance of 10z,when the amount of light is low. In such cases, light source,optical system, detector and electronic circuit systems allhave some impact on noise. Similar to the cases of high fluxnoise, generally, the average value obtained from calculationof RMS for each 100 nm segments in the wavelength rangeof 1200 � 2200 nm ordinarily must not be more than 1.0×10－3 and individual values must not exceed 2.0× 10－3.

5. Application to qualitative or quantitative analysisUnlike in the infrared range, mainly harmonic overtones

and combinations manifest as spectrum in the near-infraredrange. Such absorbance spectrums are often observed asoverlay of absorption bands of functional groups and atomicgroups. The near-infrared spectrometry, therefore, differsfrom conventional analysis methods and it is usually neces-sary to establish analysis methods that correspond to eachapplication, by preparing model analysis methods usingmethodologies of chemometrics, such as multivariateanalysis.

Characteristics of near-infrared absorption spectrum mustbe emphasized and effects of complexities of spectrums, aswell as overlay of absorption bands must be reduced byperforming mathematical preprocesses, such as primary orsecondary spectral differentiation processes or normaliza-tions, which becomes one of vital procedures in establishinganalysis methods that use methodologies of chemometrics.While there are many chemometrics methodologies andmathematical preprocessing methods for data, appropriatecombinations must be selected that suit the purposes of


intended analysis.Evaluation of validity based on analysis parameters is

ordinarily required for the analysis validation when estab-lishing a near-infrared analysis method. Selection ofparameters that are appropriate for applications must bemade for its intended use. Furthermore, following issuesmust be considered, in conformity with attributes of thenear-infrared spectrometry.

(i) Whether or not wavelengths (wave numbers) intendedfor the particular analysis method, are suitable for evalua-tion of characteristics of a sample in performing analysisunder given conditions.

(ii) Whether or not the method is adequately robust todeal with variables such as handling of samples (for instancefill condition for fine particle samples, etc.) and configura-tion matrix.

(iii) Whether or not about the same level of accuracy orprecision can be obtained, in comparison with the existingand established analysis methods, which are available asstandards.

(iv) Sustaining and managing performance of an analysismethod, once established, are critical. Continuous and sys-tematic maintenance and inspection work must therefore beimplemented. Furthermore, it must be determined whetheror not appropriate evaluation procedures are available todeal with change controls or implementation of re-validationon changes made in manufacturing processes or raw mate-rials, as well as changes arising from replacement of majorcomponents in equipment.

(v) Whether or not there are appropriate evaluationprocedures in place to verify validity of transferring im-plementation of an analysis, which presupposes the use of aspecific equipment, from such originally intended equipmentto another equipment (model transfer) for the purpose ofsharing the analysis method.5.1. Qualitative analysis

Qualitative analysis, such as verification of substances, isperformed after preparing a reference library that includesinter-lot variations within tolerance range and chemometricsmethodologies, such as multivariate analysis, have beenestablished. Minute quality characteristic variations betweenlots can also be estimated by using this method.

Furthermore, multivariate analysis includes direct analysismethods that consider wavelengths (wave numbers) andabsorptions as variables, such as wavelength correlationmethod, residual sum of squares, range sum of squares,along with factor analysis method, cluster analysis method,discriminant analysis method, as well as SIMCA (soft in-dependent modeling of class analogy).

It is also possible to consider the overall near-infraredabsorption spectrum as a single pattern and to identifyparameters obtained by applying multivariate analysismethods or characteristic wavelength (wave number) peaksof the sample substance as indices for monitoring, for thepurpose of manufacturing process control for active sub-stances or preparations.5.2. Quantitative analysis

Quantitative analysis uses spectrums of sample groups andanalysis values obtained through the existing and establishedanalysis methods, to obtain quantitative models withmethodologies of chemometrics. These are used to calculateconcentrations of individual ingredients and material valuesof samples being measured, using conversion formulas.

Chemometrics methodologies for obtaining quantitativemodels include multiple regression analysis method, mainingredient regression analysis method and PLS (partial leastsquares) regression analysis method.

In cases where the composition of a sample is simple, con-centrations of ingredients in the sample that are subject toanalysis can be calculated, by plotting a calibration curveusing the absorbance of a specific wavelength (wave number)or the correlating relationship between the parameters andconcentration, using samples for preparation of calibrationcurves with known concentrations (calibration curvemethod).

6. Reference1) General Rules for Near-infrared Spectrophotometric

Analysis, JIS K 0134 (2002), Japanese Industrial Stand-ards

2) Near-infrared Spectrophotometry, 2.2.40, EuropeanPharmacopoeia 5.0 (2005)

3) Near-infrared Spectrophotometry, <1119>, US Phar-macopoeia 30 (2007)

pH Test for GastrointestinalMedicine

In this test, medicine for the stomach and bowels, which issaid to control stomach acid, is stirred in a fixed amount ofthe 0.1 mol/L hydrochloric acid for a fixed duration, andthe pH value of this solution is obtained. The pH value of astomach medicine will be based on the dose and the dosageof the medicine (when the dosage varies, a minimum dosageis used) and expressed in the pH value obtained from the testperformed by the following procedure.

1. Preparation of SampleSolid medicine which conforms to the general regulations

for medicine (the powdered medicine section) can be used asa sample. When the medicine is in separate packages, thecontent of 20 or more packages is accurately weighed to cal-culate the average mass for one dose and mixed evenly tomake a sample. For granules and similar types in separatepackages, among the solid medicine which does not conformto the general regulations for medicine (the powdered medi-cine section), the content of 20 or more packages is accu-rately weighed to calculate the average mass for one doseand is then powdered to make sample. For granules andsimilar types not in separate packages, among solid medicinewhich does not conform to the general regulations for medi-cine (the powdered medicine section), 20 doses or more arepowdered to make a sample. For capsules and tablets, 20doses or more are weighed accurately to calculate theaverage mass for one dose or average mass and then pow-dered to make a sample.

Liquid medicine is generously mixed to make a sample.

2. ProcedurePut 50 mL of the 0.1 mol/L hydrochloric acid with the

molarity coefficient adjusted to 1.000, or equivalent 0.1mol/L hydrochloric acid with its volume accurately meas-ured in a 100-mL beaker. Stir this solution with a magneticstirrer and a magnetic stirrer rotator (35 mm length, 8 mmdiameter) at the speed of about 300 revolutions per minute.


While stirring, add the accurately weighed one-dose sample.After 10 minutes, measure the pH value of the solution usingthe pH Determination. The solution temperature should bemaintained at 37± 29C throughout this operation.

System SuitabilityIn order to ensure the reliability on the results of drug ana-

lyses, it is essential to verify that the test method to be ap-plied to the test, including the method prescribed in theJapanese Pharmacopoeia (JP), can give the results adequatefor its intended use using the analytical system in the labora-tory in which the test is to be performed, then to carry outsystem suitability testing for confirming that the analyticalsystem maintains the state suitable for the quality test.

1. Definition and role of system suitability``System Suitability'' is the concept for ensuring that the

performance of the analytical system is as suitable for theanalysis of the drug as was at the time when the verificationof the test method was performed using the system.

Usually, system suitability testing should be carried out atevery series of drug analysis. The test procedures and accep-tance criteria of system suitability testing must be prescribedin the test methods of drugs. The results of drug analyses arenot acceptable unless the requirements of system suitabilityhave been met.

System suitability testing is an integral part of testmethods using analytical instruments, and based on the con-cept that the equipments, electronic data processing systems,analytical operations, samples to be analyzed and operatorsconstitute an integral system that can be evaluated, when thetest procedures and acceptance criteria of system suitabilitytesting are prescribed in the test methods.

2. Points to consider in setting system suitabilityParameters of system suitability testing to be prescribed in

the test method depend on the intended use and type of ana-lytical method. Since system suitability testing is to be car-ried out in a routine manner, it is preferable to select theparameters necessary for ensuring that the analytical systemmaintains the state suitable for the analysis of the drug andto prescribe its test procedure able to carry out easily andrapidly.

For example, in the case of quantitative purity tests usingliquid chromatography or gas chromatography, the evalua-tion of parameters such as ``System performance'' (to con-firm the ability to analyze target substance specifically),``System repeatability'' (to confirm that the degree of varia-tion in the analytical results of target substance in replicateinjections is within the allowable limit) and ``Test for re-quired detectability'' (to confirm the linearity of chromato-graphic response around the specification limit) are usuallyrequired.

The followings are supplements to the section of systemsuitability prescribed in ``Liquid Chromatography''.2.1. System repeatability of HPLC and GC2.1.1. Allowable limit of system repeatability

It is described in the section of system suitability in ``Liq-uid Chromatography'' that ``In principle, total number ofreplicate injections should be 6'', and ``The allowable limitof ``System repeatability'' should be set at an appropriate

level based on the data when suitability of the method forthe evaluation of quality of the drug was verified, and theprecision necessary for the quality test''.

Based on the above description, an allowable limit of sys-tem repeatability for 6 replicate injections should be set inconsideration with the following descriptions. However, inthe case that the test method prescribed in the JP monographis used for the test, the allowable limit of system repeatabili-ty prescribed in the monograph should be applied.

(i) Assay for drug substance (for drug substance with thecontent nearby 100z): An adequate allowable limit shouldbe set at the level that the chromatographic system is able togive the precision suitable for the evaluation of variation inthe content of active ingredient within and among the batch-es of drug substance. For example, the allowable limit of``not more than 1.0z'' is usually recommended for the drugsubstances whose width of content specification are notmore than 5z, as is in the case of content specification of98.0 � 102.0z which is often observed in the assay using liq-uid chromatography.

(ii) Assay for drug products: An adequate allowablelimit should be set considering the width of content specifi-cation of the drug product and the allowable limit prescribedin the assay of drug substance (when the drug product is ana-lyzed by a method with the same chromatographic condi-tions as those used for the analysis of drug substance).

(iii) Purity test for related substances: An adequateallowable limit should be set considering the concentrationof active ingredients in the solution used for the system suita-bility testing. In the case that a solution with active ingredi-ent concentration of 0.5 � 1.0z is used for the test of systemrepeatability, an allowable limit of ``not more than 2.0z'' isusually recommended.

Recommendations for allowable limits described aboveshould not be applicable to gas chromatography.2.1.2. Method for decreasing the number of replicate injec-tions without losing the quality of system repeatability test-ing

It is described in the section of system suitability in ``Liq-uid Chromatography'' that ``In principle, total number ofreplicate injections should be 6. However, in the case that along time is necessary for one analysis, such as the analysisusing the gradient method, or the analysis of samples con-taining late eluting components, it may be acceptable todecrease the number of replicate injections by adopting newallowable limit of ``System repeatability'' which can guaran-tee a level of ``System repeatability'' equivalent to that at 6replicate injections.''

In consideration of the above description, a method fordecreasing the number of replicate injections without losingthe quality of system repeatability testing is adopted. Onecan set the test for system repeatability with reduced numberof replicate injections by utilizing this method, if necessary,and can also apply it as an alternative for the methodprescribed in a monograph.

The following table shows the allowable limits to be at-tained in the test at 3 � 5 replicate injections (n＝ 3 � 5) tokeep the quality test equivalent to that of test at n＝ 6.

However, it should be kept in mind that since decrease inthe number of replicate injections results in increase in theweight of each injection, it becomes more important to per-form the test by the experienced operator, and to maintainthe equipment in a suitable state.

2146

Table Allowable limits to be attained in the test at 3 � 5 replicate injections (n＝ 3 � 5) to keep the quality of test equivalentto that of test at n＝ 6*

Allowable limit (RSD)

Allowable limitprescribed in the test

of n＝ 61z 2z 3z 4z 5z 10z

Allowablelimit to be

attained

n＝ 5 0.88z 1.76z 2.64z 3.52z 4.40z 8.81z

n＝ 4 0.72z 1.43z 2.15z 2.86z 3.58z 7.16z

n＝ 3 0.47z 0.95z 1.42z 1.89z 2.37z 4.73z

* The probability for inadequate analytical systems to meet the requirements of system suitability testing, is supposed to be5z.


3. Points to consider at the change of analytical system(Change control of analytical system)

When the test method and analytical system verified iscontinuously used for the quality test without any change, itis sufficient to confirm the compliance to the requirementsof system suitability at every series of drug analysis.

However, when the test is performed for a long period, asituation in which some changes in the analytical system areinevitable, may occur. These changes don't affect the qualityof the product itself, but they affect the scale in the evalua-tion of product quality. If the change in the analytical systemmay induce a significant deviation of the scale, it may lead tothe acceptance of products with inadequate quality and/orthe rejection of products with adequate quality. Thus, at thetime of change in the analytical system, it is necessary tocheck whether the change is appropriate or not, to avoid thedeviation of the scale in the evaluation of product quality.

In the case of the change of test method, it is required toperform an adequate validation depending on the extent ofthe change.

On the other hand, in the case of the change of analyticalsystem in a laboratory, such as renewal of apparatus orcolumn of liquid chromatography, and the change of opera-tor, it is necessary to perform at least system suitability test-ing using the system after change, and to confirm that theequivalency of the results before and after change.

In the case that equivalent results would not be obtainedafter change, for example, when a renewal of column of liq-uid chromatograph may induce a significant change ofelution pattern, such as the reversal of elution order betweentarget ingredient of the test and substance for checking reso-lution, it is required to perform a revalidation of the analyti-cal system for the test using new column, since it is uncertainwhether the specificity and/or other validation characteris-tics necessary for estimating target ingredient is kept or not.

Test for Trace Amounts ofAluminum in Trans Parenteral

Nutrition (TPN) SolutionsTrans parenteral nutrition solutions (TPNs) are nutrient

preparations for intravenous injection. Since toxic effects tothe central nervous system, bone, etc. due to trace amounts

of aluminum have recently been reported in several coun-tries, testing methods for trace amounts of aluminum con-taminating TPNs are required for the official standard. Thefollowing three analytical methods are available: High-Performance Liquid Chromatography using a fluorescencephotometric detector (HPLC with fluorescence detection),Inductivity Coupled Plasma-Atomic Emission Spectrometry(ICP-AES method), Inductivity Coupled Plasma-MassSpectrometry (ICP-MS method). Detection sensitivity byHPLC with fluorescence detection is about 1 mg/L (ppb),while ICP-AES fitted with special apparatus and ICP-MShave higher sensitivity.

Since TPNs are nutrient preparations, they contain manynutrients such as sugars, amino acids, electrolytes, etc., invarious compositions. Thus, care is needed in the selectionof a suitable analytical method, because these coexistingcomponents may affect the measurement of trace amountsof aluminum.

In view of the general availability of HPLC apparatus, thepresent general information describes procedures for the de-termination of trace levels of aluminum in TPNs by meansof HPLC with a fluorescence photometric detector, usingtwo kinds of fluorescent chelating agents, i.e., Quinolinolcomplexing method, Lumogallion complexing method.

1. Quinolinol complexing methodAfter forming a complex of aluminum ion in the sample

solution with quinolinol, the assay for aluminum by HPLCfitted with a fluorescence photometer is performed.1.1. Preparation of sample solution

Pipet 1 mL of the sample (TPNs) exactly, and after adding10 mL of water for aluminum test, make up the sample solu-tion to 10 mL exactly by adding the mobile phase.1.2. Preparation of a series of standard solutions forcalibration curve

Pipet 1 mL of water for aluminum test exactly, and afteradding 10 mL each of standard solutions of aluminum (1)�(5), make up the standard solutions for calibration curve to10 mL (Aluminum concentration: 0, 1.25, 2.5, 5.0, and 10.0ppb).1.3. Standard testing method

Pipet 0.1 mL each of the sample solution and standard so-lutions, and perform the test by HPLC under the followingconditions. Calculate the aluminum cotent in the sample so-lution using a calibration curve method.Operating conditions�


Detector: Fluorescence photometer(excitation wavelength:380 nm, emission wavelength: 520 nm)

Column: A stainless steel column 4.6 mm in inside diame-ter and 15 cm in length, packed with phenylsilanized silicagel for liquid chromatography (5 mm in particle diameter).

Column temperature: A constant temperature of about409C.

Mobile phase: A mixture of 8-quinolinol in acetonitrile (3in 100) and diluted 0.5 mol/L ammonium acetate TS (2 in 5)(1:1).

Flow rate: Adjust the flow rate so that the retention timeof aluminum/8-quinolinol complex is about 9 minutes.System suitability�

The correlation coefficient of the calibration curve, whichis prepared using a series of standard solutions, is not lessthan 0.99.

Furthermore there is an alternative method, in which thechelating agent 8-quinolinol is not included in the mobilephase. In this method also, aluminum is detected as a com-plex with 8-quinolinol in the sample solution by using HPLCfitted with fluorescence photometer. But it is necessary toform a more stable aluminum/8-quinolinol complex in thesample solution, because the chelating agent is not includedin the mobile phase. Further, since the analytical wavelengthfor the fluorescence detection is different from that in thestandard method, excitation WL: 370 nm, emission WL: 504nm, the detection sensitivity is different. Thus, it is appropri-ate to obtain the calibration curve between 0 � 25 ppb of alu-minum. Other than the above- mentioned differences, thesize of column, column temperature, and the mobile phaseare also different from those used in the standard method, sosuitable analytical conditions should be established for per-forming precise and reproducible examinations of traceamounts of aluminum in the sample specimen.

2. Lumogallion complexing methodAfter forming a complex of aluminum ion in the sample

specimen with the fluorescent reagent of lumogallion, the so-lution is examined by HPLC fitted with a fluorescence pho-tometer.2.1. Preparation of sample solution

Pipet 70 mL of the sample specimen (TPN) exactly, add0.15 mL of lumogallion hydrochloric acid TS and 0.6 mL ofbuffer solution for aluminum test, pH 7.2 exactly, then mixthe solution. After this solution has been allowed to standfor 4 hours at 409C, it can be used for the measurement as asample solution.2.2. Preparation of a series of standard solutions forcalibration curve

Pipet 1 mL each of standard aluminum solutions (1) � (5)exactly, and add diluted nitric acid for aluminum test (1 in100) to make exactly 100 mL. Pipet 70 mL each of these solu-tions exactly, and add exactly 0.15 mL of lumogallion hy-drochloric acid TS and exactly 0.6 mL of buffer solution foraluminum test, pH 7.2 then allow to stand for 4 hours at409C to make a series of standard solutions for obtaining thecalibration curve (Aluminum: 0, 1.07, 2.13, 4.27, and 8.54ppb).2.3. Standard examination method

Take 0.1 mL each of the sample solution and standardaluminum solutions for the calibration curve, and performHPLC analysis under the following conditions. Calculate thealuminum content in the sample solution by using a calibra-

tion curve method.Operating conditions�

Detector: Fluorescence photometer(excitation wavelength505 nm, emission wavelength 574 nm)

Column: A stainless steel column 6.0 mm in inside diame-ter and 10 cm in length, packed with octylsilanized silica gelfor liquid chromatography (5 mm in particle diameter).

Column temperature: A constant temperature of about409C.

Mobile phase: Take 100 mL of 2-propanol, and add adiluted 1 mol/L acetic acid-sodium acetate buffer solution ofpH 5.0 (1 in 10) to make 1000 mL.

Flow rate: Adjust the flow rate so that the retention timeof aluminum/lumogallion complex is about 5 minutes.System suitability�

The correlation coefficient of the calibration curve, whichis prepared using a series of standard solutions, is not lessthan 0.99.

3. Notes(i) Regarding water, solvents, reagents, vessels and other

tools used for the examination, select those not contaminat-ed with aluminum. Further, keep the testing environmentclean and free from dust in the testing room.

(ii) Before the measurement, it is necessary to confirmthat the characteristic properties of the sample do not affectthe formation of the complex.

(iii) Reference substances of river water for analysis oftrace elements, distributed by the Japan Society for Analyti-cal Chemistry, contain certified amounts of aluminum:JSAC 0301-1 and JSAC 0302 (a known amount of alumi-num is artificially added to JSAC 0301-1).

4. Standard Solutions, Reagents and Test SolutionsOther than the standard solutions, reagents and test solu-

tions specified in the Japanese Pharmacopoeia, thosedescribed below can be used in this test.

(i) N,N-Bis(2-hydroxyethyl)-2-aminoethane sulfonic acidC6H15NO5S White crystals or powder.

(ii) Hydrochloric acid for aluminum test Same as thereagent Hydrochloric acid. Further, it contains not morethan 1 ppb of aluminum.

(iii) Lumogallion [5-Chloro-2-hydroxy-3(2,4-dihydrox-yphenylazo)benzenesulfonic acid] C12H9ClN2O6S Red-brownto dark brown powder. Further, it contains not more than 1ppm of aluminum.

(iv) Lumogallion hydrochloric acid TS Dissolve 0.86 gof lumogallion in 300 mL of 2-propanol, and add 350 mL ofdiluted Hydrochloric acid for aluminum test (9 in 50) andWater for aluminum test to make 1000 mL exactly.

(v) Nitric acid for aluminum test Same as the reagentNitric acid. Further, it contains not more than 1 ppb of alu-minum.

(vi) pH buffer solution for aluminum test, pH 7.2 Dis-solve 106.6 g of N,N-bis(2-hydroxyethyl)-2-aminoethane sul-fonic acid in 800 mL of Water for aluminum test, adjust thepH 7.2 by using Tetramethylammonium hydroxide aqueoussolution, and add Water for aluminum test to make 1000mL.

(vii) Standard aluminum solution Pipet a constantvolume each of Water for aluminum test or the Standardaluminum stock solution, dilute and adjust the aluminumconcentration to 0, 1.25, 2.5, 5.0, and 10 ppm by usingdiluted Nitric acid for aluminum test (1 in 100), to make


Standard aluminum solutions (1) � (5).(viii) Tetramethylammonium hydroxide TS (CH3)4NOH

It is a 25z aqueous solution, prepared for aluminum test.Further, it contains not more than 1 ppb of aluminum.

(ix) Water for aluminum test It contains not more than1 ppb of aluminum.

Validation of Analytical ProceduresThe validation of an analytical procedure is the process of

confirming that the analytical procedure employed for a testof pharmaceutics is suitable for its intended use. In otherword, the validation of an analytical procedure requires usto demonstrate scientifically that risks in decision by testingcaused by errors from analytical steps are acceptably small.The performance of an analytical procedure is established byvarious kinds of validation characteristics. The validity of aproposed analytical procedure can be shown by demonstrat-ing experimentally that the validation characteristics of theanalytical procedure satisfy the standards set up according tothe acceptable limits of testing.

When an analytical procedure is to be newly carried in theJapanese Pharmacopoeia, when a test carried in theJapanese Pharmacopoeia is to be revised, and when the testcarried in the Japanese Pharmacopoeia is to be replaced witha new test according to regulations in general notices, ana-lytical procedures employed for these tests should be validat-ed according to this document.

1. Required data for analytical procedures to be carried inthe Japanese Pharmacopoeia1.1. Outline

This section should provide a brief explanation of theprinciple of a proposed analytical procedure, identify thenecessity of the analytical procedure and its advantage com-pared with other procedures, and summarize the validation.When an analytical procedure is revised, the limitation of thecurrent analytical procedure and the advantage offered bythe new analytical procedure should be described.1.2. Analytical procedure

This section should contain a complete description of theanalytical procedure to enable skilled persons to evaluatecorrectly the analytical procedure and replicate it if neces-sary. Analytical procedures include all important operatingprocedures for performing analyses, the preparation ofstandard samples, reagents and test solutions, precautions,procedures to verify system suitability (e.g. the verificationof the separating performance of a chromatographic sys-tem), formulas to obtain results, the number of replicationsand so forth. Any instruments and apparatus that are notstated in the Japanese Pharmacopoeia should be described indetail. The physical, chemical or biological characteristics ofany new reference standards should be clarified and their tes-ting methods should be established.1.3. Data showing the validity of analytical procedures

This section should provide complete data showing thevalidity of the analytical procedures. This includes the ex-perimental designs to determine the validation characteris-tics, experimental data, calculation results and results ofhypothesis tests.

2. Validation characteristicsThe definition of typical validation characteristics to be

assessed in validation of analytical procedures and examplesof assessing procedures are given below.

The terminology and definitions of the validation charac-teristics may possibly vary depending upon the fields towhich analytical procedures are applied. The terminologyand definitions shown in this document are established forthe purpose of the Japanese Pharmacopoeia. Typicalmethods for assessing the validation characteristics areshown in the item of assessment. Various kinds of methodsto determine the validation characteristics have beenproposed and any methods that are widely accepted will beaccepted for the present purpose. However, since values ofthe validation characteristics may possibly depend uponmethods of determination, it is required to present themethods of determining the validation characteristics, thedata and calculation methods in sufficient detail.

Although robustness is not listed as a validation character-istic, it should be considered during the development of ana-lytical procedures. Studying the robustness may help to im-prove analytical procedures and to establish appropriateanalytical conditions including precautions.2.1. Accuracy/Trueness2.1.1. Definition

The accuracy is a measure of the bias of observed valuesobtained by an analytical procedure. The accuracy is ex-pressed as the difference between the average value obtainedfrom a large series of observed values and the true value.2.1.2. Assessment

The estimate of accuracy of an analytical method is ex-pressed as the difference between the total mean of observedvalues obtained during investigation of the reproducibilityand the true value. The theoretical value is used as the truevalue (e.g., in the case of titration methods, etc.). Whenthere is no theoretical value or it is difficult to obtain a theo-retical value even though it exists, a certified value or a con-sensus value may be used as the true value. When an analyti-cal procedure for a drug product is considered, the observedvalue of the standard solution of the drug substance may beused as the consensus value.

It may be inferred from specificity data that an analyticalprocedure is unbiased.

The estimate of accuracy and a 95z confidence interval ofthe accuracy should be calculated using the standard errorbased on the reproducibility (intermediate precision). Itshould be confirmed that the confidence interval includeszero or that the upper or lower confidence limits are withinthe range of the accuracy required of the analytical proce-dure.2.2. Precision2.2.1. Definition

The precision is a measure of the closeness of agreementbetween observed values obtained independently from multi-ple samplings of a homogenous sample and is expressed asthe variance, standard deviation or relative standard devia-tion (coefficient of variation) of observed values.

The precision should be considered at three levels withdifferent repetition conditions; repeatability, intermediateprecision and reproducibility.

(i) Repeatability/Intra-assay precisionThe repeatability expresses the precision of observed

values obtained from multiple samplings of a homogenous


sample over a short time interval within a laboratory, by thesame analyst, using the same apparatus and instruments, lotsof reagents and so forth (repeatability conditions).

(ii) Intermediate precisionThe intermediate precision expresses the precision of ob-

served values obtained from multiple samplings of ahomogenous sample by changing a part of or all of the oper-ating conditions including analysts, experimental dates, ap-paratus and instruments and lots of reagents within a labora-tory (intermediate precision condition).

(iii) ReproducibilityThe reproducibility expresses the precision of observed

values obtained from multiple samplings of a homogenoussample in different laboratories (reproducibility condition).2.2.2. Assessment

A sufficient volume of a homogenous sample should beprepared before studying the precision. The solution is as-sumed to be homogenous. When it is difficult to obtain ahomogenous sample, the following samples may be used ashomogenous samples; e.g., a large amount of drug productsor mixture of drug substance and vehicles that are crushedand mixed well until they can be assumed to be homogenous.

Suitable experimental designs such as one-way layout maybe employed when more than one level of precision is to beinvestigated simultaneously. A sufficient number of repeti-tions, levels of operating conditions and laboratories shouldbe employed. Sources of variations affecting analyticalresults should be evaluated as thoroughly as possible throughthe validation.

It is required to show the variance, standard deviation andrelative standard deviation (coefficient of variation) of eachlevel of precision. The 90z confidence interval of the vari-ance and corresponding intervals of the standard deviationand relative standard deviation should also be established.The validity of the proposed analytical procedure for its in-tended use may be confirmed by comparing obtained valueswith the required values of the analytical procedure.Whether the proposed analytical procedure is acceptablemay normally be decided based on the reproducibility.2.3. Specificity2.3.1. Definition

The specificity is the ability of an analytical procedure tomeasure accurately an analyte in the presence of componentsthat may be expected to be present in the sample matrix. Thespecificity is a measure of discriminating ability. Lack ofspecificity of an analytical procedure may be compensatedby other supporting analytical procedures.2.3.2. Assessment

It should be confirmed that the proposed analytical proce-dure can identify an analyte or that it can accurately measurethe amount or concentration of an analyte in a sample. Themethod to confirm the specificity depends very much uponthe purpose of the analytical procedure. For example, thespecificity may be assessed by comparing analytical resultsobtained from a sample containing the analyte only withresults obtained from samples containing excipients, relatedsubstances or degradation products, and including or exclud-ing the analyte. If reference standards of impurities are un-available, samples that are expected to contain impurities ordegradation products may be used (e.g. samples after ac-celerated or stress tests).

2.4. Detection limit2.4.1. Definition

The detection limit is the lowest amount or concentrationof the analyte in a sample that is detectable, but not neces-sarily quantifiable.2.4.2. Assessment

The detection limit should be normally determined so thatproducer's and consumer's risks are less than 5z. The detec-tion limit may be calculated using the standard deviation ofresponses of blank samples or samples containing an analyteclose to the detection limit and the slope of the calibrationcurve close to the detection limit. The following equation isan example to determine the detection limit using the stand-ard deviation of responses of blank samples and the slope ofthe calibration curve.

DL＝ 3.3s/slope

DL: detection limits: the standard deviation of responses of blank samplesslope: slope of the calibration curve

The noise level may be used as the standard deviation ofresponses of blank samples in chromatographic methods. Itshould be ensured that the detection limit of the analyticalprocedure is lower than the specified limit for testing.2.5. Quantitation limit2.5.1. Definition

The quantitation limit is the lowest amount or concentra-tion of the analyte in a sample that can be determined. Theprecision expressed as the relative standard deviation of sam-ples containing an analyte at the quantitation limit is usually10z.2.5.2. Assessment

The quantitation limit may be calculated using the stand-ard deviation of responses of blank samples or samples con-taining an analyte close to the quantitation limit and theslope of the calibration curve close to the quantitation limit.The following equation is an example to determine the quan-titation limit using the standard deviation of responses ofblank samples and the slope of the calibration curve.

QL＝ 10s/slope

QL: quantitation limits: the standard deviation of responses of blank samplesslope: slope of the calibration curve

The noise level may be used as the standard deviation ofresponses of blank samples in chromatographic methods. Itshould be ensured that the quantitation limit of the analyti-cal procedure is lower than the specified limit for testing.2.6. Linearity2.6.1. Definition

The linearity is the ability of an analytical procedure toelicit responses linearly related to the amount or concentra-tion of an analyte in samples. A well-defined mathematicaltransformation may sometimes be necessary to obtain alinear relationship.2.6.2. Assessment

Responses are obtained after analyzing samples with vari-ous amounts or concentrations of an analyte according todescribed operating procedures. The linearity may be eval-uated in terms of the correlation coefficient, and the slopeand y-intercept of the regression line. It may be also helpfulfor evaluating the linearity to plot residual errors from the

2150

Table Lists of validation characteristics required to beevaluated in tests of each type

Type of test Type I Type II Type III

Validationcharacteristics

Quantitationtest

Limittest

Accuracy/Trueness －＋－＋

PrecisionRepeatability －＋－＋

Intermediate precision －－* －－*Reproducibility －＋* －＋*

Specificity** ＋＋＋＋

Detection limit －－＋－

Quantitation limit －＋－－

Linearity －＋－＋

Range －＋－＋

－ Usually need not to be evaluated.＋ Usually need to be evaluated.* Either intermediate precision or reproducibility should be

evaluated depending upon circumstances in which analyticalprocedures or tests are performed. The latter should be normallyevaluated in the validation of analytical procedures proposed tobe included in the Japanese Pharmacopoeia.

** The lack of the specificity of an analytical procedure may becompensated by other relevant analytical procedures


regression line against the amount or concentration and toconfirm that there is no particular tendency in the graph.Samples with five different amounts or concentrations of ananalyte should be usually investigated.2.7. Range2.7.1. Definition

The range for the validation of analytical procedures is theinterval between the lower and upper limits of the amount orconcentration of an analyte providing sufficient accuracyand precision. The range for the validation of analyticalprocedures for an analytical procedure with linearity is theinterval between the lower and upper limits providingsufficient accuracy, precision and linearity.2.7.2. Assessment

When the range for the validation of analytical proceduresis investigated, 80 to 120z of specified limits of testingshould be usually considered. The accuracy, precision andlinearity should be evaluated using samples containing thelower and upper limits and in the middle of the range.

3. Categories of tests employing analytical proceduresTests covered with this document are roughly classified

into three categories shown below according to their pur-poses. The table lists the normally required validation char-acteristics to be evaluated in the validation of analyticalprocedures used in these tests. This list should be consideredto represent typical validation characteristics. A different ap-proach to validating analytical procedures should be consi-dered depending upon the characteristics of analytical proce-dures and their intended use.

(i) Type I Identification. Tests for identifying majorcomponents in pharmaceuticals according to their character-istics.

(ii) Type II Impurity tests. Tests for determination ofimpurities in pharmaceuticals.

(iii) Type III Tests for assaying drug substances, active

ingredients, and major components in pharmaceuticals.(Additives such as stabilizing agents and preservatives areincluded in major components.) Tests for determining per-formance of pharmaceuticals, such as dissolution testing.

4. Terminology used in the validation of analytical proce-dures

(i) Analytical procedure: This document covers analyti-cal procedures applied to identification, and ones that pro-vides responses depending upon the amount or concentra-tion of analytes in samples.

(ii) Laboratory: The laboratory means an experimentalroom or facility where tests are performed. In this documentdifferent laboratories are expected to perform an analyticalprocedure using different analysts, different experimentalapparatus and instruments, different lots of reagents and soforth.

(iii) Number of replications: The number of replicationsis one that is described in analytical procedures. An observedvalue is often obtained by more than one measurement inorder to achieve good precision of analytical procedures.Analytical procedures including the number of replicationsshould be validated. This is different from repetition in thevalidation of analytical procedures to obtain accuracy orprecision.

(iv) Observed value: The value of a characteristic ob-tained as the result of performing an analytical procedure.

(v) Consumer's risk: This is the probability that productsout of the specification of tests are decided to be acceptedafter testing. It is usually expressed as b, and is called theprobability of type II error or the probability of false nega-tive in impurity tests.

(vi) Producer's risk: This is the probability that productssatisfying the specification of tests are decided to be rejectedafter testing. It is usually expressed as a, and is called theprobability of type I error or the probability of false positivein impurity tests.

(vii) Robustness: The robustness is a measure of thecapacity to remain unaffected by small but deliberate varia-tions in analytical conditions. The stability of observedvalues may be studied by changing various analytical condi-tions within suitable ranges including pH values of solutions,reaction temperature, reaction time or amount of reagentsadded. When observed values are unstable, the analyticalprocedure should be improved. Results of studying robust-ness may be reflected in the developed analytical procedureas precautions or significant digits describing analytical con-ditions.

(viii) Test: Tests mean various tests described in generaltests and official monographs in the Japanese Phar-macopoeia such as impurity tests and assay. They includessampling methods, specification limits and analytical proce-dures.

21512151JP XVI General Information / Solid-state Properties

G2 Solid-state Properties

Laser Diffraction Measurement ofParticle Size

This test is harmonized with the European Pharmacopoeiaand the U.S. Pharmacopoeia.

The laser light diffraction technique used for the determi-nation of particle-size distribution is based on the analysis ofthe diffraction pattern produced when particles are exposedto a beam of monochromatic light. Historically, the earlylaser diffraction instruments only used scattering at smallangles. However, the technique has since been broadened toinclude laser light scattering in a wider range and applicationof the Mie theory, in addition to the Fraunhofer approxima-tion and anomalous diffraction.

The technique cannot distinguish between scattering bysingle particles and scattering by clusters of primary parti-cles, i.e. by agglomerates or aggregates. As most particulatesamples contain agglomerates or aggregates and as the focusof interest is generally on the size distribution of primaryparticles, the clusters are usually dispersed into primary par-ticles before measurement.

For non-spherical particles, an equivalent sphere-size dis-tribution is obtained because the technique assumes sphericalparticles in its optical model. The resulting particle-size dis-tribution may differ from those obtained by methods basedon other physical principles (e.g. sedimentation, sieving).

This chapter provides guidance for the measurement ofsize distributions of particles in different dispersed systems,for example, powders, sprays, aerosols, suspensions, emul-sions, and gas bubbles in liquids, through analysis of theirangular light-scattering patterns. It does not address specificrequirements of particle-size measurement of specificproducts.

1. PrincipleA representative sample, dispersed at an adequate concen-

tration in a suitable liquid or gas, is passed through a beamof monochromatic light, usually a laser. The light scatteredby the particles at various angles is measured by a multi-element detector. Numerical values representing the scatter-ing pattern are then recorded for subsequent analysis. Thesescattering pattern values are then transformed, using anappropriate optical model and mathematical procedure, toyield the proportion of total volume to a discrete number ofsize classes, forming a volumetric particle-size distribution.

2. InstrumentThe instrument is located in an environment where it is not

affected by electrical noise, mechanical vibrations, tempera-ture fluctuations, humidity or direct bright light. An exam-ple of a set-up of a laser light diffraction instrument is givenin Fig. 1. Other equipment may be used.

The instrument comprises a laser light source, beamprocessing optics, a sample measurement region (or cell), aFourier lens, and a multi-element detector for measuring thescattered light pattern. A data system is also required fordeconvolution of the scattering data into a volumetric size

distribution and associated data analysis and reporting.The particles can enter the laser beam in 2 positions. In the

conventional case the particles enter the parallel beam beforethe collecting lens and within its working distance. In so-called reversed Fourier optics the particles enter behind thecollecting lens and thus, in a converging beam. The advan-tage of the conventional set-up is that a reasonable pathlength for the sample is allowed within the working distanceof the lens. The second set-up allows only small path lengthsbut enables measurement of scattered light at lager angles,which is useful when submicron particles are present.

The interaction of the incident light beam and the ensem-ble of dispersed particles results in a scattering pattern withdifferent light intensities at various angles. The total angularintensity distribution, consisting of both direct and scatteredlight, is then focused onto a multi-element detector by a lensor a series of lenses. These lenses create a scattering patternthat, within limits, does not depend on the location of theparticles in the light beam. Hence, the continuous angularintensity distribution is converted into a discrete spatial in-tensity distribution on a set of detector elements.

It is assumed that the measured scattering pattern of theparticle ensemble is identical to the sum of the patterns fromall individual single scattering particles presented in randomrelative positions. Note that only a limited angular range ofscattered light is collected by the lens(es) and, therefore, bythe detector.

3. Development of the methodThe measurement of particle size by laser diffraction can

give reproducible data, even in the sub-micron region,provided the instrument used and the sample tested are care-fully controlled to limit variability of the test conditions (e.g.dispersion medium, method of preparation of the sampledispersion).

Traditionally, the measurement of particle size using laserdiffraction has been limited to particles in the range of ap-proximately 0.1 mm to 3 mm. Because of recent advances inlens and equipment design, newer instruments are capable ofexceeding this range routinely. With the validation report theuser demonstrates the applicability of the method for its in-tended use.3.1. Sampling

The sampling technique must be adequate to obtain arepresentative sample of a suitable volume for the particle-size measurement. Sample splitting techniques such as rotat-ing riffler or the cone and quartering method may be ap-plied.3.2. Evaluation of the dispersion procedure

Inspect the sample to be analyzed, visually or with the aidof a microscope, to estimate its size range and particle shape.The dispersion procedure must be adjusted to the purpose ofthe measurement. The purpose may be such that it is prefera-ble to deagglomerate clusters into primary particles as far aspossible, or it may be desirable to retain clusters as intact aspossible. In this sense, the particles of interest may be eitherprimary particles or clusters.

For the development of a method it is highly advisable tocheck that comminution of the particles does not occur, andconversely, that dispersion of particles or clusters is satisfac-tory. This can usually be done by changing the dispersingenergy and monitoring the change of the particle-size distri-bution. The measured size distribution must not change sig-

2152

Fig. 1 Example of a set-up of a laser light diffraction instrument

2152 JP XVISolid-state Properties / General Information

nificantly when the sample is well dispersed and the particlesare neither fragile nor soluble. Moreover, if the manufactur-ing process (e.g. crystallization, milling) of the material haschanged, the applicability of the method must by verified(e.g. by microscopic comparison).

Sprays, aerosols and gas bubbles in a liquid should bemeasured directly, provided that their concentration is ade-quate, because sampling or dilution generally alters the par-ticle-size distribution.

In other cases (such as emulsions, pastes and powders),representative samples may be dispersed in suitable liquids.Dispersing aids (wetting agents, stabilizers) and/or mechani-cal forces (e.g. agitation, sonication) are often applied fordeagglomeration or deaggregation of clusters and stabiliza-tion of the dispersion. For these liquid dispersions, a recir-culating system is most commonly used, consisting of an op-tical measuring cell, a dispersion bath usually equipped withstirrer and ultrasonic elements, a pump, and tubing. Non-recirculating, stirred cells are useful when only smallamounts of a sample are available or when special dispersionliquids are used.

Dry powders can also be converted into aerosols throughthe use of suitable dry powder dispersers, which applymechanical force for deagglomeration or deaggregation.Generally, the dispersers use the energy of compressed gas orthe differential pressure of a vacuum to disperse the particlesto an aerosol, which is blown through the measuring zone,usually into the inlet of a vacuum unit that collects the parti-cles. However, for free flowing, coarser particles or granulesthe effect of gravity may be sufficient to disperse the parti-cles adequately.

If the maximum particle size of the sample exceeds themeasuring range of the instrument, the material that is toocoarse can be removed by sieving and the mass and percen-tage of removed material are reported. However, after pre-sieving, note that the sample is no longer representative, un-less otherwise proven.3.3. Optimization of the liquid dispersion

Liquids, surfactants, and dispersing aids used to dispersepowders must:

(i) be transparent at the laser wavelength and practicallyfree from air bubbles or particles;

(ii) have a refractive index that differs from that of the

test material;(iii) be non-solvent of the test material (pure liquid or

pre-filtered, saturated solution);(iv) not alter the size of the test materials (e.g. by solubil-

ity, solubility enhancement, or recrystallization effects);(v) favor easily formation and stability of the dispersion;(vi) be compatible with the materials used in the instru-

ment (such as O-rings, gaskets, tubing, etc.);(vii) possess a suitable viscosity to facilitate recircula-

tion, stirring and filtration.Surfactants and/or dispersing aids are often used to wet

the particles and to stabilize the dispersion. For weak acidsand weak bases, buffering of the dispersing medium at lowor high pH respectively can assist in identifying a suitabledispersant.

A preliminary check of the dispersion quality can beperformed by visual or microscopic inspection. It is alsopossible to take fractional samples out of a well-mixed stockdispersion. Such stock dispersions are formed by adding aliquid to the sample while mixing it with, for example, aglass rod, a spatula or a vortex mixer. Care must be taken toensure the transfer of a representative sample and thatsettling of larger particles does not occur. Therefore a sam-ple paste is prepared or sampling is carried out quickly froma suspension maintained under agitation.3.4. Optimization of the gas dispersion

For sprays and dry powder dispersions, a compressed gasfree from oil, water and particles may be used. To removesuch materials from the compressed gas, a dryer with a filtercan be used. Any vacuum unit should be located away fromthe measurement zone, so that its output does not disturb themeasurement.3.5. Determination of the concentration range

In order to produce an acceptable signal-to-noise ratio inthe detector, the particle concentration in the dispersionmust exceed a minimum level. Likewise, it must be below amaximum level in order to avoid multiple scattering. Theconcentration range is influenced by the width of the laserbeam, the path length of the measurement zone, the opticalproperties of the particles, and the sensitivity of the detectorelements.

In view of the above, measurements must be performed atdifferent particle concentrations to determine the appropri-


ate concentration range for any typical sample of material.(Note: in different instruments, particle concentrations areusually represented by differently scaled and differentlynamed numbers, e.g. obscuration, optical concentration,proportional number of total mass).3.6. Determination of the measuring time

The time of measurement, the reading time of the detectorand the acquisition frequency is determined experimentallyin accordance with the required precision. Generally, thetime for measurement permits a large number of detectorscans or sweeps at short time intervals.3.7. Selection of an appropriate optical model

Most instruments use either the Fraunhofer or the Mietheory, though other approximation theories are sometimesapplied for calculation of the scattering matrix. The choiceof the theoretical model depends on the intended applicationand the different assumptions (size, absorbance, refractiveindex, roughness, crystal orientation, mixture, etc.) madefor the test material. If the refractive index values (real andimaginary parts for the used wavelength) are not exactlyknown, then the Fraunhofer approximation or the Mietheory with a realistic estimate of the refractive index can beused. The former has the advantages that it is simple and itdoes not need refractive index values; the latter usually pro-vides less-biased particle-size distributions for small parti-cles. For instance, if the Fraunhofer model is used for sam-ples containing an appreciable amount of small, transparentparticles, a significantly larges amount of small particles maybe calculated. In order to obtain traceable results, it is essen-tial to document the refractive index values used, since smalldifferences in the values assumed for the real and imaginarypart of the complex refractive index may cause significantdifferences in the resulting particle-size distributions. Smallvalues of the imaginary part of the refractive index (about0.01 � 0.1i) are often applied to allow the correction of theabsorbance for the surface roughness of the particles. Itshould be noted, in general, that the optical properties of thesubstance to be tested, as well as the structure (e.g. shape,surface roughness and porosity) bear upon the final result.3.8. Validation

Typically, the validity of a procedure may be assessed bythe evaluation of its specificity, linearity, range, accuracy,precision and robustness. In particle-size analysis by laserlight diffraction, specificity as defined by ICH is not applica-ble as it is not possible to discriminate different componentsinto a sample, as is neither possible to discriminate betweenagglomerates from dispersed particles unless properlycomplemented by microscopic techniques. Exploring a linerrelationship between concentration and response, or amathematical model for interpolation, is not applicable tothis procedure. Rather than evaluating linearity, this methodrequires the definition of a concentration range within whichthe result of the measurements does not vary significantly.Concentrations below that range produce an error due to apoor signal to noise ratio, while concentrations above thatrange produce an error due to multiple scattering. The rangedepends mostly in the instrument hardware. Accuracyshould be confirmed through an appropriate instrumentqualification and comparison with microscopy, while preci-sion may be assessed by means of a repeatability determina-tion.

The attainable repeatability of the method mainly dependson the characteristics of the material (milled/not milled,

robust/fragile, width of its size distribution, etc.), whereasthe required repeatability depends on the purpose of themeasurement. Mandatory limits cannot be specified in thischapter, as repeatabilities (different sample preparations)may vary appreciably from one substance to another.However, it is good practice to aim at acceptance criteria forrepeatability such as RSD (z)≦ 10z [n＝ 6] for any cen-tral value of the distribution (e.g. for x50). Values at the sidesof the distribution (e.g. x10 and x90) are oriented towards lessstringent acceptance criteria such as RSD≦ 15z [n＝ 6].Below 10 mm, these values must be doubled. Robustness maybe tested during the selection and optimization of the disper-sion media and forces. The change of the dispersing energymay be monitored by the change in the particle-size distribu-tion.

4. Measurement4.1. Precautions

(i) never look into the direct path of the laser beam or itsreflections;

(ii) earth all instrument components to prevent ignitionof solvents or dust explosions;

(iii) check the instrument set-up (e.g. warm-up, requiredmeasuring range and lens, appropriate working distance,position of the detector, no direct bright daylight);

(iv) in the case of wet dispersions, avoid air bubbles,evaporation of liquid, schlieren or other inhomogeneities inthe dispersion; similarly, avoid improper mass-flow from thedisperser or turbulent air-flow in the case of dry dispersions;such effects can cause erroneous particle-size distributions.4.2. Measurement of the light scattering of dispersed sam-ple(s)

After proper alignment of the optical part of the instru-ment, a blank measurement of the particle-free dispersionmedium must be performed using the same method as thatused for the measurement of the sample. The backgroundsignal must be below an appropriate threshold. The detectordata are saved in order to substract them later from the dataobtained with the sample. The sample dispersion is measuredaccording to the developed method.

For each detector element, an average signal is calculated,sometimes together with its standard deviation. The magni-tude of the signal from each detector element depends uponthe detection area, the light intensity and the quantum ef-ficiency. The co-ordinates (size and position) of the detectorelements together with the focal distance of the lens deter-mine the range of scattering angles for each element. Mostinstruments also measure the intensity of the central (unscat-tered) laser beam. The ratio of the intensity of a dispersedsample to that in its absence (the blank measurement) indi-cates the proportion of scattered light and hence the particleconcentration.4.3. Conversion of scattering pattern into particle-size dis-tribution

This deconvolution step is the inverse of the calculation ofa scattering pattern for a given particle-size distribution. Theassumption of spherical particle shape is particularly im-portant as most algorithms use the mathematical solution forscattering from spherical particles. Furthermore, the meas-ured data always contain some random and systematicerrors, which may vitiate the size distributions. Severalmathematical procedures have been developed for use in theavailable instruments. They contain some weighting of devi-


ations between measured and calculated scattering patterns(e.g. least squares), some constraints (e.g. non-negativity foramounts of particles), and/or some smoothing of the sizedistribution curve.

The algorithms used are specific to each make and modelof equipment, and are proprietary. The differences in thealgorithms between different instruments may give rise todifferences in the calculated particle-size distributions.4.4. Replicates

The number of replicate measurements (with individualsample preparations) to be performed, depends on therequired measurement precision. It is recommended to setthis number in a substance-specific method.

5. Reporting of resultsThe particle-size distribution data are usually reported as

cumulative undersize distribution and/or as density distribu-tion by volume. The symbol x is used to denote the particlesize, which in turn is defined as the diameter of a volume-equivalent sphere. Q3(x) denotes the volume fraction under-size at the particle size x. In a graphical representation, x isplotted on the abscissa and the dependent variable Q3 on theordinate. Most common characteristic values are calculatedfrom the particle-size distribution by interpolation. Theparticle sizes at the undersize values of 10z, 50z, and 90z(denoted as x10, x50, and x90 respectively) are frequently used.x50 is also known as the median particle size. It is recognizedthat the symbol d is also widely used to designate the particlesize, thus the symbol x may be replaced by d.

Moreover, sufficient information must be documentedabout the sample, the sample preparation, the dispersionconditions, and the cell type. As the results depend on theparticular instrument, data analysis program, and opticalmodel used, these details must also be documented.

6. Control of the instrument performanceUse the instrument according to the manufacturer's in-

structions and carry out the prescribed qualifications at anappropriate frequency, according to the use of the instru-ment and substances to be tested.6.1. Calibration

Laser diffraction systems, although assuming idealizedproperties of the particles, are based on first principles oflaser light scattering. Thus, calibration in the strict sense isnot required. However, it is still necessary to confirm thatthe instrument is operating correctly. This can be undertakenusing any certified reference material that is acceptable inindustrial practice. The entire measurement procedure isexamined, including sample collection, sample dispersion,sample transport through the measuring zone, measurement,and the deconvolution procedure. It is essential that the totaloperational procedure is fully described.

The preferred certified reference materials consist ofspherical particles of a known distribution. They must becertified as to the mass-percentage size distribution by anabsolute technique, if available, and used in conjunctionwith an agreed, detailed operation procedure. It is essentialthat the real and imaginary parts of the complex refractiveindex of the material are indicated if the Mie theory isapplied in data analysis. The representation of the particle-size distribution by volume will equal that of the distributionby mass, provided that the density of the particles is thesame for all size fractions.

The response of a laser diffraction instrument is consi-

dered to meet the requirements if the mean value of x50 fromat least 3 independent measurements does not deviate bymore than 3z from the certified range of values of the certi-fied reference material. The mean values for x10 and x90 mustnot deviate by more than 5z from the certified range ofvalues. Below 10 mm, these values must be doubled.

Although the use of materials consisting of sphericalparticles is preferable, non-spherical particles may also beemployed. Preferably, these particles have certified or typi-cal values from laser diffraction analysis performed accord-ing to an agreed, detailed operating procedure. The use ofreference values from methods other than laser diffractionmay cause a significant bias. The reason for this bias is thatthe different principles inherent in the various methods maylead to different sphere-equivalent diameters for the samenon-spherical particle.

Although the use of certified reference materials ispreferred, other well-defined reference materials may also beemployed. They consist of substances of typical compositionand particle-size distribution for a specified class of sub-stances. Their particle-size distribution has proven to bestable over time. The results must comply with previouslydetermined data, with the same precision and bias as for thecertified reference material.6.2. Qualification of the system

In addition to the calibration, the performance of theinstrument must be qualified at regular time intervals or asfrequently as appropriate. This can be undertaken using anysuitable reference material as mentioned in the previousparagraph.

The qualification of the system is based on the conceptthat the equipment, electronics, software and analyticaloperations constitute an integral system, which can be eval-uated as an entity. Thus the entire measurement procedure isexamined, including sample collection, sample dispersion,sample transport through the measuring zone, and the meas-urement and deconvolution procedure. It is essential that thetotal operational procedure is fully described.

In general, unless otherwise specified in the individualmonograph, the response of a laser diffraction instrument isconsidered to meet the requirements if the x50 value does notdeviate by more than 10z from the range of values of thereference material. If optionally the values at the sides of thedistribution are evaluated (e.g. x10 and x90), then these valuesmust not deviate by more than 15z from the certified rangeof values. Below 10 mm, these values must be doubled.

Note 1: For calibration of the instrument stricter requirements arelaid down in 6.1. Calibration.Note 2: Laser Diffraction Measurement of Particle Size complieswith ISO13320-1 (1999) and 9276-1 (1998).

Powder FinenessThis classification is harmonized with the European Phar-

macopoeia and the U.S. Pharmacopoeia.

A simple descriptive classification of powder fineness isprovided in this chapter. Sieving is most suitable where amajority of the particles are larger than about 75 mm,although it can be used for some powders having smallerparticle sizes where the method can be validated. Light


diffraction is also a widely used technique for measuring thesize of a wide range of particles.

Where the cumulative distribution has been determined byanalytical sieving or by application of other methods, parti-cle size may be characterized in the following manner:

x90: particle size corresponding to 90z of the cumulativeundersize distribution

x50: median particle size (ie: 50z of the particles aresmaller and 50z of the particles are larger)

x10: particle size corresponding to 10z of the cumulativeundersize distribution

It is recognized that the symbol d is also widely used todesignate these values. Therefore, the symbols d90, d50, d10

may be used.The following parameters may be defined based on the

cumulative distribution.Qr(x ): cumulative distribution of particles with a dimen-

sion less than or equal to x where the subscript r reflects thedistribution type

r Distribution type

0 Number

1 Length

2 Area

3 Volume

Therefore, by definition:

Qr(x )＝ 0.90 when x＝ x90

Qr(x )＝ 0.50 when x＝ x50

Qr(x )＝ 0.10 when x＝ x10

An alternative but less informative method of classifyingpowder fineness is by use of the descriptive terms in the fol-lowing table.

Classification of powders by fineness

Descriptiveterm

x50 (mm)Cumulative distribution by volumebasis, Q3(x)

Coarse ＞355 Q3(355)＜0.50

Moderatelyfine

180�355 Q3(180)＜0.50 and Q3(355)≧0.50

Fine 125�180 Q3(125)＜0.50 and Q3(180)≧0.50

Very fine ≦125 Q3(125)≧0.50

Powder FlowThis test is harmonized with the European Pharmacopoeia

and the U.S. Pharmacopeia.

The widespread use of powders in the pharmaceutical in-dustry has generated a variety of methods for characterizingpowder flow. Not surprisingly, scores of references appear in

the pharmaceutical literature, attempting to correlate thevarious measures of powder flow to manufacturing proper-ties. The development of such a variety of test methods wasinevitable; powder behavior is multifaceted and thus compli-cates the effort to characterize powder flow. The purpose ofthis chapter is to review the methods for characterizing pow-der flow that have appeared most frequently in the phar-maceutical literature. In addition, while it is clear that nosingle and simple test method can adequately characterizethe flow properties of pharmaceutical powders, this chapterproposes the standardization of test methods that may bevaluable during pharmaceutical development.

Four commonly reported methods for testing powder floware (1) angle of repose, (2) compressibility index or Hausnerratio, (3) flow rate through an orifice, and (4) shear cell. Inaddition, numerous variations of each of these basicmethods are available. Given the number of test methodsand variations, standardizing the test methodology, wherepossible, would be advantageous.

With this goal in mind, the most frequently used methodsare discussed below. Important experimental considerationsare identified and recommendations are made regardingstandardization of the methods. In general, any method ofmeasuring powder flow should be practical, useful,reproducible, sensitive, and yield meaningful results. It bearsrepeating that no one simple powder flow method will ade-quately or completely characterize the wide range of flowproperties experienced in the pharmaceutical industry. Anappropriate strategy may well be the use of multiple stan-dardized test methods to characterize the various aspects ofpowder flow as needed by the pharmaceutical scientist.

1. Angle of reposeThe angle of repose has been used in several branches of

science to characterize the flow properties of solids. Angle ofrepose is a characteristic related to interparticulate friction,or resistane to movement between particles. Angle of reposetest results are reported to be very dependent upon themethod used. Experimental difficulties arise due to segrega-tion of material and consolidation or aeration of the powderas the cone is formed. Despite its difficulties, the methodcontinues to be used in the pharmaceutical industry, and anumber of examples demonstrating its value in predictingmanufacturing problems appear in the literature.

The angle of repose is the constant, three-dimensionalangle (relative to the horizontal base) assumed by a cone-likepile of material formed by any of several different methods(described briefly below).1.1. Basic methods for angle of repose

A variety of angle of repose test methods are reported inthe literature. The most common methods for determiningthe static angle of repose can be classified based on two im-portant experimental variables:

(i) The height of the ``funnel'' through which the powderpasses may be fixed relative to the base, or the height may bevaried as the pile forms.

(ii) The base upon which the pile forms may be of fixeddiameter or the diameter of the powder cone may be allowedto vary as the pile forms.1.2. Variations in angle of repose methods

In addition to the above methods, variations of them havebeen used to some extent.

(i) Drained angle of repose: This is determined by


allowing an excess quantity of material positioned above afixed diameter base to ``drain'' from the container. Forma-tion of a cone of powder on the fixed diameter base allowsdetermination of the drained angle of repose.

(ii) Dynamic angle of repose: This is determined by fill-ing a cylinder (with a clear, flat cover on one end) and rotat-ing it at a specified speed. The dynamic angle of repose is theangle (relative to the horizontal) formed by the flowingpowder. The internal angle of kinetic friction is defined bythe plane separating those particles sliding down the toplayer of the powder and those particles that are rotating withthe drum (with roughened surface).1.3. Angle of repose general scale of flowability

While there is some variation in the qualitative descriptionof powder flow using the angle of repose, much of the phar-maceutical literature appears to be consistent with the classi-fication by Carr1), which is shown in Table 1. There are ex-amples of formulations with an angle of repose in the rangeof 40 to 50 degrees that manufactured satisfactorily. Whenthe angle of repose exceeds 50 degrees, the flow is rarely ac-ceptable for manufacturing purposes.

Table 1 Flow properties and correspondingangles of repose1)

Flow property Angle of repose (degrees)

Excellent 25 � 30Good 31 � 35Fair aid not needed 36 � 40

Passable may hang up 41 � 45Poor most agitate,

vibrate46 � 55

Very poor 56 � 65Very, very poor ＞66

1.4. Experimental considerations for angle of reposeAngle of repose is not an intrinsic property of the powder,

that is to say, it is very much dependent upon the methodused to form the cone of powder. On this subject, the exist-ing literature raises these important considerations:

(i) The peak of the cone of powder can be distorted bythe impact of powder from above. By carefully building thepowder cone, the distortion caused by impact can beminimized.

(ii) The nature of the base upon which the powder coneis formed influences the angle of repose. It is recommendedthat the powder cone be formed on a ``common base'',which can be achieved by forming the cone of powder on alayer of powder. This can be done by using a base of fixeddiameter with a protruding outer edge to retain a layer ofpowder upon which the cone is formed.1.5. Recommended procedure for angle of repose

Form the angle of repose on a fixed base with a retaininglip to retain a layer of powder on the base. The base shouldbe free of vibration. Vary the height of the funnel to care-fully build up a symmetrical cone of powder. Care should betaken to prevent vibration as the funnel is moved. Thefunnel height should be maintained approximately 2 � 4 cmfrom the top of the powder pile as it is being formed in orderto minimize the impact of falling powder on the tip of thecone. If a symmetrical cone of powder cannot be successful-

ly or reproducibly prepared, this method is not appropriate.Determine the angle of repose by measuring the height of thecone of powder and calculating the angle of repose, a, fromthe following equation:

tan a＝ height/(base× 0.5)

2. Compressibility index and Hausner ratioIn recent years the compressibility index and the closely

related Hausner ratio have become the simple, fast andpopular methods of predicting powder flow characteristics.The compressibility index bas been proposed as an indirectmeasure of bulk density, size and shape, surface area, mois-ture content, and cohesiveness of materials because all ofthese can influence the observed compressibility index. Thecompressibility index and the Hausner ratio are determinedby measuring both the bulk volume and tapped volume of apowder.2.1. Basic methods for compressibility index and Hausnerratio

While there are some variations in the method of deter-mining the compressibility index and Hausner ratio, the bas-ic procedure is to measure (1) the unsettled apparent volume,Vo, and (2) the final tapped volume, Vf, of the powder aftertapping the material until no further volume changes occur.The compressibility index and the Hausner ratio are calcu-lated as follows:

Compressibility Index＝ (Vo－ Vf)/Vo× 100Hausner Ratio＝ Vo/Vf

Alternatively, the compressibility index and Hausner ratiomay be calculated using measured values for bulk density(rbulk) and tapped density (rtapped) as follows:

Compressibility Index＝ (rtapped－ rbulk)/rtapped× 100Hausner Ratio＝ rtapped/rbulk

In a variation of these methods, the rate of consolidationis sometimes measured rather than, or in addition to, thechange in volume that occurs on tapping. For the compres-sibility index and the Hausner ratio, the generally acceptedscale of flowability is given in Table 2.

Table 2 Scale of flowability1)

Compressibilityindex (z) Flow character Hausner ratio

≦10 Excellent 1.00 � 1.1111 � 15 Good 1.12 � 1.1816 � 20 Fair 1.19 � 1.2521 � 25 Passable 1.26 � 1.3426 � 31 Poor 1.35 � 1.4532 � 37 Very poor 1.46 � 1.59＞38 Very, very poor ＞1.60

2.2. Experimental considerations for the compressibilityindex and Hausner ratio

Compressibility index and Hausner ratio are not intrinsicproperties of the powder, that is to say, they are dependentupon the methodology used. The existing literature pointsout several important considerations affecting the determi-nation of the (1) unsettled apparent volume, Vo, (2) the finaltapped volume, Vf, (3) the bulk density, rbulk, and (4) thetapped density, rtapped:


(i) The diameter of the cylinder used(ii) The number of times the powder is tapped to achieve

the tapped density(iii) The mass of material used in the test(iv) Rotation of the sample during tapping

2.3. Recommended procedure for compressibility indexand Hausner ratio

Use a 250-mL volumetric cylinder with a test sampleweight of 100 g. Smaller weights and volumes may be used,but variations in the method should be described with theresults. An average of three determinations is recommended.

3. Flow through an orificeThe flow rate of a material depends upon many factors,

some of which are particle-related and some related to theprocess. Monitoring the rate of flow of material through anorifice has been proposed as a better measure of powderflowability. Of particular significance is the utility ofmonitoring flow continuously since pulsating flow patternshave been observed even for free flowing materials. Changesin flow rate as the container empties can also be observed.Empirical equations relating flow rate to the diameter of theopening, particle size, and particle density have been deter-mined. However, determining the flow rate through anorifice is useful only with free-flowing materials.

The flow rate through an orifice is generally measured asthe mass per time flowing from any of a number of types ofcontainers (cylinders, funnels, hoppers). Measurement of theflow rate can be in discrete increments or continuous.3.1. Basic methods for flow through an orifice

There are a variety of methods described in the literature.The most common for determining the flow rate through anorifice can be classified based on three important experimen-tal variables:

(1) The type of container used to contain the powder.Common containers are cylinders, funnels and hoppers fromproduction equipment.

(2) The size and shape of the orifice used. The orifice di-ameter and shape are critical factors in determining powderflow rate.

(3) The method of measuring powder flow rate. Flowrate can be measured continuously using an electronicbalance and with some sort of recording device (strip chartrecorder, computer). It can also be measured in discrete sam-ples (for example, the time it takes for 100 g of powder topass through the orifice to the nearest tenth of a second orthe amount of powder passing through the orifice in 10seconds to the nearest tenth of a gram).3.2. Variations in methods for flow through an orifice

Either mass flow rate or volume flow rate can be deter-mined. Mass flow rate is the easier of the methods, but itbiases the results in favor of high-density materials. Since diefill is volumetric, determining volume flow rate may bepreferable. A vibrator is occasionally attached to facilitateflow from the container, however, this appears to complicateinterpretation of results. A moving orifice device has beenproposed to more closely simulate rotary press conditions.The minimum diameter orifice through which powder flowscan also be identified.3.3. General scale of flowability for flow through anorifice

No general scale is available because flow rate is criticallydependent on the method used to measure it. Comparison

between published results is difficult.3.4. Experimental considerations for flow through anorifice

Flow rate through an orifice is not an intrinsic property ofthe powder. It is very much dependent upon the methodolo-gy used. The existing literature points out several importantconsiderations affecting these methods:

(i) The diameter and shape of the orifice(ii) The type of container material (metal, glass, plastic)(iii) The diameter and height of the powder bed.

3.5. Recommended procedure for flow through an orificeFlow rate through an orifice can be used only for materials

that have some capacity to flow. It is not useful for cohesivematerials. Provided that the height of the powder bed (the`head' of powder) is much greater than the diameter of theorifice, the flow rate is virtually independent of the powderhead. Use a cylinder as the container because the cylindermaterial should have little effect on flow. This configurationresults in flow rate being determined by the movement ofpowder over powder rather than powder along the wall ofthe container. Powder flow rate often increases when theheight of the powder column is less than two times the diam-eter of the column. The orifice should be circular and thecylinder should be free of vibration. General guidelines fordimensions of the cylinder are as follows:

(i) Diameter of opening ＞6 times the diameter of theparticles

(ii) Diameter of the cylinder ＞2 times the diameter ofthe opening

Use of a hopper as the container may be appropriate andrepresentative of flow in a production situation. It is not ad-visable to use a funnel, particularly one with a stem, becauseflow rate will be determined by the size and length of thestem as well as the friction between the stem and the powder.A truncated cone may be appropriate, but flow will be in-fluenced by the powder�wall friction coefficient, thus,selection of an appropriate construction material is im-portant.

For the opening in the cylinder, use a flat-faced bottomplate with the option to vary orifice diameter to providemaximum flexibility and better ensure a powder-over-pow-der flow pattern. Rate measurement can be either discrete orcontinuous. Continuous measurement using an electronicbalance can more effectively detect momentary flow ratevariations.

4. Shear cell methodsIn an effort to put powder flow studies and hopper design

on a more fundamental basis, a variety of powder sheartesters and methods that permit more thorough and preciselydefined assessment of powder flow properties have been de-veloped. Shear cell methodology has been used extensively inthe study of pharmaceutical materials. From these methods,a wide variety of parameters can be obtained, including theyield loci representing the shear stress-shear strain relation-ship, the angle of internal friction, the unconfined yieldstrength, the tensile strength, and a variety of derivedparameters such as the flow factor and other flowability in-dices. Because of the ability to more precisely control experi-mental parameters, flow properties can also be determinedas a function of consolidation load, time, and other environ-mental conditions. The methods have been successfully usedto determine critical hopper and bin parameters.


4.1. Basic methods for shear cellOne type of shear cell is the cylindrical shear cell which is

split horizontally, forming a shear plane between the lowerstationary base and the upper movable portion of the shearcell ring. After powder bed consolidation in the shear cell(using a well-defined procedure), the force necessary to shearthe powder bed by moving the upper ring is determined.Annular shear cell designs offer some advantages over thecylindrical shear cell design, including the need for less mate-rial. A disadvantage, however, is that because of its design,the powder bed is not sheared as uniformly because materialon the outside of the annulus is sheared more than materialin the inner region. A third type of shear cell (plate-type)consists of a thin sandwich of powder between a lowerstationary rough surface and an upper rough surface that ismoveable.

All of the shear cell methods have their advantages anddisadvantages, but a detailed review is beyond the scope ofthis chapter. As with the other methods for characterizingpowder flow, many variations are described in the literature.A significant advantage of shear cell methodology in generalis a greater degree of experimental control. The methodologygenerally is rather time-consuming and requires significantamounts of material and a well-trained operator.4.2. Recommendations for shear cell

The many existing shear cell configurations and testmethods provide a wealth of data and can be used very effec-tively to characterize powder flow. They are also helpful inthe design of equipment such as hoppers and bins.

Because of the diversity of available equipment and ex-perimental procedures, no specific recommendations regard-ing methodology are presented in this chapter. It is recom-mended that the results of powder flow characterizationusing shear cell methodology include a complete descriptionof equipment and methodology used.

References1) Carr R.L.: Evaluating flow properties of solids. Chem.

Eng. 1965; 72: 163�168.

Solid and Particle DensitiesDensity of a solid or a powder as a state of aggregation

has different definitions depending on the way of includingof the interparticulate and intraparticulate voids that existbetween the particles or inside the powder. Different figuresare obtained in each case, and there are different practicalmeanings. Generally, there are three levels of definitions ofthe solid or powder density.

(1) Crystal density: It is assumed that the system is ho-mogeneous with no intraparticulate void. Crystal density isalso called true density.

(2) Particle density: The sealed pores or the experimen-tally non-accessible open pores is also included as a part ofthe volumes of the solid or the powder.

(3) Bulk density: The interparticulate void formed in thepowder bed is also included as a part of the volumes of thesolid or the powder. Bulk density is also called apparent den-sity. Generally, the powder densities at loose packing and attapping are defined as the bulk density and the tapped den-sity, respectively.

Generally, the densities of liquid or gas are affected onlyby temperature and pressure, but the solid or powder densityis affected by the state of aggregation of the molecules or theparticles. Therefore, the solid or powder densities naturallyvary depending on crystal structure or crystallinity of thesubstance concerned, and also varies depending on themethod of preparation or handling if the sample is amor-phous form or partially amorphous. Consequently, even in acase that two solids or powders are chemically identical, itmay be possible that the different figures of density are ob-tained if their crystal structures are different. As the solid orpowder particle densities are important physical propertiesfor the powdered pharmaceutical drugs or the powdered rawmaterials of drugs, the Japanese Pharmacopoeia specifieseach density determination as ``Powder Particle Density De-termination'' for the particle density and as ``Determinationof Bulk and Tapped Densities'' for the bulk density.

The solid or powder densities are expressed in mass perunit volume (kg/m3), and generally expressed in g/cm3 (1 g/cm3＝ 1000 kg/m3).

Crystal DensityThe crystal density of a substance is the average mass per

unit volume, exclusive of all voids that are not a fundamen-tal part of the molecular packing arrangement. It is an in-trinsic property concerning the specific crystal structure ofthe substance, and is not affected by the method of determi-nation. The crystal density can be determined either by cal-culation or by simple measurement.A. The calculated crystal density is obtained using:

1) For example, the crystallographic data (volume andcomposition of the unit cell) obtained by indexingthe perfect crystal X-ray diffraction data fromsingle crystal or the powder X-ray diffraction data.

2) Molecular mass of the substance.B. The measured crystal density is obtained as the mass to

volume ratio after measuring the single crystal mass andvolume.

Particle DensityThe particle density takes account both the crystal density

and the intraparticulate porosity (sealed and/or experimen-tally non-accessible open pores) as a part of the particlevolume. The particle density depends on the value of thevolume determined, and the volume in turn depends on themethod of measurement. Particle density can be determinedeither by gas displacement pycnometry or mercury porosi-metry, but the Japanese Pharmacopoeia specifies the pycno-metry as the ``Powder Particle Density Determination''.A. The pycnometric density is obtained by assuming that

the volume of the gas displaced, which is measured withthe gas displacement pycnometer, is equivalent to thatof a known mass of the powder. In pycnometric densitymeasurements, any volume with the open pores accessi-ble to the gas is not included as a part of volume of thepowder, but the sealed pores or pores inaccessible to thegas is included as a part of the volume of the powder.Due to the high diffusivity of helium which can pene-trate to most open pores, it is recommendable as themeasurement gas of particle density. Therefore, the py-cnometric particle density of a finely milled powder isgenerally not very different from the crystal density.Hence, the particle density by this method is the best es-timate of the true density of an amorphous or partially

21592159JP XVI General Information / Biotechnological/Biological Products

crystalline sample, and can be widely used for manufac-turing control of the processed pharmaceutical powdersamples.

B. The mercury porosimetric density is also called granulardensity. This method also includes the sealed pores as apart of the volumes of the solid or the powder, but ex-cludes the volume only from the open pores larger thansome size limit. This pore size limit or minimal accessdiameter depends on the maximal mercury intrusionpressure applied during the measurement and undernormal operating pressure, the mercury does not pene-trate the finenest pores accessible to helium. Since thismethod is capable of measuring the density which cor-responds to the pore size limit at each mercury intrusionpressure, the various granular densities can be obtainedfrom one sample.

Bulk Density and Tapped DensityThe bulk density of a powder includes the contribution of

interparticulate void volume as a part of the volume of thepowder. Therefore, the bulk density depends on both thepowder particle density and the space arrangement of parti-cles in the power bed. Further, since the slightest disturbanceof the bed may result in variation of the space arrangement,it is often very difficult to determine the bulk density withgood reproducibility. Therefore, it is essential to specify howthe determination was made upon reporting the bulk density.

The Japanese Pharmacopoeia specifies ``Determination ofBulk and Tapped Densities''.A. The bulk density is determined by measuring the appar-

ent volume of a known mass of powder sample that hasbeen passed through a screen in a graduated cylinder(constant mass method). Separately, the Phar-macopoeia specifies the method of determining bulkdensity by measuring the mass of powder in a vesselhaving a known volume (constant volume method).

B. The tapped density is obtained by mechanically tappinga measuring cylinder containing a powder sample. Afterdetermining the initial bulk volume, carry out tappingunder a fixed measurement condition (tapping rate anddrop height), and the measurement is carried outrepeatedly until the bulk volume variation obtained atconsecutive two measurements is within an acceptablerange (constant mass method). Separately, the Phar-macopoeia specifies the method of determining thetapped density by measuring the mass of a fixed volumeof the tapped powder (constant volume method).

G3 Biotechnological/BiologicalProducts

Amino Acid AnalysisThis test is harmonized with the European Pharmacopoeia


Amino acid analysis refers to the methodology used to de-termine the amino acid composition or content of proteins,peptides, and other pharmaceutical preparations. Proteinsand peptides are macromolecules consisting of covalently

bonded amino acid residues organized as a linear polymer.The sequence of the amino acids in a protein or peptidedetermines the properties of the molecule. Proteins areconsidered large molecules that commonly exist as foldedstructures with a specific conformation, while peptides aresmaller and may consist of only a few amino acids. Aminoacid analysis can be used to quantify protein and peptides, todetermine the identity of proteins or peptides based on theiramino acid composition, to support protein and peptidestructure analysis, to evaluate fragmentation strategies forpeptide mapping, and to detect atypical amino acids thatmight be present in a protein or peptide. It is necessary tohydrolyze a protein/peptide to its individual amino acidconstituents before amino acid analysis. Following protein/peptide hydrolysis, the amino acid analysis procedure can bethe same as that practiced for free amino acids in other phar-maceutical preparations. The amino acid constituents of thetest sample are typically derivatized for analysis.

ApparatusMethods used for amino acid analysis are usually based on

a chromatographic separation of the amino acids present inthe test sample. Current techniques take advantage of theautomated chromatographic instrumentation designed foranalytical methodologies. An amino acid analysis instrumentwill typically be a low-pressure or high-pressure liquid chro-matograph capable of generating mobile phase gradientsthat separate the amino acid analytes on a chromatographiccolumn. The instrument must have postcolumn derivatiza-tion capability, unless the sample is analyzed using precol-umn derivatization. The detector is usually an ultraviolet-visible or fluorescence detector depending on the derivatiza-tion method used. A recording device (e.g., integrator) isused for transforming the analog signal from the detectorand for quantitation. It is preferred that instrumentation bededicated particularly for amino acid analysis.

General PrecautionsBackground contamination is always a concern for the

analyst in performing amino acid analysis. High purity rea-gents are necessary (e.g., low purity hydrochloric acid cancontribute to glycine contamination). Analytical reagents arechanged routinely every few weeks using only high-pressureliquid chromatography (HPLC) grade solvents. Potentialmicrobial contamination and foreign material that might bepresent in the solvents are reduced by filtering solvents be-fore use, keeping solvent reservoirs covered, and not placingamino acid analysis instrumentation in direct sunlight.

Laboratory practices can determine the quality of theamino acid analysis. Place the instrumentation in a lowtraffic area of the laboratory. Keep the laboratory clean.Clean and calibrate pipets according to a maintenance sched-ule. Keep pipet tips in a covered box; the analysts may nothandle pipet tips with their hands. The analysts may wearpowder-free latex or equivalent gloves. Limit the number oftimes a test sample vial is opened and closed because dustcan contribute to elevated levels of glycine, serine, andalanine.

A well-maintained instrument is necessary for acceptableamino acid analysis results. If the instrument is used on aroutine basis, it is to be checked daily for leaks, detector andlamp stability, and the ability of the column to maintainresolution of the individual amino acids. Clean or replace allinstrument filters and other maintenance items on a routine

21602160 JP XVIBiotechnological/Biological Products / General Information

schedule.

Reference Standard MaterialAcceptable amino acid standards are commercially availa-

ble for amino acid analysis and typically consist of an aque-ous mixture of amino acids. When determining amino acidcomposition, protein or peptide standards are analyzed withthe test material as a control to demonstrate the integrity ofthe entire procedure. Highly purified bovine serum albuminhas been used as a protein standard for this purpose.

Calibration of InstrumentationCalibration of amino acid analysis instrumentation typi-

cally involves analyzing the amino acid standard, which con-sists of a mixture of amino acids at a number of concentra-tions, to determine the response factor and range of analysisfor each amino acid. The concentration of each amino acidin the standard is known. In the calibration procedure, theanalyst dilutes the amino acid standard to several differentanalyte levels within the expected linear range of the aminoacid analysis technique. Then, replicates at each of thedifferent analyte levels can be analyzed. Peak areas obtainedfor each amino acid are plotted versus the known concentra-tion for each of the amino acids in the standard dilution.These results will allow the analyst to determine the range ofamino acid concentrations where the peak area of a givenamino acid is an approximately linear function of the aminoacid concentration. It is important that the analyst preparethe samples for amino acid analysis so that they are withinthe analytical limits (e.g., linear working range) of the tech-nique employed in order to obtain accurate and repeatableresults.

Four to six amino acid standard levels are analyzed to de-termine a response factor for each amino acid. The responsefactor is calculated as the average peak area or peak heightper nmol of amino acid present in the standard. A calibra-tion file consisting of the response factor for each aminoacid is prepared and used to calculate the concentration ofeach amino acid present in the test sample. This calculationinvolves dividing the peak area corresponding to a givenamino acid by the response factor for that amino acid to givethe nmol of the amino acid. For routine analysis, a single-point calibration may be sufficient; however, the calibrationfile is updated frequently and tested by the analysis of ana-lytical controls to ensure its integrity.

RepeatabilityConsistent high quality amino acid analysis results from

an analytical laboratory require attention to the repeatabilityof the assay. During analysis of the chromatographic separa-tion of the amino acids or their derivatives, numerous peakscan be observed on the chromatogram that correspond to theamino acids. The large number of peaks makes it necessaryto have an amino acid analysis system that can repeatedlyidentify the peaks based on retention time and integrate thepeak areas for quantitation. A typical repeatability evalua-tion involves preparing a standard amino acid solution andanalyzing many replicates (i.e., six analyses or more) of thesame standard solution. The relative standard deviation(RSD) is determined for the retention time and integratedpeak area of each amino acid. An evaluation of the repeata-bility is expanded to include multiple assays conducted overseveral days by different analysts. Multiple assays includethe preparation of standard dilutions from starting materials

to determine the variation due to sample handling. Often theamino acid composition of a standard protein (e.g., bovineserum albumin) is analyzed as part of the repeatability evalu-ation. By evaluating the replicate variation (i.e., RSD), thelaboratory can establish analytical limits to ensure that theanalyses from the laboratory are under control. It is desira-ble to establish the lowest practical variation limits to ensurethe best results. Areas to focus on to lower the variability ofthe amino acid analysis include sample preparation, highbackground spectral interference due to quality of reagentsand/or laboratory practices, instrument performance andmaintenance, data analysis and interpretation, and analystperformance and habits. All parameters involved are fullyinvestigated in the scope of the validation work.

Sample PreparationAccurate results from amino acid analysis require purified

protein and peptide samples. Buffer components (e.g., salts,urea, detergents) can interfere with the amino acid analysisand are removed from the sample before analysis. Methodsthat utilize postcolumn derivatization of the amino acids aregenerally not affected by buffer components to the extentseen with precolumn derivatization methods. It is desirableto limit the number of sample manipulations to reducepotential background contamination, to improve analyterecovery, and to reduce labor. Common techniques used toremove buffer components from protein samples include thefollowing methods: (1) injecting the protein sample onto areversed-phase HPLC system, eluting the protein with avolatile solvent containing a sufficient organic component,and drying the sample in a vacuum centrifuge; (2) dialysisagainst a volatile buffer or water; (3) centrifugal ultrafiltra-tion for buffer replacement with a volatile buffer or water;(4) precipitating the protein from the buffer using an organicsolvent (e.g., acetone); and (5) gel filtration.

Internal StandardsIt is recommended that an internal standard be used to

monitor physical and chemical losses and variations duringamino acid analysis. An accurately known amount of inter-nal standard can be added to a protein solution prior to hy-drolysis. The recovery of the internal standard gives the gen-eral recovery of the amino acids of the protein solution. Freeamino acids, however, do not behave in the same way asprotein-bound amino acids during hydrolysis because theirrates of release or destruction are variable. Therefore, theuse of an internal standard to correct for losses during hy-drolysis may give unreliable results. It will be necessary totake this point under consideration when interpreting theresults. Internal standards can also be added to the mixtureof amino acids after hydrolysis to correct for differences insample application and changes in reagent stability and flowrates. Ideally, an internal standard is an unnaturally occur-ring primary amino acid that is commercially available andinexpensive. It should also be stable during hydrolysis, itsresponse factor should be linear with concentration, and itneeds to elute with a unique retention time without overlap-ping other amino acids. Commonly used amino acid stand-ards include norleucine, nitrotyrosine, and a-aminobutyricacid.

Protein HydrolysisHydrolysis of protein and peptide samples is necessary for

amino acid analysis of these molecules. The glassware used


for hydrolysis must be very clean to avoid erroneous results.Glove powders and fingerprints on hydrolysis tubes maycause contamination. To clean glass hydrolysis tubes, boiltubes for 1 hour in 1 mol/L hydrochloric acid or soak tubesin concentrated nitric acid or in a mixture of concentratedhydrochloric acid and concentrated nitric acid (1:1). Cleanhydrolysis tubes are rinsed with high-purity water followedby a rinse with HPLC grade methanol, dried overnight in anoven, and stored covered until use. Alternatively, pyrolysisof clean glassware at 5009C for 4 hours may also be used toeliminate contamination from hydrolysis tubes. Adequatedisposable laboratory material can also be used.

Acid hydrolysis is the most common method for hydrolyz-ing a protein sample before amino acid analysis. The acidhydrolysis technique can contribute to the variation of theanalysis due to complete or partial destruction of severalamino acids. Tryptophan is destroyed; serine and threonineare partially destroyed; methionine might undergo oxida-tion; and cysteine is typically recovered as cystine (but cys-tine recovery is usually poor because of partial destruction orreduction to cysteine). Application of adequate vacuum(≦less than 200 mm of mercury or 26.7 Pa) or introductionof an inert gas (argon) in the headspace of the reaction vesselcan reduce the level of oxidative destruction. In peptidebonds involving isoleucine and valine the amido bonds ofIle-Ile, Val-Val, Ile-Val, and Val-Ile are partially cleaved;and asparagine and glutamine are deamidated, resulting inaspartic acid and glutamic acid, respectively. The loss oftryptophan, asparagine, and glutamine during an acid hy-drolysis limits quantitation to 17 amino acids. Some of thehydrolysis techniques described are used to address theseconcerns. Some of the hydrolysis techniques described (i.e.,Methods 4-11) may cause modifications to other aminoacids. Therefore, the benefits of using a given hydrolysistechnique are weighed against the concerns with the tech-nique and are tested adequately before employing a methodother than acid hydrolysis.

A time-course study (i.e., amino acid analysis at acid hy-drolysis times of 24, 48, and 72 hours) is often employed toanalyze the starting concentration of amino acids that arepartially destroyed or slow to cleave. By plotting the ob-served concentration of labile amino acids (i.e., serine andthreonine) versus hydrolysis time, the line can be extrapo-lated to the origin to determine the starting concentration ofthese amino acids. Time-course hydrolysis studies are alsoused with amino acids that are slow to cleave (e.g., isoleucineand valine). During the hydrolysis time course, the analystwill observe a plateau in these residues. The level of thisplateau is taken as the residue concentration. If the hydroly-sis time is too long, the residue concentration of the samplewill begin to decrease, indicating destruction by the hydroly-sis conditions.

An acceptable alternative to the time-course study is tosubject an amino acid calibration standard to the same hy-drolysis conditions as the test sample. The amino acid in freeform may not completely represent the rate of destruction oflabile amino acids within a peptide or protein during the hy-drolysis. This is especially true for peptide bonds that areslow to cleave (e.g., Ile-Val bonds). However, this techniquewill allow the analyst to account for some residue destruc-tion. Microwave acid hydrolysis has been used and is rapidbut requires special equipment as well as special precautions.The optimal conditions for microwave hydrolysis must be

investigated for each individual protein/peptide sample. Themicrowave hydrolysis technique typically requires only a fewminutes, but even a deviation of one minute may give inade-quate results (e.g., incomplete hydrolysis or destruction oflabile amino acids). Complete proteolysis, using a mixture ofproteases, has been used but can be complicated, requiresthe proper controls, and is typically more applicable to pep-tides than proteins.

Note: During initial analyses of an unknown protein, ex-periments with various hydrolysis time and temperature con-ditions are conducted to determine the optimal conditions.

Method 1Acid hydrolysis using hydrochloric acid containing phenol

is the most common procedure used for protein/peptide hy-drolysis preceding amino acid analysis. The addition ofphenol to the reaction prevents the halogenation of tyrosine.

Hydrolysis Solution 6 mol/L hydrochloric acid contain-ing 0.1z to 1.0z of phenol.

Procedure�Liquid Phase Hydrolysis Place the protein or peptide

sample in a hydrolysis tube, and dry. [Note: The sample isdried so that water in the sample will not dilute the acid usedfor the hydrolysis.] Add 200 mL of Hydrolysis Solution per500 mg of lyophilized protein. Freeze the sample tube in a dryice-acetone bath, and flame seal in vacuum. Samples aretypically hydrolyzed at 1109C for 24 hours in vacuum orinert atmosphere to prevent oxidation. Longer hydrolysistimes (e.g., 48 and 72 hours) are investigated if there is aconcern that the protein is not completely hydrolyzed.

Vapor Phase Hydrolysis This is one of the most commonacid hydrolysis procedures, and it is preferred for microanal-ysis when only small amounts of the sample are available.Contamination of the sample from the acid reagent is alsominimized by using vapor phase hydrolysis. Place vials con-taining the dried samples in a vessel that contains an ap-propriate amount of Hydrolysis Solution. The HydrolysisSolution does not come in contact with the test sample.Apply an inert atmosphere or vacuum (≦less than 200 mm ofmercury or 26.7 Pa) to the headspace of the vessel, and heatto about 1109C for a 24-hour hydrolysis time. Acid vaporhydrolyzes the dried sample. Any condensation of the acid inthe sample vials is minimized. After hydrolysis, dry the testsample in vacuum to remove any residual acid.

Method 2Tryptophan oxidation during hydrolysis is decreased by

using mercaptoethanesulfonic acid (MESA) as the reducingacid.

Hydrolysis Solution 2.5 mol/L MESA solution.Vapor Phase Hydrolysis About 1 to 100 mg of the pro-

tein/peptide under test is dried in a hydrolysis tube. The hy-drolysis tube is placed in a larger tube with about 200 mL ofthe Hydrolysis Solution. The larger tube is sealed in vacuum(about 50 mm of mercury or 6.7 Pa) to vaporize the Hydroly-sis Solution. The hydrolysis tube is heated to 1709C to 1859Cfor about 12.5 minutes. After hydrolysis, the hydrolysis tubeis dried in vacuum for 15 minutes to remove the residualacid.

Method 3Tryptophan oxidation during hydrolysis is prevented by

using thioglycolic acid (TGA) as the reducing acid.Hydrolysis Solution A solution containing 7 mol/L hy-


drochloric acid, 10z of trifluoroacetic acid, 20z of thiogly-colic acid, and 1z of phenol.

Vapor Phase Hydrolysis About 10 to 50 mg of the pro-tein/peptide under test is dried in a sample tube. The sampletube is placed in a larger tube with about 200 mL of theHydrolysis Solution. The larger tube is sealed in vacuum(about 50 mm of mercury or 6.7 Pa) to vaporize the TGA.The sample tube is heated to 1669C for about 15 to 30minutes. After hydrolysis, the sample tube is dried in vacu-um for 5 minutes to remove the residual acid. Recovery oftryptophan by this method may be dependent on the amountof sample present.

Method 4Cysteine-cystine and methionine oxidation is performed

with performic acid before the protein hydrolysis.Oxidation Solution The performic acid is prepared fresh

by mixing formic acid and 30 percent hydrogen peroxide(9:1), and incubated at room temperature for 1 hour.

Procedure The protein/peptide sample is dissolved in 20mL of formic acid, and heated at 509C for 5 minutes; then100 mL of the Oxidation Solution is added. The oxidation isallowed to proceed for 10 to 30 minutes. In this reaction,cysteine is converted to cysteic acid and methionine is con-verted to methionine sulfone. The excess reagent is removedfrom the sample in a vacuum centrifuge. This technique maycause modifications to tyrosine residues in the presence ofhalides. The oxidized protein can then be acid hydrolyzedusing Method 1 or Method 2.

Method 5Cysteine-cystine oxidation is accomplished during the liq-

uid phase hydrolysis with sodium azide.Hydrolysis Solution 6 mol/L hydrochloric acid contain-

ing 0.2z of phenol, to which is added sodium azide to ob-tain a final concentration of 0.2z (w/v). The added phenolprevents halogenation of tyrosine.

Liquid Phase Hydrolysis The protein/peptide hydrolysisis conducted at about 1109C for 24 hours. During the hy-drolysis, the cysteine-cystine present in the sample is con-verted to cysteic acid by the sodium azide present in theHydrolysis Solution. This technique allows better tyrosinerecovery than Method 4, but it is not quantitative formethionine. Methionine is converted to a mixture of the par-ent methionine and its two oxidative products, methioninesulfoxide and methionine sulfone.

Method 6Cysteine-cystine oxidation is accomplished with dimethyl

sulfoxide (DMSO).Hydrolysis Solution 6 mol/L hydrochloric acid contain-

ing 0.1z to 1.0z of phenol, to which DMSO is added toobtain a final concentration of 2z (v/v).

Vapor Phase Hydrolysis The protein/peptide hydrolysisis conducted at about 1109C for 24 hours. During the hy-drolysis, the cysteine-cystine present in the sample is con-verted to cysteic acid by the DMSO present in the HydrolysisSolution. As an approach to limit variability and compensatefor partial destruction, it is recommended to evaluate thecysteic acid recovery from oxidative hydrolyses of standardproteins containing 1 to 8 mol of cysteine. The response fac-tors from protein/peptide hydrolysates are typically about30z lower than those for nonhydrolyzed cysteic acid stand-ards. Because histidine, methionine, tyrosine, and trypto-

phan are also modified, a complete compositional analysis isnot obtained with this technique.

Method 7Cysteine-cystine reduction and alkylation is accomplished

by a vapor phase pyridylethylation reaction.Reducing Solution Transfer 83.3 mL of pyridine, 16.7 mL

of 4-vinylpyridine, 16.7 mL of tributylphosphine, and 83.3mL of water to a suitable container, and mix.

Procedure Add the protein/peptide (between 1 and 100mg) to a hydrolysis tube, and place in a larger tube. Transferthe Reducing Solution to the large tube, seal in vacuum(about 50 mm of mercury or 6.7 Pa), and incubate at about1009C for 5 minutes. Then remove the inner hydrolysis tube,and dry it in a vacuum desiccator for 15 minutes to removeresidual reagents. The pyridylethylated protein/peptide canthen be acid hydrolyzed using previously described proce-dures. The pyridylethylation reaction is performed simul-taneously with a protein standard sample containing 1 to 8mol of cysteine to improve accuracy in the pyridylethyl-cysteine recovery. Longer incubation times for the pyri-dylethylation reaction can cause modifications to the a-amino terminal group and the e-amino group of lysine in theprotein.


by a liquid phase pyridylethylation reaction.Stock Solutions Prepare and filter three solutions: 1

mol/L Tris hydrochloride (pH 8.5) containing 4 mmol/Ldisodium dihydrogen ethylendiamine tetraacetate (Stock So-lution A), 8 mol/L guanidine hydrochloride (Stock SolutionB), and 10z of 2-mercaptoethanol in water (Stock SolutionC ).

Reducing Solution Prepare a mixture of Stock SolutionB and Stock Solution A (3:1) to obtain a buffered solutionof 6 mol/L guanidine hydrochloride in 0.25 mol/L Tris hy-drochloride.

Procedure Dissolve about 10 mg of the test sample in 50mL of the Reducing Solution, and add about 2.5 mL of StockSolution C. Store under nitrogen or argon for 2 hours atroom temperature in the dark. To achieve the pyridylethyla-tion reaction, add about 2 mL of 4-vinylpyridine to the pro-tein solution, and incubate for an additional 2 hours at roomtemperature in the dark. The protein/peptide is desalted bycollecting the protein/peptide fraction from a reversed-phaseHPLC separation. The collected sample can be dried in avacuum centrifuge before acid hydrolysis.


by a liquid phase carboxymethylation reaction.Stock Solutions Prepare as directed for Method 8.Carboxymethylation Solution Prepare a solution con-

taining 100 mg of iodoacetamide per mL of ethanol (95).Buffer Solution Use the Reducing Solution, prepared as

directed for Method 8.Procedure Dissolve the test sample in 50 mL of the

Buffer Solution, and add about 2.5 mL of Stock Solution C.Store under nitrogen or argon for 2 hours at room tempera-ture in the dark. Add the Carboxymethylation Solution in aratio 1.5 fold per total theoretical content of thiols, and in-cubate for an additional 30 minutes at room temperature inthe dark. [Note: If the thiol content of the protein is


unknown, then add 5 mL of 100 mmol/L iodoacetamide forevery 20 nmol of protein present.] The reaction is stopped byadding excess of 2-mercaptoethanol. The protein/peptide isdesalted by collecting the protein/peptide fraction from areversed-phase HPLC separation. The collected sample canbe dried in a vacuum centrifuge before acid hydrolysis. TheS-carboxyamidomethyl-cysteine formed will be converted toS-carboxymethylcysteine during acid hydrolysis.

Method 10Cysteine-cystine is reacted with dithiodiglycolic acid or

dithiodipropionic acid to produce a mixed disulfide. [Note:The choice of dithiodiglycolic acid or dithiodipropionic aciddepends on the required resolution of the amino acid analy-sis method.]

Reducing Solution A solution containing 10 mg ofdithiodiglycolic acid (or dithiodipropionic acid) per mL of0.2 mol/L sodium hydroxide.

Procedure Transfer about 20 mg of the test sample to ahydrolysis tube, and add 5 mL of the Reducing Solution.Add 10 mL of isopropyl alcohol, and then remove all of thesample liquid by vacuum centrifugation. The sample is thenhydrolyzed using Method 1. This method has the advantagethat other amino acid residues are not derivatized by sidereactions, and the sample does not need to be desalted priorto hydrolysis.

Method 11Asparagine and glutamine are converted to aspartic acid

and glutamic acid, respectively, during acid hydrolysis.Asparagine and aspartic acid residues are added and repre-sented by Asx, while glutamine and glutamic acid residuesare added and represented by Glx. Proteins/peptides can bereacted with bis(1,1-trifluoroacetoxy)iodobenzene (BTI) toconvert the asparagine and glutamine residues to diamino-propionic acid and diaminobutyric acid residues, respec-tively, upon acid hydrolysis. These conversions allow theanalyst to determine the asparagine and glutamine content ofa protein/peptide in the presence of aspartic acid and glu-tamic acid residues.

Reducing Solutions Prepare and filter three solutions: asolution of 10 mmol/L trifluoroacetic acid (Solution A), asolution of 5 mol/L guanidine hydrochloride and 10 mmol/L trifluoroacetic acid (Solution B), and a freshly preparedsolution of N,N-dimethylformamide containing 36 mg ofBTI per mL (Solution C ).

Procedure In a clean hydrolysis tube, transfer about 200mg of the test sample, and add 2 mL of Solution A or Solu-tion B and 2 mL of Solution C. Seal the hydrolysis tube invacuum. Heat the sample at 609C for 4 hours in the dark.The sample is then dialyzed with water to remove the excessreagents. Extract the dialyzed sample three times with equalvolumes of n-butyl acetate, and then lyophilize. The proteincan then be acid hydrolyzed using previously describedprocedures. The a,b-diaminopropionic and a,g-diaminobu-tyric acid residues do not typically resolve from the lysineresidues upon ion-exchange chromatography based onamino acid analysis. Therefore, when using ion-exchange asthe mode of amino acid separation, the asparagine andglutamine contents are the quantitative difference in theaspartic acid and glutamic acid content assayed withunderivatized and BTI-derivatized acid hydrolysis. [Note:The threonine, methionine, cysteine, tyrosine, and histidineassayed content can be altered by BTI derivatization; a hy-

drolysis without BTI will have to be performed if the analystis interested in the composition of these other amino acidresidues of the protein/peptide.]

Methodologies of Amino Acid Analysis General PrinciplesMany amino acid analysis techniques exist, and the choice

of any one technique often depends on the sensitivity re-quired from the assay. In general, about one-half of theamino acid analysis techniques employed rely on the separa-tion of the free amino acids by ion-exchange chromatogra-phy followed by postcolumn derivatization (e.g., with nin-hydrin or o-phthalaldehyde). Postcolumn detection tech-niques can be used with samples that contain small amountsof buffer components, such as salts and urea, and generallyrequire between 5 and 10 mg of protein sample per analysis.The remaining amino acid techniques typically involve pre-column derivatization of the free amino acids (e.g., phenylisothiocyanate; 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate or o-phthalaldehyde; (dimethylamino)azoben-zenesulfonyl chloride; 9-fluorenylmethylchloroformate; and,7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole) followed by rever-sed-phase HPLC. Precolumn derivatization techniques arevery sensitive and usually require between 0.5 and 1.0 mg ofprotein sample per analysis but may be influenced by buffersalts in the samples. Precolumn derivatization techniquesmay also result in multiple derivatives of a given amino acid,which complicates the result interpretation. Postcolumnderivatization techniques are generally influenced less byperformance variation of the assay than precolumn derivati-zation techniques.

The following Methods may be used for quantitativeamino acid analysis. Instruments and reagents for theseprocedures are available commercially. Furthermore, manymodifications of these methodologies exist with differentreagent preparations, reaction procedures, chromatographicsystems, etc. Specific parameters may vary according to theexact equipment and procedure used. Many laboratories willutilize more than one amino acid analysis technique toexploit the advantages offered by each. In each of theseMethods, the analog signal is visualized by means of a dataacquisition system, and the peak areas are integrated forquantification purposes.

Method 1�Postcolumn Ninhydrin Detection GeneralPrinciple

Ion-exchange chromatography with postcolumn ninhydrindetection is one of the most common methods employed forquantitative amino acid analysis. As a rule, a Li-basedcation-exchange system is employed for the analysis of themore complex physiological samples, and the faster Na-based cation-exchange system is used for the more simplisticamino acid mixtures obtained with protein hydrolysates(typically containing 17 amino acid components). Separationof the amino acids on an ion-exchange column is accom-plished through a combination of changes in pH and cationstrength. A temperature gradient is often employed toenhance separation.

When the amino acid reacts with ninhydrin, the reactanthas characteristic purple or yellow color. Amino acids, ex-cept imino acid, give a purple color, and show the maximumabsorption at 570 nm. The imino acids such as proline give ayellow color, and show the maximum absorption at 440 nm.The postcolumn reaction between ninhydrin and amino acideluted from column is monitored at 440 and 570 nm, and the


chromatogram obtained is used for the determination ofamino acid composition.

Detection limit is considered to be 10 pmol for most of theamino acid derivatives, but 50 pmol for proline. Responselinearity is obtained in the range of 20 to 500 pmol with cor-relation coefficients exceeding 0.999. To obtain good com-position data, samples larger than 1 mg before hydrolysis arebest suited for this amino acid analysis of protein/peptide.

Method 2�Postcolumn OPA Fluorometric DetectionGeneral Principle

o-Phthalaldehyde (OPA) reacts with primary amines inthe presence of thiol compound, to form highly fluorescentisoindole products. This reaction is utilized for the postcol-umn derivatization in analysis of amino acids by ion-exchange chromatography. The rule of the separation is thesame as Method 1. Instruments and reagents for this form ofamino acid analysis are available commercially. Manymodifications of this methodology exist.

Although OPA does not react with secondary amines(imino acids such as proline) to form fluorescent substances,the oxidation with sodium hypochlorite allows secondaryamines to react with OPA. The procedure employs astrongly acidic cation-exchange column for separation offree amino acids followed by postcolumn oxidation withsodium hypochlorite and postcolumn derivatization usingOPA and thiol compound such as N-acetyl-L-cysteine and 2-mercaptoethanol. The derivatization of primary amino acidsare not noticeably affected by the continuous supply ofsodium hypochlorite.

Separation of the amino acids on an ion-exchange columnis accomplished through a combination of changes in pHand cation strength. After postcolumn derivatization ofeluted amino acids with OPA, the reactant passes throughthe fluorometric detector. Fluorescence intensity of OPA-derivatized amino acids are monitored with an excitationwavelength of 348 nm and an emission wavelength of 450nm.

Detection limit is considered to be a few tens of picomolelevel for most of the amino acid derivatives. Responselinearity is obtained in the range of a few picomole level to afew tens of nanomole level. To obtain good compositionaldata, the starting with greater than 500 ng of sample beforehydrolysis is best suited for the amino acid analysis of pro-tein/peptide.

Method 3�Precolumn PITC Derivatization GeneralPrinciple

Phenylisothiocyanate (PITC) reacts with amino acids toform phenylthiocarbamyl (PTC) derivatives which can bedetected with high sensitivity at 245 nm. Therefore, precol-umn derivatization of amino acids with PITC followed by areversed-phase HPLC separation with UV detection is usedto analyze the amino acid composition.

After the reagent is removed under vacuum, the deriva-tized amino acids can be stored dry and frozen for severalweeks with no significant degradation. If the solution for in-jection is kept cold, no noticeable loss in chromatographicresponse occurs after three days.

Separation of the PTC-amino acids on a reversed-phaseHPLC with ODS column is accomplished through a combi-nation of changes in concentrations of acetonitrile andbuffer ionic strength. PTC-amino acids eluted from thecolumn are monitored at 254 nm.

Detection limit is considered to be 1 pmol for most of theamino acid derivatives. Response linearity is obtained in therange of 20 to 500 pmol with correlation coefficientsexceeding 0.999. To obtain good compositional data, sam-ples larger than 500 ng of protein/peptide before hydrolysisis best suited for this amino acid analysis of proteins/peptides.

Method 4�Precolumn AQC Derivatization GeneralPrinciple

Precolumn derivatization of amino acids with 6-amino-quinolyl-N-hydroxysuccinimidyl carbamate (AQC) followedby reversed-phase HPLC separation with fluorometric detec-tion is used.

AQC reacts with amino acids to form stable, fluorescentunsymmetric urea derivatives (AQC-amino acids) which arereadily amenable to analysis by reversed-phase HPLC.Therefore, precolumn derivatization of amino acids withAQC followed by reversed-phase HPLC separation is usedto analyze the amino acid composition.

Separation of the AQC-amino acids on an ODS column isaccomplished through a combination of changes in concen-trations of acetonitrile and salt. Selective fluorescence detec-tion of the derivatives with excitation wavelength at 250 nmand emission wavelength at 395 nm allows for the directinjection of the reaction mixture with no significant interfer-ence from the only major fluorescent reagent by-product,6-aminoquinoline. Excess reagent is rapidly hydrolyzed(t1/2＜ 15 seconds) to yield 6-aminoquinoline, N-hydroxy-succinimide and carbon dioxide, and after 1 minute no fur-ther derivatization can take place.

Peak areas for AQC-amino acids are essentially un-changed for at least 1 week at room temperature, and thederivatives have more than sufficient stability to allow forovernight automated chromatographic analysis.

Detection limit is considered to be ranging from ca. 40 to320 fmol for each amino acid, except for Cys. Detectionlimit for Cys is approximately 800 fmol. Response linearityis obtained in the range of 2.5 to 200 mmol/L with correla-tion coefficients exceeding 0.999. Good compositional datacould be obtained from the analysis of derivatized proteinhydrolysates containing as little as 30 ng of protein/peptide.

Method 5�Precolumn OPA Derivatization GeneralPrinciple

Precolumn derivatization of amino acids with o-phthalal-dehyde (OPA) followed by reversed-phase HPLC separationwith fluorometric detection is used. This technique does notdetect amino acids that exist as secondary amines (e.g.,proline).

OPA in conjunction with a thiol reagent reacts withprimary amine groups to form highly fluorescent isoindoleproducts. 2-Mercaptoethanol or 3-mercaptopropionic acidcan be used as thiol. OPA itself does not fluoresce and con-sequently produces no interfering peaks. In addition, itssolubility and stability in aqueous solution, along with therapid kinetics for the reaction, make it amenable to automat-ed derivatization and analysis using an autosampler to mixthe sample with the reagent. However, lack of reactivity withsecondary amino acids has been a predominant drawback.This method does not detect amino acids that exist assecondary amines (e.g., proline). To compensate for thisdrawback, this technique may be combined with anothertechnique described in Method 7 or Method 8.


Precolumn derivatization of amino acids with OPA is fol-lowed by a reversed-phase HPLC separation. Because of theinstability of the OPA-amino acid derivative, HPLC separa-tion and analysis are performed immediately followingderivatization. The liquid chromatograph is equipped with afluorometric detector for the detection of derivatized aminoacids. Fluorescence intensity of OPA-derivatized aminoacids is monitored with an excitation wavelength of 348 nmand an emission wavelength of 450 nm.

Detection limits as low as 50 fmol via fluorescence havebeen reported, although the practical limit of analysisremains at 1 pmol.

Method 6�Precolumn DABS-Cl Derivatization GeneralPrinciple

Precolumn derivatization of amino acids with(dimethylamino)azobenzenesulfonyl chloride (DABS-Cl)followed by reversed-phase HPLC separation with visiblelight detection is used.

DABS-Cl is a chromophoric reagent employed for thelabeling of amino acids. Amino acids labeled with DABS-Cl(DABS-amino acids) are highly stable and show the maxi-mum absorption at 436 nm.

DABS-amino acids, all 19 naturally occurring amino acidsderivatives, can be separated on an ODS column of a rever-sed-phase HPLC by employing gradient systems consistingof acetonitrile and aqueous buffer mixture. SeparatedDABS-amino acids eluted from the column are detected at436 nm in the visible region.

This Method can analyze the imino acids such as prolinetogether with the amino acids at the same degree of sensi-tivity, DABS-Cl derivatization method permits the simul-taneous quantification of tryptophan residues by previoushydrolysis of the protein/peptide with sulfonic acids such asmercaptoethanesulfonic acid, p-toluenesulfonic acid ormethanesulfonic acid described under Method 2 in ``ProteinHydrolysis''. The other acid-labile residues, asparagine andglutamine, can also be analysed by previous conversion intodiaminopropionic acid and diaminobutyric acid, respec-tively, by treatment of protein/peptide with BTI describedunder Method 11 in ``Protein Hydrolysis''.

The non-proteinogenic amino acid, norleucine cannot beused as internal standard in this method, as this compound iseluted in a chromatographic region crowded with peaks ofprimary amino acids. Nitrotyrosine can be used as an inter-nal standard, because it is eluted in a clean region.

Detection limit of DABS-amino acid is about 1 pmol. Aslittle as 2 to 5 pmol of an individual DABS-amino acid canbe quantitatively analysed with reliability, and only 10 to 30ng of the dabsylated protein hydrolysate is required for eachanalysis.

Method 7�Precolumn FMOC-Cl Derivatization GeneralPrinciple

Precolumn derivatization of amino acids with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed byreversed-phase HPLC separation with fluorometric detec-tion is used.

FMOC-Cl reacts with both primary and secondary aminoacids to form highly fluorescent products. The reaction ofFMOC-Cl with amino acid proceeds under mild conditionsin aqueous solution and is completed in 30 seconds. Thederivatives are stable, only the histidine derivative showingany breakdown. Although FMOC-Cl is fluorescent itself,

the reagent excess and fluorescent side-products can beeliminated without loss of FMOC-amino acids.

FMOC-amino acids are separated by a reversed-phaseHPLC using an ODS column. The separation is carried outby gradient elution varied linearly from a mixture of aceto-nitrile methanol and acetic acid buffer (10:40:50) to a mix-ture of acetonitrile and acetic acid buffer (50:50), and 20amino acid derivatives are separated in 20 minutes. Eachderivative eluted from the column is monitored by a fluoro-metric detector set at an excitation wavelength of 260 nmand an emission wavelength of 313 nm.

The detection limit is in the low fmol range. A linearityrange of 0.1 to 50 mmol/L is obtained for most of the aminoacids.

Method 8�Precolumn NBD-F Derivatization GeneralPrinciple

Precolumn derivatization of amino acids with 7-fluoro-4-nitrobenzo-2-oxa-1.3-diazole (NBD-F) followed by reversed-phase HPLC separation with fluorometric detection is used.

NBD-F reacts with both primary and secondary aminoacids to form highly fluorescent products. Amino acids arederivatized with NBD-F by heating to 609C for 5 minutes.

NBD-amino acid derivatives are separated on an ODScolumn of a reversed-phase HPLC by employing gradientelution system consisting of acetonitrile and aqueous buffermixture, and 17 amino acid derivatives are separated in 35minutes. e-Aminocaproic acid can be used as an internalstandard, because it is eluted in a clean chromatographicregion. Each derivative eluted from the column is monitoredby a fluorometric detector set at an excitation wavelength of480 nm and an emission wavelength of 530 nm.

The sensitivity of this method is almost the same as forprecolumn OPA derivatization method (Method 5), exclud-ing proline to which OPA is not reactive, and might be ad-vantageous for NBD-F against OPA. The detection limit foreach amino acid is about 10 fmol. Profile analysis wasachieved for about 1.5 mg of protein hydrolysates in the finalprecolumn labeling reaction mixture for HPLC.

Data Calculation and AnalysisWhen determining the amino acid content of a protein/

peptide hydrolysate, it should be noted that the acid hydroly-sis step destroys tryptophan and cysteine. Serine and threo-nine are partially destroyed by acid hydrolysis, while isoleu-cine and valine residues may be only partially cleaved.Methionine can undergo oxidation during acid hydrolysis,and some amino acids (e.g., glycine and serine) are commoncontaminants. Application of adequate vacuum (≦0.0267kPa) or introduction of inert gas (argon) in the headspace ofthe reaction vessel during vapor phase hydrolysis can reducethe level of oxidative destruction. Therefore, the quantitativeresults obtained for cysteine, tryptophan, threonine, isoleu-cine, valine, methionine, glycine, and serine from a protein/peptide hydrolysate may be variable and may warrant fur-ther investigation and consideration.

CalculationsAmino Acid Mole Percent This is the number of specific

amino acid residues per 100 residues in a protein. This resultmay be useful for evaluating amino acid analysis data whenthe molecular weight of the protein under investigation isunknown. This information can be used to corroborate theidentity of a protein/peptide and has other applications.


Carefully identify and integrate the peaks obtained as di-rected for each Procedure. Calculate the mole percent foreach amino acid present in the test sample by the formula:

100rU/r,in which rU is the peak response, in nmol, of the amino acidunder test; and r is the sum of peak responses, in nmol, forall amino acids present in the test sample. Comparison of themole percent of the amino acids under test to data fromknown proteins can help establish or corroborate the identityof the sample protein.

Unknown Protein Samples This data analysis techniquecan be used to estimate the protein concentration of anunknown protein sample using the amino acid analysis data.Calculate the mass, in mg, of each recovered amino acid bythe formula:

mMW/1000,in which m is the recovered quantity, in nmol, of the aminoacid under test; and MW is the average molecular weight forthat amino acid, corrected for the weight of the water molec-ule that was eliminated during peptide bond formation. Thesum of the masses of the recovered amino acids will give anestimate of the total mass of the protein analyzed after ap-propriate correction for partially and completely destroyedamino acids. If the molecular weight of the unknown proteinis available (i.e., by SDS-PAGE analysis or mass spectros-copy), the amino acid composition of the unknown proteincan be predicted. Calculate the number of residues of eachamino acid by the formula:

m/(1000M/MWT),in which m is the recovered quantity, in nmol, of the aminoacid under test; M is the total mass, in mg, of the protein;and MWT is the molecular weight of the unknown protein.

Known Protein Samples This data analysis technique canbe used to investigate the amino acid composition and pro-tein concentration of a protein sample of known molecularweight and amino acid composition using the amino acidanalysis data. When the composition of the protein beinganalyzed is known, one can exploit the fact that some aminoacids are recovered well, while other amino acid recoveriesmay be compromised because of complete or partial destruc-tion (e.g., tryptophan, cysteine, threonine, serine, methio-nine), incomplete bond cleavage (i.e., for isoleucine andvaline) and free amino acid contamination (i.e., by glycineand serine).

Because those amino acids that are recovered bestrepresent the protein, these amino acids are chosen to quan-tify the amount of protein. Well-recovered amino acidsare, typically, aspartate-asparagine, glutamate-glutamine,alanine, leucine, phenylalanine, lysine, and arginine. Thislist can be modified based on experience with one's ownanalysis system. Divide the quantity, in nmol, of each of thewell-recovered amino acids by the expected number ofresidues for that amino acid to obtain the protein contentbased on each well-recovered amino acid. Average the pro-tein content results calculated. The protein content deter-mined for each of the well-recovered amino acids should beevenly distributed about the mean. Discard protein contentvalues for those amino acids that have an unacceptable de-viation from the mean. Typically ≧greater than 5z varia-tion from the mean is considered unacceptable. Recalculatethe mean protein content from the remaining values to ob-tain the protein content of the sample. Divide the content ofeach amino acid by the calculated mean protein content to

determine the amino acid composition of the sample byanalysis.

Calculate the relative compositional error, in percentage,by the formula:

100m/mS,in which m is the experimentally determined quantity, innmol per amino acid residue, of the amino acid under test;and mS is the known residue value for that amino acid. Theaverage relative compositional error is the average of theabsolute values of the relative compositional errors of theindividual amino acids, typically excluding tryptophan andcysteine from this calculation. The average relative composi-tional error can provide important information on the stabil-ity of analysis run over time. The agreement in the aminoacid composition between the protein sample and the knowncomposition can be used to corroborate the identity andpurity of the protein in the sample.

Basic Requirements for Viral Safetyof Biotechnological/Biological

Products listed in JapanesePharmacopoeia

IntroductionThe primary role of specification of biotechnological/

biological products listed in Japanese Pharmacopoeia (JP) isnot only for securing quality control or consistency of thequality but also for assuring their efficacy and safety. In themeantime, the requirements to assure quality and safety ofdrugs have come to be quite strict recently, and a rigid atti-tude addressing safety assurance is expected for biotechno-logical/biological products. The key points for quality andsafety assurance of biotechnological/biological products areselection and appropriate evaluation of source material, ap-propriate evaluation of manufacturing process and main-tenance of manufacturing consistency, and control of spe-cific physical properties of the products. Now, how to assurequality and safety of such drugs within a scope of JP hascome to be questioned. This General Information describeswhat sorts of approaches are available to overcome theseissues.

It is desired that quality and safety assurance of JP listedproducts are achieved by state-of-the-art methods and con-cepts which reflect progress of science and accumulation ofexperiences. This General Information challenges to showthe highest level of current scientific speculation. It is ex-pected that this information will contribute to promotion ofscientific understanding of quality and safety assurance ofnot only JP listed products but also the other biotechnologi-cal/biological products and to promotion of active discus-sion of each Official Monograph in JP.

1. Fundamental measures to ensure viral safety of JP listedbiotechnological/biological products

The biotechnological/biological product JP includes theproducts derived from living tissue and body fluid (urine,blood, etc.) of mammals, etc. Protein drugs derived fromcell lines of human or animal origin (e.g., recombinant DNAdrug, cell culture drug) are also included. The fundamentalmeasures required for comprehensive viral safety of JP listed


biotechnological/biological products are as follows: 1) ac-quaintance of possible virus contamination (source of con-tamination); 2) careful examination of eligibility of raw ma-terials and their sources, e.g. human/animal, and thoroughanalysis and screening of the sample chosen as a substratefor drug production (e.g., pooled body fluid, cell bank, etc.)to determine any virus contamination and determination oftype and nature of the virus, if contaminated; 3) evaluationto determine virus titer and virus-like particles hazardous tohuman, if exists; 4) selection of production related material(e.g., reagent, immune antibody column) free from infecti-ous or pathogenic virus; 5) performance of virus free test atan appropriate stage of manufacturing including the finalproduct, if necessary; 6) adoption of effective viral clearancemethod in the manufacturing process to remove/inactivatevirus. Combined method sometimes achieves higher level ofclearance; 7) development of a deliberate viral clearancescheme; 8) performance of the test to evaluate viral removaland inactivation. It is considered that the stepwise and sup-plemental adoption of the said measures will contribute toensure viral safety and its improvement.

2. Safety assurance measures described in the OfficialMonograph and this General Information

As mentioned in above 1, this General Informationdescribes, in package, points to be concerned with and con-crete information on the measures taken for viral safety ofJP listed products. Except where any specific caution isprovided in Official Monograph of a product in question,Official Monograph provides in general that ``Any raw ma-terial, substrate for drug production and production relatedmaterial used for production of drug should be derived fromhealthy animals and should be shown to be free of latentvirus which is infectious or pathogenic to human'', ``Cellline and culture method well evaluated in aspects of ap-propriateness and rationality on viral safety are used forproduction, and the presence of infectious or pathogenic la-tent virus to human in process related materials derived fromliving organisms should be denied''. and ``biotechnological/biological drug should be produced through a manufactur-ing process which is capable of removing infectious orpathogenic virus'', etc., to raise awareness on viral safetyand on necessity to conduct test and process evaluation forviral safety.

3. Items and contents described in this General InformationAs for viral safety of protein drug derived from cell line of

human or animal origin, there is a Notice in Japan entitled``Viral safety evaluation of biotechnology products derivedfrom cell lines of human or animal origin'' (Iyakushin No.329 issued on February 22, 2000 by Director, Evaluation andLicensing Division, Pharmaceutical and Medical SafetyBureau, Ministry of Health and Welfare) to reflect the inter-nationally harmonized ICH Guideline, and as for bloodplasma protein fraction preparations, there is a documententitled ``Guideline for ensuring viral safety of blood plasmaprotein fraction preparations''. This General Informationfor ensuring viral safety of JP listed biotechnological/bio-logical products has been written, referencing the contents ofthose guidelines, to cover general points and their details tobe concerned for ensuring viral safety of not only JP listedbiotechnological/biological products but also all productswhich would be listed in JP in future, i.e., biologicalproducts derived from living tissue and body fluid, such as

urine, and protein drugs derived from cell line of human oranimal origin (Table 1).3.1. Purpose

The purpose of this document is to propose the compre-hensive concepts of the measures to be taken for ensuringviral safety of biotechnological/biological products derivedfrom living tissue or body fluid of mammals, etc. and ofprotein drugs derived from cell lines of human or animalorigin. That is to say, this document describes the measuresand the points of concern on the items, such as considera-tion of the source of virus contamination; appropriateevaluation on eligibility at selecting the raw material and onqualification of its source, e.g. human or animal; virustest, and its analysis and evaluation at a stage of cell sub-strate for drug production; appropriate evaluation tochoose product related materials derived from living organ-isms (e.g. reagent, immune antibody column, etc.); con-duct of necessary virus test on the product at an appropriatestage of manufacturing; development of viral clearancetest scheme; performance and evaluation of viral clear-ance test. This document is also purposed to comprehen-sively describe in details that supplemental and combiningadoption of the said measures will contribute to secure viralsafety and its improvement.3.2. Background

One of the most important issues to be cautioned forsafety of a biological product, which is directly derived fromhuman or animal, or of a protein drug, which is derivedfrom cell line of human or animal origin (recombinant DNAderived product, cell culture derived product, etc.), is risk ofvirus contamination. Virus contamination may cause serioussituation at clinical use once it occurs. Virus contaminationmay be from a raw material or from a cell substrate for drugproduction, or may be from an adventitious factor intro-duced to the manufacturing process.

JP listed biological drugs or protein drugs derived fromcell line have achieved drastic contribution to the medicalsociety, and to date, there has not been any evidence of anysafety problem on them caused by virus. But, social require-ment of health hazard prevention is strong, and it is nowvery important to prevent accidental incidence, takingsecurity measures carefully supported by scientific ration-ality. It is always great concern among the persons involvedthat under what sort of viewpoint and to what extent wehave to pursue for ensuring viral safety of a biotechnolo-gical/biological product. Before discussing these issues, twofundamental points have to be reconfirmed. One is that; wehave to consider scientific, medical, and social profiles adrug has. In other words, ``Medicine is a social asset which isutilized in medical practice paying attention to the risk andbenefit from the standpoints of science and society''. It is thedestine and the mission of the medical/pharmaceuticalsociety to realize prompt and stable supply of such a socialasset, drug, among the medical work front to bring gospel tothe patients.

The other is that; issue of viral safety is independent fromsafety of the components of a drug per se (narrow sense ofsafety). It is important to consider that this is the matter ofgeneral safety of drug (broad sense of safety). In case of adrug which has been used for a long time in the medicalfront, such as a JP listed product, its broad sense of safety isconsidered to have been established epidemiologically, andits usage past records have a great meaning. However,

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Table 1 Items described in General Informationfor Viral Safety Assurance of JP listedBiotechnological/Biological Product

I. Introduction1. Fundamental measures to ensure viral safety of JP list-

ed biotechnological/biological products2. Safety assurance measures described in the Official

Monograph and this General Information3. Items and contents described in this General Informa-

tionII. General Matters

1. Purpose2. Background3. Unknown risk on the measures taken for ensuring viral

safety4. Applicable range5. Possible viral contamination to a JP listed biotechno-

logical/biological product (source of virus contamina-tion)

6. Basis for ensuring viral safety7. Limit of virus test8. Roles of viral clearance studies

III. Raw material/substrate for drug production1. Issues relating to animal species and its region as a

source of raw materialsubstrate for drug productionand countermeasures to be taken thereto

2. Qualification evaluation test on human or animal as asource of raw material/substrate for drug production

IV. Points of concern with respect to manufacturing andvirus testing

1. Virus test conducted in advance of purification process2. Virus test as an acceptance test of an intermediate

material, etc.3. Virus test on a final product

V. Process evaluation on viral clearance1. Rationale, objective and general items to be concerned

with respect to viral clearance process evaluation2. Selection of virus3. Design of viral clearance studies4. Interpretation of viral clearance studies

1) Evaluation of viral clearance factor2) Calculation of viral clearance index3) Interpretation of results and items to be concerned at

evaluationVI. Statistics

1. Statistical considerations for assessing virus assays2. Reproducibility and confidence limit of viral clearance

studiesVII. Re-evaluation of viral clearanceVIII. Measurement for viral clearance studies

1. Measurement of virus infective titer2. Testing by nucleic-acid amplification test (NAT)

IX. Reporting and preservationX. Others


different from safety of drug per se (its components), takinginto account any possibility of virus contamination, we haveto say that only the results accumulated can not alwaysassure viral safety of a drug used in future. Accordingly, thebasis for securing broad sense of viral safety of JP listedbiotechnological/biological products is to pay every atten-

tion to the measures to take for prevention, while evaluatingthe accumulated results.

Adopting strict regulations and conducting tests at maxi-mum level to the extent theoretically considered may be theways off assuring safety, but applying such way generally,without sufficient scientific review of the ways and evalua-tion of usage results, causes excessive requirement of regula-tion and test not having scientific rationality. As the results,effective and prompt supply of an important drug, alreadyhaving enough accumulation of experiences, to the medicalwork front will be hampered, and the drug, a social asset,may not to be utilized effectively. Medicine is a sword usedin medical field having double-edge named effectiveness andsafety. Effectiveness and safety factors have to be derived asthe fruits of leading edge of science, and relatively evaluatedon a balance sheet of usefulness. Usefulness evaluationshould not be unbalanced in a way that too much emphasisis placed on safety concern without back-up of appropriatescientific rationality. A drug can play an important role as asocial asset only when well balanced appropriate scientificusefulness evaluation in addition to social concern of the ageare given. In other words, drug is a common asset utilized bysociety for medication as a fruit of science of the age, andthe key point of its utilization lies on a balance of risk andbenefit produced from scientific and social evaluation. So,those factors have to be taken into account when target andpursuance levels for ensuring viral safety of a JP listedbiotechnological/biological product are reviewed.

And, in general, the risk and benefit of drugs should beconsidered with the relative comparison to alternative drugsor medical treatment. The usefulness of certain drug shouldbe reviewed finally after the competitive assessment on therisk and benefit on the alternative drugs, relevant drugs and/or alternative medical treatment.

Under such background, the purpose of this article is todescribe the scientific and rational measures to be taken forensuring viral safety of JP listed biotechnological/biologicalproducts. Giving scientific and rational measures mean that;appropriate and effective measures, elaborated from the cur-rent scientific level, are given to the issues assumable underthe current scientific knowledge. In other words, possiblecontaminant virus is assumed to have the natures of genus,morph, particle size, physical/chemical properties, etc.which are within the range of knowledge of existingvirology, and is those assumed to exist in human and animal,tissue and body fluid, which are the source of biotechnolo-gical/biological product, reagent, material, additives, etc.Accordingly, viral clearance studies using a detectionmethod which target those viruses have to be designed.3.3. Unknown risk on the measures taken for ensuringviral safety

There are known and unknown risks.It is easy to determine a test method and an evaluation

standard on the known risk, which exists in the drug per se(pharmaceutical component) or inevitably exists due to aquality threshold, and quantification of such risk is possible.In other words, it is easy to evaluate the known risk on abalance sheet in relation to the benefit, and we can say thatvaluation even in this respect has been established to someextent.

On the other hand, as for the unknown risk which is in-evitable for ensuring viral safety, the subject of the risk cannot be defined and quantitative concept is hard to introduce,


and, therefore, taking a counter measure and evaluating itseffect are not so easy. Therefore, this is the subject to bechallenged calling upon wisdom of the related parties amongthe society of drug.

Talking about the unknown risk, there are view pointsthat say `Ìt is risky because it is unknown.'' and ``What arethe unknowns, and how do we cope with them in ensuringsafety?''.

The view of `Ìt is risky because it is unknown.'' is alreadynothing but a sort of evaluation result, and directly connectsto a final decision if it can be used as a drug. Such evalu-ation/decision has to be made based upon a rational, scien-tific or social judgment.

For example, in the case that `Ìn a manufacturing processof drug, virus, virus-like particle or retrovirus was detected,but its identification could not be confirmed, and, therefore,its risk can not be denied.'', the evaluation of `Ìt is riskybecause it is unknown.'' is scientifically rational and reason-able. On the other hand, however, if we reach a decision of`Ìt is risky because it is unknown.'' due to the reason that`Ìn a manufacturing process of drug, virus, virus-like parti-cle or retrovirus was not detected, but there is a `concern'that something unknown may exist.'', it can not be said thatsuch evaluation is based upon a rational, scientific or socialjudgment. It goes without saying that the utmost care has tobe taken for viral safety, but the substance of `concern' hasto be at least clearly explainable. Otherwise, the `concern'may result in causing contradiction in the meaningful mis-sion to utilize a social asset, drug, in medical practice.

From scientific view point, we should not be narrow mind-ed by saying `ìt is risky'' because ``there is a `concern' thatsomething unknown may exists'', but challenge to clarify thesubject of ``What is unknown, and how to cope with it forensuring safety'' using wisdom. What is important at thetime is to define ``what is unknown'' based upon currentscientific knowledge. Only through this way, is it possiblefor us to elaborate the measures for ensuring safety.

Once we chase up the substance of unknown risk for viralsafety without premises of ``what is unknown'', `ùnknown''will be an endless question because it theoretically remainsunresolved forever. If this kind of approach is taken, theissue and the measure can not be scientifically connected toeach other, which will result in the excessive requirement ofregulation and of test to be conducted. Yet, it is unlikely thatthe measure which has no relation with science will be effec-tive to the subject of ``What is unknown is unknown.''

For example, ``what is unknown'' at the `èvaluation of apurification process which can completely clear up everyvirus that contaminated in a manufacturing process'' shouldbe the subject of ``what sort of existing virus that contami-nated is unknown'', not on the subject of ``what sort ofvirus that exist in the world is unknown. In the former sub-ject, the premise of the study is based on all the knowledgeon viruses including DNA/RNA-virus, virus with/withoutenvelope, particle size, physical/chemical properties, etc.The premise is that the virus contaminated should be withinrange of existing wisdom and knowledge of virus such asspecies, type, nature, etc., even though the virus that con-taminated is unknown. Under such premise, when evalua-tion is made on a purification process to decide its capabilityof clearing a derived virus, which is within the range of exist-ing wisdom and learning, specific viral clearance studiesdesigned to combine a few model viruses with different na-

tures, such as type of nucleic acid, with/without envelope,particle size, physical/chemical properties, etc., would beenough to simulate every sort of the virus already known,and will be a ``good measure for ensuring safety''.

The issue of ``the sort of viruses that exist in the world isunknown'' may be a future study item, but it is not an ap-propriate subject for the viral clearance test. Further, even ifthe subject of `ùnknown viruses, which have a particle sizesmaller than that of currently known viruses, may exists'' or`ùnknown viruses, which have special physical/chemicalproperties that can not be matched to any of the currentlyknown viruses, may exists'' is set up as an armchair theory,any experimental work can not be pursued under the currentscientific level, since such virus model is not available. Fur-ther, any viral clearance test performed by using the current-ly available methods and technologies will be meaningless``for ensuring safety'', since particle size or natures of suchspeculated virus are unknown. Likewise, any counter meas-ures can not be taken on the subject of `ùnknown virus,which can not be detected by currently available screeningmethod, may exist'', and conducting any virus detection testat any stage will be useless ``for ensuring safety''.

The requirement of regulations or tests excessively overscientific rationality will raise human, economical and tem-poral burden to the pharmaceutical companies, and will ad-versely affect prompt, effective and economical supply of adrug to the medical front. As drug is a sort of social asset,which has to be scientifically evaluated, how to assure max-imization of its safety by means of scientifically rational ap-proach at minimum human, economical and time resourcesis important.

It is also important to reconfirm that achievement of thoseissues is on the premise that appropriate measures are takenon the supply source of drugs. For example, in a case of `Ìna manufacturing process of drug, virus, virus-like particle orretrovirus was not detected, but there is a `concern' thatsomething unknown may exist.'', appropriateness of thetest, which resulted in the judgment that ``virus, virus-likeparticle or retrovirus was not detected in a process of drugproduction'', should be a prerequisite premise when judgedby science standard ate the time. If there is any question onthe premise, it is quite natural that the question of ``there is a`concern' that unknown something may exist.'' will be effec-tive.3.4. Applicable range

This General Information is on JP listed biologicalproducts, derived from living tissue or body fluid, and pro-tein drugs, derived from human or animal cell line, that inJapan. In the case of protein drugs derived from human oranimal cell line, the products developed and approved beforeenforcement of the Notice Iyakushin No. 329 entitled ``Viralsafety evaluation of biotechnology products derived fromcell lines of human or animal origin'' should have been treat-ed under the Notice had there been one, and it is inevitablethat some products approved after the Notice might not havebeen sufficiently treated. It is expected that such biodrug willbe sufficiently examined to meet such General Informationbefore being listed in JP. On the other hand, blood prepara-tions listed in the biological products standard and coveredby ``Guideline for securing safety of blood plasma proteinfraction preparations against virus'', are out of the scope ofthis General Information. Further, in case of a relativelylower molecular biogenous substance, such as amino acid,


saccharide and glycerin, and of gelatin, which is even classi-fied as infectious or pathogenic polymer, there are cases thatviral contamination can not be considered due to itsmanufacturing or purification process, and that potent viralinactivation/removal procedure that can not be applied toprotein, can be used, and, therefore, it is considered reason-able to omit such substances from the subject for applica-tion. However, some part of this General Information maybe used as reference. Further, a comprehensive assurancemeasure for viral safety is recommendable on a biotechnolo-gical/biological product not listed in JP using this documentas a reference so long as it is similar to the biotechnological/biological product JP.3.5. Possible viral contamination to a JP listed biotech-nological/biological product (source of virus contamination)

Promoting awareness of virus contamination to a JP listedbiotechnological/biological product (source of virus con-tamination) and citing countermeasure are important foreradicating any possible virus contamination and raisingprobability of safety assurance. Many biotechnological/biological products are produced from a ``substrate'' whichis derived from human or animal tissue, body fluid, etc. asan origin/raw material, and in purification or pharmaceuti-cal processing of such products column materials or addi-tives, which are living organism origin, are occasionallyused. Accordingly, enough safety measures should be takenagainst diffusion of the contaminant virus. Further, as men-tioned in Notice Iyakushin No. 329, any protein drug de-rived from cell lines of human or animal origin should becarefully examined with respect to the risk of virus contami-nation through the cell line, the cell substrate for drugproduction, and through the manufacturing process appliedthereafter.

``Substrate for drug production'' is defined as a startingmaterial which is at a stage where it is deemed to be in aposition to ensure quality/safety of an active substance. The``substrate for drug production'' is sometimes tissue, bodyfluid, etc. of human or animal per se and pooled materialsuch as urine, and sometimes a material after some treat-ment. In many cases, it is considered rational that startingpoint of full-scale test, evaluation and control should be atthe stage of ``substrate for drug production''. The morestrict levels of test, evaluation and control achieved at thestage of ``substrate for drug production'' can more ration-alize evaluation and control of the raw material or individuallevel of upper stream. On the contrary, strict evaluation andcontrol of the raw material or individual level at an upperstream stage can rationalize tests, evaluation or quality con-trol at the stage of ``substrate for drug production''.

The measures taken for ensuring viral safety on abiotechnological/biological product currently listed in JPcan be assumed from the provisions of manufacturingmethod, specification and test methods of each preparation.However, unitary principles or information with respect tothe measures to be taken for ensuring viral safety, totallyreviewing the entire process up to the final product rationallyand comprehensively, including source/raw material/sub-strate, purification process, etc. have not been clarified. Themost important thing for ensuring viral safety is to takethorough measures to eliminate the risk of virus contamina-tion at any stage of source animal, raw material and sub-strate. Although not the cases of a biotechnological/biologi-cal product, known examples of a virus contamination from

a raw material/substrate for drug production in old timesare Hepatitis A Virus (HAV) or Hepatitis C Virus (HCV)contamination in blood protein fraction preparations. It isalso well known that Human Immunodeficiency Virus (HIV)infection caused by blood plasma protein fraction prepara-tions occurred in 1980s. The aim of this General Informationis to show concrete guidelines for comprehensive viral safetyassurance of the JP listed biotechnological/biological prod-ucts. The pathogenic infectious viruses, currently known tocontaminate to raw materials, etc. of drug and have to becautioned, are HIV, HAV, Hepatitis B Virus (HBV), HCV,Human T-Lymphotropic Virus (HTLV-I/II), Human Par-vovirus B19, Cytomegalovirus (CMV), etc. Biotechnolo-gical/biological products produced from raw material/cellsubstrate derived from tissue or body fluid of human oranimal origin always have a risk of contamination of patho-genic or other latent virus. Therefore, safety measuresshould be thoroughly taken. There is also the case that amaterial, other than the biological component such as rawmaterial/substrate, causes virus contamination. Using enzy-matic or monoclonal antibody column or using albumin etc.as a stabilizer, is the example of the case, in which cautionhas to be taken on risk of virus contamination from thesource animal or cell. Further, there is a possibility of con-tamination from environment or personnel in charge ofproduction or at handling of the product. So, caution has tobe taken on these respects as well.

In case of protein drugs derived from cell line of human oranimal origin, there may be cases where latent or persistentinfectious viruses (e.g., herpesvirus) or endogenous retro-viruses exist in the cell. Further, adventitious viruses may beintroduced through the routes such as: 1) derivation of a cellline from an infected animal; 2) use of a virus to drive a cellline; 3) use of a contaminated biological reagent (e.g.,animal serum components); 4) contamination during cellhandling. In the manufacturing process of drug, an adven-titious virus may contaminate to the final product throughthe routes, such as 1) contamination through a reagent of liv-ing being origin, such as serum component, which is used forculturing, etc.; 2) use of a virus for introduction of a specificgene expression to code an objective protein; 3) contamina-tion through a reagent used for purification such asmonoclonal antibody affinity column; 4) contaminationthrough an additive used for formulation production; 5)contamination at handling of cell and culture media, etc. Itis reported that monitoring of cell culture parameter may behelpful for early detection of an adventitious viral contami-nation.3.6. Basis for ensuring viral safety

Viral safety of a biotechnological/biological product pro-duced from a raw material/substrate, which derived fromtissue, body fluid, cell line, etc. of human or animal origin,can be achieved by supplemental and appropriate adoptionof the following plural methods.(1) Acquaintance of possible virus contamination (source

of contamination).(2) Careful examination of eligibility of the raw material

and its source, i.e., human or animal, thorough analysisand screening of the sample chosen as the substrate fordrug production to determine virus contamination andthrough examination of the type of virus and its nature,if contaminated.

(3) Evaluation to determine hazardous properties of the vi-


rus or virus-like particle to human, if exists.(4) Choosing a product related material of living organism

origin (e.g., reagent, immune anti-body column, etc.)which is free from infectious or pathogenic virus.

(5) Conduct virus free test at an appropriate stage ofmanufacturing including the final product, if necessary.

(6) Adoption of an effective method to remove/inactivatethe virus in the manufacturing process for viral clear-ance. Combined processes sometimes achieve higherlevel of viral clearance.

(7) Develop a deliberate viral clearance scheme.(8) Conduct test and evaluation to confirm removal/inacti-

vation of the virus.Manufacturer is responsible for explaining rationality of

the way of approach adopted among the comprehensivestrategy for viral safety on each product and its manufactur-ing process. At the time, the approach described in thisGeneral Information shall be applicable as far as possible.3.7. Limit of virus test

Virus test has to be conducted to define existence of virus,but it should be noted that virus test alone can not reach aconclusion of inexistence of virus nor sufficient to securesafety of the product. Examples of a virus not being detectedare as follows: 1) Due to statistical reason, there is an inher-ent quantitative limit, such as detection sensitivity at lowerconcentration depends upon the sample size. 2) Generally,every virus test has a detection limit, and any negative resultof a virus test can not completely deny existence of a virus.3) A virus test applied is not always appropriate in terms ofspecificity or sensitivity for detection of a virus which existsin the tissue or body fluid of human or animal origin.

Virus testing method is improved as science and tech-nology progress, and it is important to apply scientificallythe most advanced technology at the time of testing so that itcan be possible to raise the assurance level of virus detection.It should be noted, however, that the limit as mentionedabove can not always be completely overcome. Further, riskof virus contamination in a manufacturing process can notbe completely denied, and, therefore, it is necessary toelaborate the countermeasure taken these effects into ac-count.

Reliable assurance of viral free final product can not beobtained only by negative test results on the raw material/substrate for drug production or on the product in general,it is also necessary to demonstrate inactivation/removalcapability of the purification process.3.8. Roles of viral clearance studies

Under the premises as mentioned in the preceding clausethat there is a limit of a virus test, that there is a possibilityof existence of latent virus in a raw material/substrate fordrug production and that there is a risk of entry of a non-endogenous virus in a manufacturing process, one of theimportant measures for viral safety is how to remove or inac-tivate the virus, which exists in a raw material, etc. and cannot be detected, or the virus, which is contingently contami-nated in a manufacturing process. The purpose of viralclearance study is to experimentally evaluate the viralremoval/inactivation capability of a step that mounted in amanufacturing process. So, it is necessary to conduct an ex-perimental scale spike test using an appropriate virus that isselected by taking account the properties, such as particlesize, shape, with or without envelope, type of nucleic acid(DNA type, RNA type), heat and chemical treatment toler-

ance, etc., with an aim to determine removal/inactivationcapability of the virus that can not be detected in a raw mate-rial or contingently contaminated.

As mentioned above, the role of viral clearance study is tospeculate removal/inactivation capability of a processthrough a model test, and it contributes to give scientificbasis to assure that a biotechnological/biological product ofhuman or animal origin has reached an acceptable level inaspect of viral safety.

At a viral clearance study, it is necessary to adopt an ap-propriate approach method which is definitive and rationaland can assure viral safety of a final product, taking intoconsideration the source and the properties of the raw mate-rial/substrate as well as the manufacturing process.

4. Raw material/substrate for drug production4.1. Issues relating to animal species and its region as asource of raw material/substrate for drug production andcountermeasures to be taken thereto

For manufacturing JP listed biotechnological/biologicalproducts, which require measures for viral safety, a raw ma-terial/substrate derived mainly from human, bovine, swineor equine is used, and it is obvious that such human andanimal has to be healthy nature. A wild animal should beavoided, and it is recommended to use animals derived froma colony controlled by an appropriate SPF (SpecificPathogen-Free) condition and bred under a well deignedhygienic control, including appropriate control for preven-tion of microbial contamination and contaminationmonitoring system. If a meat standard for food is available,an animal meeting this standard has to be used. The type ofvirus to be concerned about depend on animal species, but itmay be possible to narrow down the virus for investigationby means of examining the hygiene control, applicability ofa meat standard for food, etc. On the other hand, even withthe animals of the same species, a different approach may benecessary depending upon the region where the specimen fora raw material/substrate is taken. For example, in case ofobtaining raw material/substrate from blood or other spe-cific region, it is necessary to be aware of the risk level, virusmultiplication risk, etc. which may specifically exists de-pending upon its region. Such approach may be differentfrom those applied to body waste such as urine, milk, etc. asa source of raw material/substrate. Further, caution has tobe taken on transmissible spongiform encephalopathy (TSE)when pituitary gland, etc. is used as a raw material. Thisreport does not include detailed explanation on TSE, butrecommendations are to use raw material derived from 1)animals originated in the countries (area) where incidence ofTSE has not been reported; 2) animals not infected by TSE;or 3) species of animal which has not been reported on TSE.It is recommended to discuss the matters concerned withTSE with the regulatory authority if there is any unclearpoint.

Followings are the raw material/substrate used formanufacturing biotechnological/biological products inJapan.(1) Biological products derived from human

Blood plasma, placenta, urine, etc. derived from humanare used as the sources of raw material of biotechnological/biological product. As for these raw materials, there are 2cases: 1) Appropriateness can be confirmed by interview orby examination of the individual who supplies each raw ma-


terial, and 2) Such sufficient interview or examination of theindividual can not be made due to type of raw material. Incase that sufficient examination of individual level is notpossible, it is necessary to perform test to deny virus con-tamination at an appropriate manufacturing stage, for ex-ample, the stage to decide it as a substrate for drug produc-tion.(2) Biological products derived from animal besides human

Heparin, gonadotropin, etc. are manufactured from bloodplasma or from various organs of bovine, swine and equine.(3) Protein drug derived from cell line of human or animalorigin

In the case of protein drugs derived from cell line of hu-man or animal origin, a cell line of human or animal is theraw material per se, and the substrate for drug production isa cell bank prepared from cloned cell line (master cell bankor working cell bank). Examination at cell bank level is con-sidered enough for viral safety qualification, but it goeswithout saying that the more appropriate and rationalqualification evaluation test of cell bank can be realizedwhen more information is available on the virus of thesource animal or on prehistory of driving the cell line, thebase of cell bank.4.2. Qualification evaluation test on human or animal as asource of raw material/substrate for drug production(1) Biological products derived from human

Body fluid etc. obtained from healthy human must beused for biological products production. Further, in casethat interview or examination of the individual, who suppliesthe raw material, can be possible and is necessary, interviewunder an appropriate protocol and a serologic test well eval-uated in aspects of specificity, sensitivity and accuracy haveto be performed, so that only the raw material, which is de-nied latent HBV, HCV and HIV, will be used. In addition tothe above, it is necessary to test for gene of HBV, HCV andHIV by a nucleic amplification test (NAT) well evaluated inaspects of specificity, sensitivity and accuracy.

In case of the raw material (e.g., urine), which can not betested over the general medical examination of the individualwho supplies the material, or of the raw material which isirrational to conduct individual test, the pooled raw mate-rial, as the substrate for drug production, has to be conduct-ed at least to deny existence of HBV, HCV and HIV, using amethod well evaluated in aspects of specificity, sensitivityand accuracy, such as the antigen test or NAT.(2) Biological products derived from animal besides human

The animal used for manufacturing biological product hasto be under appropriate health control, and has to be con-firmed of its health by various tests. Further, it is necessarythat the population, to which the animal belongs, has beenunder an appropriate breeding condition, and that no abnor-mal individual has been observed in the population. Further,it is necessary to demonstrate information or scientific basiswhich can deny known causes infection or disease to human,or to deny such animal inherent latent virus by serologic testor by nucleic amplification test (NAT). The infectious virusthat is known to be common between human and animal,and known to cause infection in each animal are tentativelylisted in Table 2. It is necessary that the table is completedunder careful examination, and denial of all of them, bymeans of tests on individual animal, tissue, body fluid, etc.as a raw material, or on pooled raw material (as a direct sub-strate for drug production), is not always necessary. Table 2

can be used as reference information, in addition to theother information, such as; source of animal, health condi-tion, health and breeding control, conformity to the meatstandard for food, etc., to elaborate to which virus whatkind of test has to be performed, and for which virus it is notalways necessary to test for, etc. It is important to clarifyand record the basis of choosing the virus and the test con-ducted thereof.(3) Protein drug derived from cell line of human or animalorigin

It is important to conduct thorough investigation on latentendogenous and non-endogenous virus contamination in amaster cell bank (MCB), which is the cell substrate for drugproduction, in accordance with the Notice Iyakushin No.329 entitled ``Viral safety evaluation of biotechnology prod-ucts derived from cell lines of human or animal origin''. Fur-ther, it is necessary to conduct an appropriate adventitiousvirus test (e.g., in vitro and in vivo test) and a latentendogenous virus test on the cell at the limit of in vitro cellage (CAL) for drug production. Each WCB as a starting cellsubstrate for drug production should be tested for adventiti-ous virus either by direct testing or by analysis of cells at theCAL, initiated from the WCB. When appropriate non-en-dogenous virus tests have been performed on the MCB andcells cultured up to or beyond the CAL have been derivedfrom the WCB and used for testing for the presence of ad-ventitious viruses, similar tests need not be performed on theinitial WCB.

5. Points of concern with respect to manufacturing andvirus testing

To ensure viral safety of a biological product derived fromtissue, body fluid etc. of human or animal origin, it is neces-sary to exclude any possibility of virus contamination from araw material, such as tissue and body fluid, or a substrate,paying attention to the source of virus contamination asmentioned in above 3.5, and to adopt appropriate manufac-turing conditions and technologies in addition to enhance-ment of manufacturing environment, so that virus contami-nation in the course of process and handling and from oper-ators, facilities and environment can be minimized.

In addition to the above, effective virus test and viral inac-tivation/removal technology, which are reflected by rapidprogress of science, have to be introduced. Adoption of twoor more steps with different principles is recommended forvirus inactivation/removal process. Further, it is importantto minimize any possible virus derivation by using a reagent,which quality is equivalent to that of a drug. Examples ofvirus inactivation/removal processes are heating (It isreported that almost viruses are inactivated by heating at55 � 609C for 30 minutes with exceptions of hepatitis virus,etc. and that dry heating at 609C for 10 � 24 hours is effec-tive in case of the products of blood or urine origin.), treatment with organic solvent/surfactant (S/D treatment), membrane filtration (15 � 50 nm), acid treatment, irradiation (g-irradiation, etc.), treatment with columnchromatograph (e.g. affinity chromatography, ion-exchangechromatography), fractionation (e.g. organic solvent orammonium sulfate fractionation), extraction.5.1. Virus test conducted in advance of purificationprocess(1) Biological products derived from human

In many cases, samples for virus test before purification

2173

Table 2 Infectious viruses known to be commonbetween human and animal and known to

cause infection to each animal

bovine swine sheep goat equine

Cowpox virus w

Paravaccinia virus w w w w

Murray valley encephalitisvirus w w

Louping ill virus w w w w

Wesselsbron virus w

Foot-and-mouth diseasevirus w w

Japanese encephalitisvirus w

Vesicular stomatitis virus w

Bovine papular stomatitisvirus w

Orf virus w

Borna disease virus w w

Rabies virus w w w w w

Influenza virus w

Hepatitis E virus w

Encephalomyocarditisvirus w w

Rotavirus w

Eastern equineencephalitis virus w

Western equineencephalitis virus w

Venezuelan equineencephalitis virus w

Morbillivirus w

Hendra virus w

Nipah virus w

Transmissible gastroente-ritis virus w

Porcine respiratorycoronavirus w

Porcine epidemicdiarrhea virus w

Hemagglutinatingencephalomyelitis virus w

Porcine respiratory andreproductive syndrome virus w

Hog cholera virus w

Parainfluenza virus Type 3 w

Talfan/Teschen diseasevirus w

Reovirus w

Endogenous retrovirus w

Porcine adenovirus w

Porcine circovirus w

Porcine parvovirus w

Porcine poxvirus w

Porcine cytomegalovirus w

Pseudorabies virus w

Russian spring-summerencephalitis virus w w

Rift Valley fever virus w w

Crimean-Congo hemor-rhagic fever virus(Nairovirus)

w w w

Torovirus w


process are body fluid or tissue of individual collected as araw material, or its pooled material or extraction as a sub-strate. As mentioned in 4.2 (1), it is necessary to deny latentHBV, HCV and HIV by the test evaluated enough in aspectsof specificity, sensitivity and accuracy. Even in a case that anon-purified bulk before purification process is producedfrom a substrate, it is not always necessary to conduct virustest again at the stage before purification, so long as thepresence of any latent virus can be denied at the stage of sub-strate by an appropriate virus test, with cases where the non-purified bulk is made from the substrate by adding any rea-gent etc. of living organisms origin are an exception.(2) Biological products derived from animal besides human

Similar to (1) above, samples for virus test before purifica-tion process are, in many cases, body fluid or tissue of in-dividual collected as a raw material, or its pooled material orextraction as a substrate. In these cases, it is necessary tohave a data, which can deny latent virus of probable cause ofhuman infection or disease as mentioned in the above 4.2(2), or to have a result of serologic test or nucleic amplifica-tion test (NAT) evaluated enough in aspects of specificity,sensitivity and accuracy. The concept, which is applied to acase that non-purified bulk before purification process isproduced from substrate, is the same as those provided inthe above 4.2 (1).(3) Protein drug derived from cell line of human or animalorigin

Generally, substrate in this case is cell bank, and the sam-ple for testing before purification process is a harvested cellafter cell culturing or unprocessed bulk which consists ofsingle or pooled complex culture broth. The unprocessedbulk may be sometimes culture broth without cell. Denial oflatent virus, which is determined by virus test at a MCB orWCB level, does not always deny latent virus in unprocessed


bulk after culturing. Further, it is noted that the viral test atthe CAL is meaningful as a validation but can not guaranteedefinite assurance of latent virus denial, since the test is gen-erally performed only once. In case of using a serum or acomponent of blood origin in a culture medium, definitedenial of latent virus at the level of unprocessed bulk can notbe assured so long as the viral test has not been conducted oneach lot at the CAL, since lot renewal can be a variable fac-tor on viral contamination.

A representative sample of the unprocessed bulk, removedfrom the production reactor prior to further processing,represents one of the most suitable levels at which the pos-sibility of adventitious virus contamination can be deter-mined with a high probability of detection. Appropriate test-ing for viruses should be performed at the unprocessed bulklevel unless virus testing is made more sensitive by initial par-tial processing (e.g., unprocessed bulk may be toxic in testcell cultures, whereas partially processed bulk may not betoxic). In certain instances it may be more appropriate to testa mixture consisting of both intact and disrupted cells andtheir cell culture supernatants removed from the productionreactor prior to further processing.

In case of unprocessed bulk, it is required to conduct virustest on at least 3 lots obtained from pilot scale or commercialscale production. It is recommended that manufacturers de-velop programs for the ongoing assessment of adventitiousviruses in production batches. The scope, extent and fre-quency of virus testing on the unprocessed bulk should bedetermined by taking several points into consideration in-cluding the nature of the cell lines used to produce thedesired products, the results and extent of virus tests per-formed during the qualification of the cell lines, the cultiva-tion method, raw material sources and results of viral clear-ance studies. Screening in vitro tests, using one or several celllines, are generally employed to test unprocessed bulk. If ap-propriate, a NAT test or other suitable methods may beused.

Generally, harvest material in which adventitious virus hasbeen detected should not be used to manufacture theproduct. If any adventitious viruses are detected at this level,the process should be carefully checked to determine thecause of the contamination, and appropriate actions taken.5.2. Virus test as an acceptance test of an intermediate ma-terial, etc.

When a biological product is manufactured from tissue,body fluid etc. of human or animal origin, there are casesthat an intermediate material, partially processed as a rawmaterial or substrate by outside manufacturer, is purchasedand used for manufacturing. In such case, if any test to meetthis General Information has been conducted by such out-side manufacturer, it is necessary for the manufacturer ofthe biological product, who purchased the intermediate ma-terial, to examine what sort of virus test has to be conductedas acceptance tests, and to keep record on the basis ofrationality including the details of the test conducted.

On the other hand, if no test to meet this General Infor-mation has been conducted by such outside manufacturer ofthe raw material, all necessary virus free test has to be con-ducted to meet this General Information on the intermediatematerial regarding it as the direct substrate for drug produc-tion.5.3. Virus test on a final product

Virus tests to be conducted on a final product (or on a

product to reach the final product) has to be defined undercomprehensive consideration of the type of raw material orsubstrate, the result of virus test conducted on raw material/substrate, the result of evaluation on viral removal/inactiva-tion process, any possibility of virus contamination in themanufacturing process, etc. Comprehensive viral safety as-surance can only be achieved by appropriate selection of theraw material/substrate, an appropriate virus test conductedon the raw material/substrate/intermediate material, the vi-rus test conducted at an appropriate stage of manufacturing,an appropriate viral clearance test, etc. However, there arecases of having specific backgrounds, such as 1) use of theraw material derived from unspecified individual human, 2)possible existence of virus at window period, 3) specific de-tection limit of virus test, etc. and in these cases, virus con-tamination to the final product may occur if there is anydeficiency on the manufacturing process (e.g., damage ofmembrane filter) or any mix-up of the raw materials, etc. Toavoid such accidental virus contamination, it may be recom-mended to conduct nucleic amplification test (NAT) on thefinal product focusing on the most risky virus among thosethat may possibly to exist in the raw material.

6. Process evaluation on viral clearance6.1. Rationale, objective and general items to be concernedwith respect to viral clearance process evaluation

Evaluation of a viral inactivation/removal process is im-portant for ensuring safety of a biological product derivedfrom tissue or body fluid of human or animal origin. Con-ducting evaluation on viral clearance is to assure, even tosome extent, elimination of the virus, which may exist in araw material, etc. or may be derived to the process due tounexpected situation. Viral clearance studies should be madeby a carefully designed appropriate method, and has to berationally evaluated.

The objective of viral clearance studies is to assess processstep(s) that can be considered to be effective in inactivating/removing viruses and to estimate quantitatively the overalllevel of virus reduction obtained by the process. This shouldbe achieved by the deliberate addition (``spiking'') of sig-nificant amounts of a virus at different manufacturing/purification steps and demonstrating its removal or inactiva-tion during the subsequent steps. It is not necessary to evalu-ate or characterize every step of a manufacturing process ifadequate clearance is demonstrated by the use of fewersteps. It should be borne in mind that other steps in theprocess may have an indirect effect on the viral inactivation/removal achieved. Manufacturers should explain and justifythe approach used in studies for evaluating viral clearance.

The reduction of virus infectivity may be achieved byremoval of virus particles or by inactivation of viral infectiv-ity. For each production step assessed, the possible mechan-ism of loss of viral infectivity should be described withregard to whether it is due to inactivation or removal. For in-activation steps, the study should be planned in such a waythat samples are taken at different times and an inactivationcurve constructed.6.2. Selection of virus

To obtain broad range of information of viral inactiva-tion/removal, it is desirable that a model virus used for viralclearance studies should be chosen from the viruses withbroad range of characteristics in aspects of DNA/RNA, withor without envelope, particle size, significant resistance to

2175

Table 3 Example of viruses which have been used for viral clearance studies

Virus Family Genus Natural host Genome Env Size (nm) Shape Resistance

Vesicular Stomatitis Virus Rhabdo Vesiculovirus EquineBovine

RNA yes 70×150 Bullet Low

Parainfluenza Virus Paramyxo Type 1,3Respirovirus

Type 2,4Rubulavirus

Various RNA yes 100 � 200＋ Pleo-Spher Low

MuLV Retro Type Concovirus

Mouse RNA yes 80 � 110 Spherical Low

Sindbis Virus Toga Alphavirus Human RNA yes 60 � 70 Spherical Low

BVDV Flavi Pestivirus Bovine RNA yes 50 � 70 Pleo-Spher Low

Pseudorabies Virus Herpes Varicellovirus Swine DNA yes 120 � 200 Spherical Med

Poliovirus Sabin Type 1 Picorna Enterovirus Human RNA no 25 � 30 Icosahedral Med

EncephalomyocardititisVirus

Picorna Cardiovirus Mouse RNA no 25 � 30 Icosahedral Med

Reovirus Type 3 Reo Orthoreovirus Various kind RNA no 60 � 80 Spherical Med

SV 40 Papova Polyomavirus Monkey DNA no 40 � 50 Icosahedral Very high

Parvovirus: canine, por-cine

Parvo Parvovirus CaninePorcine

DNA no 18 � 24 Icosahedral Very high


physical/chemical treatment, etc. and it is necessary to com-bine about 3 model viruses to cover these characteristics.

At choice of a model virus, there are also the ways tochoose a virus closely related to or having the same charac-teristics of the virus known to exist in the raw material. Insuch case, it is in principle recommendable to choose a viruswhich demonstrates a higher resistance to inactivation/removal treatment if two or more candidate viruses are avail-able for choice. Further, a virus which can grow at a hightiter is desirable for choice, although this may not always bepossible. In addition to the above, choosing a virus, whichwill provide effective and reliable assay result at each step, isnecessary, since sample condition to be tested at each step ofa production process may influence the detection sensitivity.Consideration should also be given to health hazard whichmay pose to the personnel performing the clearance studies.

For the other items taken for consideration at choice ofvirus, the Notice, Iyakushin No. 329 can be used as a refer-ence. Examples of the virus which have been used for viralclearance studies are shown in Table 3 which was derivedfrom Iyakushin No. 329. However, the Notice, IyakushinNo. 329, is on viral safety of a product derived cell line ofhuman or animal origin, and a more appropriate model virushas to be chosen taking into account the origin/raw materialof biological products.6.3. Design of viral clearance studies

The purpose of viral clearance studies is to quantitativelyevaluate removal or inactivation capability of a process, inwhich a virus is intentionally spiked to a specific step of amanufacturing process.

Following are the precautions to be taken at planning viralclearance studies.

(1) Care should be taken in preparing the high-titer virusto avoid aggregation which may enhance physical removaland decrease inactivation thus distorting the correlation with

actual production.(2) Virus detection method gives great influence to viral

clearance factor. Accordingly, it is advisable to gain detec-tion sensitivity of the methods available in advance, and usea method with a detection sensitivity as high as possible.Quantitative infectivity assays should have adequate sensi-tivity and reproducibility in each manufacturing process,and should be performed with sufficient replicates to ensureadequate statistical validity of the result. Quantitative assaysnot associated with infectivity may be used if justified. Ap-propriate virus controls should be included in all infectivityassays to ensure the sensitivity of the method. Also, thestatistics of sampling virus when at low concentrations (forexample, number of virus is 1-1000/L) should be considered.

(3) Viral clearance studies are performed in a miniaturesize system that simulates the actual production process ofthe biotechnological/biological product used by the manu-facturer. It is inappropriate to introduce any virus into aproduction facility because of GMP constraints. Therefore,viral clearance studies should be conducted in a separatelaboratory equipped for virological work and performed bystaff with virological expertise in conjunction with produc-tion personnel involved in designing and preparing a scaled-down version of the purification process. The viral clearancestudies should be performed under the basic concept ofGLP.

(4) Each factor on a viral clearance study of a process,which is performed in miniature size, should reflect that ofactual manufacturing as far as possible, and its rationalityshould be clarified. In case of chromatograph process,length of column bed, linear velocity, ratio of bed volumeper velocity (in other words, contact time), buffer, type ofcolumn packing, pH, temperature, protein concentration,salt concentration and concentration of the objectiveproduct are all correspondent to those of the actual produc-


tion. Further, similarity of elution profile should beachieved. For the other process, similar concept should beapplied. If there is any factor which can not reflect the actualproduction, its effect to the result should be examined.

(5) It is desirable that two or more inactivation/removalprocesses of different principles are selected and examined.

(6) As for the process which is expected to inactivate/remove virus, each step should be evaluated in aspect ofclearance capability, and carefully determined if it is thestage of inactivation, removal or their combination fordesigning the test. Generally, in viral clearance test, a virus isspiked in each step which is the object of the test, and afterpassing through the process in question, the reduction levelof infectivity is evaluated. But, in some case, it is acceptedthat a high potential virus is spiked at a step of the process,and virus concentration of each succeeding step is carefullymonitored. When removal of virus is made by separation orfractionation, it is desirable to investigate how the virus isseparated or fractionated (mass balance).

(7) For assessment of viral inactivation, unprocessedcrude material or intermediate material should be spikedwith infectious virus and the reduction factor calculated. Itshould be recognized that virus inactivation is not a simple,first order reaction and is usually more complex, with a fast``phase 1'' and a slow ``phase 2''. The study should, there-fore, be planned in such a way that samples are taken atdifferent times and an inactivation curve constructed. It isrecommended that studies for inactivation include at leastone time point less than the minimum exposure time andgreater than zero, in addition to the minimum exposuretime. The reproducible clearance should be demonstrated inat least two independent studies. When there is a possibilitythat the virus is a human pathogen, it is very important thatthe effective inactivation process is designed and additionaldata are obtained. The initial virus load should be deter-mined from the virus which can be detected in the spikedstarting material. If this is not possible, the initial virus loadmay be calculated from the titer of the spiking virus prepara-tion. Where inactivation is too rapid to plot an inactivationcurve using process conditions, appropriate controls shouldbe performed to demonstrate that infectivity is indeed lost byinactivation.

(8) If antibody against virus exists in an unprocessed ma-terial, caution should be taken at clearance studies, since itmay affect the behavior of virus at viral removal or inactiva-tion process.

(9) Virus spiked in unprocessed material should besufficient enough to evaluate viral removal or inactivationcapability of the process. However, the virus ``spike'' to beadded to the unprocessed material should be as small aspossible in comparison with the sample volume of the un-processed material so as not to cause characteristic change ofthe material by addition of the virus nor to cause behavioralchange of the protein in the material.

(10) It is desirable that the virus in the sample is subjectfor quantitative determination without applying ultracen-trifuge, dialysis, storage, etc. as far as possible. However,there may be a case that any handling before quantitativetest, such as remove procedure of inhibitor or toxic sub-stance, storage for a period to realize test at a time, etc., isinevitable. If any manipulation, such as dilution, concentra-tion, filtration, dialysis, storage, etc., is applied for prepara-tion of the sample for testing, a parallel control test, which

passes through a similar manipulation, should be conductedto assess infectivity variance at the manipulation.

(11) Buffers and product (desired protein or other com-ponent contained therein) should be evaluated independentlyfor toxicity or interference in assays used to determine thevirus titer, as these components may adversely affect theindicator cells. If the solutions are toxic to the indicatorcells, dilution, adjustment of the pH, or dialysis of thebuffer containing spiked virus might be necessary. If theproduct itself has anti-viral activity, the clearance study mayneed to be performed without the product in a ``mock'' run,although omitting the product or substituting a similar pro-tein that does not have anti-viral activity could affect the be-haviour of the virus in some production steps.

(12) Many purification schemes use the same or similarbuffers or columns, repetitively. The effects of this approachshould be taken into account when analyzing the data. Theeffectiveness of virus elimination by a particular process mayvary with the stage in manufacture at which it is used.

(13) Overall reduction factors may be underestimatedwhere production conditions or buffers are too cytotoxic orvirucidal and should be discussed on a case-by-case basis.Overall reduction factors may also be overestimated due toinherent limitations or inadequate design of viral clearancestudies.

(14) It has to be noted that clearance capability of viralremoval/inactivation process may vary depending upon thetype of virus. The viral removal/inactivation process, whichdisplays viral clearance by specific principle or mechanism,may be quite effective to the virus, which meets suchmechanism of action, but not effective to the other type ofviruses. For example, S/D treatment is generally effective tothe virus with lipid membrane, but not effective to the non-enveloped virus. Further, some virus is resistant to the gen-eral heating process (55 � 609C, 30 minutes). When clearanceis expected for such virus, introduction of a further severecondition or process, which has different principle ormechanism, is necessary. Virus removal by membrane filtra-tion, which is different from S/D or heat treatment in aspectof principle, is effective to a broad range of virus that cannot pass through the membrane. Affinity chromatographyprocess, which specifically absorbs the objective protein, canthoroughly wash out the materials other than the objectiveprotein including virus etc. and is generally effective for viralremoval. Separation/fractionation of a virus from an objec-tive protein is sometimes very difficult, but there are not sorare that ion exchange chromatography, ethanol fractiona-tion, etc. is effective for clearance of a virus which can notbe sufficiently inactivated or removed by the other process.

(15) Effective clearance may be achieved by any of thefollowing: multiple inactivation steps, multiple complemen-tary separation steps, or combinations of inactivation andseparation steps. Separation methods may be dependent onthe extremely specific physico-chemical properties of a viruswhich influence its interaction with gel matrices and precipi-tation properties. However, despite these potential variables,effective removal can be obtained by a combination of com-plementary separation steps or combinations of inactivationand separation steps. Well designed separation steps, such aschromatographic procedures, filtration steps and extrac-tions, can be also effective virus removal steps provided thatthey are performed under appropriately controlled condi-tions.


(16) An effective virus removal step should givereproducible reduction of virus load shown by at least twoindependent studies.

(17) Over time and after repeated use, the ability ofchromatography columns and other devices used in thepurification scheme to clear virus may vary. Some estimateof the stability of the viral clearance after several uses mayprovide support for repeated use of such columns.

(18) The Notice, Iyakushin No. 329, would be used as areference when viral clearance studies on biological productsare designed.6.4. Interpretation of viral clearance studies6.4.1. Evaluation on viral clearance factor

Viral clearance factor is a logarithm of reduction ratio ofviral amount (infectious titer) between each step applied forviral clearance of a manufacturing process. Total viral clear-ance factor throughout the process is sum of the viral clear-ance factor of each step appropriately evaluated.

Whether each and total viral clearance factor obtained areacceptable or should not be evaluated in aspects of every vi-rus that can be realistically anticipated to derive into the rawmaterial or the manufacturing process, and its rationalityshould be recorded.

In case that existence of any viral particle is recognized ina substrate for drug production, e.g., a substrate of rodentorigin for biodrug production, it is important not only todemonstrate removal or inactivation of such virus, but alsoto demonstrate that the purification process has enoughcapability over the required level to assure safety of the finalproduct at an appropriate level. The virus amount removedor inactivated in a manufacturing process should be com-pared with the virus amount assumed to exist in the substrateetc. used for manufacturing drug, and for this purpose, it isnecessary to obtain the virus amount in the raw materials/substrate, etc. Such figure can be obtained by measuringinfectious titer or by the other method such as transmissionelectron microscope (TEM). For evaluation of overallprocess, a virus amount, far larger than that assumed to existin the amount of the raw materials/substrate which isequivalent to single administration of the final product, hasto be removed. It is quite rare that existence of virus can beassumed in a substrate for drug production, with the excep-tion of the substrate of rodent origin, and such suspiciousraw material/substrate should not be used for manufactur-ing drug with a special exceptional case that the drug in ques-tion is not available from the other process and is clinicallyindispensable, and that the information including infectiousproperties of the virus particle assumed to exist has beenclarified.6.4.2. Calculation of viral clearance index

Viral clearance factor, ``R'', for viral removal/inactiva-tion process can be calculated by the following formula.

R＝ log[(V1× T1)/(V2× T2)]

In whichR: Logarithm of reduction ratioV1: Sample volume of the unprocessed materialT1: Virus amount (titer) of the unprocessed materialV2: Sample volume of the processed materialT2: Virus amount (titer) of the processed material

At the calculation of viral clearance factor, it is recom-mendable to use the virus titer detected in the sample prepa-

ration of the unprocessed material after addition of virus,not the viral titer added to the sample preparation whereverpossible. If this is not possible, loaded virus amount is calcu-lated from virus titer of the solution used for spike.6.4.3. Interpretation of results and items to be concernedat evaluation

At the interpretation and the evaluation of the data on ef-fectiveness of viral inactivation/removal process, there arevarious factors to be comprehensively taken into account,such as appropriateness of the virus used for the test, design of the viral clearance studies, virus reduction ratioshown in logarithm, time dependence of inactivation, factors/items which give influence to the inactivation/removal process, sensitivity limit of virus assay method, possible effect of the inactivation/removal process whichis specific to certain class of viruses.

Additional items to be concerned at appropriate interpre-tation and evaluation of the viral clearance data are asfollows:(1) Behavior of virus used to the test

At interpretation of the vial clearance results, it is neces-sary to recognize that clearance mechanism may differ de-pending upon the virus used for the test. Virus used for a testis generally produced in tissue culture, but behavior of thevirus prepared in the tissue culture may be different fromthat of the native virus. Examples are possible differences ofpurity and degree of aggregation between the native and thecultured viruses. Further, change of surface properties of avirus, e.g., addition of a sucrose chain which is ascribed tospecific nature of a separation process, may give effect to theseparation. These matters should be also considered at inter-pretation of the results.(2) Design of test

Viral clearance test should have been designed taking intoaccount variation factors of the manufacturing process andscaling down, but there still remain some variance from ac-tual production scale. It is necessary to consider such vari-ance at the interpretation of the data and limitation of thetest.(3) Acceptability of viral reduction data

Total viral clearance factor is expressed as a sum oflogarithm of reduction ratio obtained at each step. The sum-mation of the reduction factor of multiple steps, particularlyof steps with little reduction (e.g., below 1 log10), may over-estimate viral removal/inactivation capability of the overallprocess. Therefore, virus titer of the order of 1 log10 or lesshas to be ignored unless justified. Further, viral clearancefactor achieved by repeated use of the same or similarmethod should be ignored for calculation unless justified.(4) Time dependence of inactivation

Inactivation of virus infectivity frequently shows biphasiccurve, which consists of a rapid initial phase and subsequentslow phase. It is possible that a virus not inactivated in a stepmay be more resistant to the subsequent step. For example,if an inactivated virus forms coagulation, it may be resistantto any chemical treatment and heating.(5) Evaluation of viral reduction ratio shown logarithm

Viral clearance factor shown in logarithm of reductionratio of virus titer can demonstrate drastic reduction ofresidual infectious virus, but there is a limit that infectioustiter can never be reduced to zero. For example, reduction ininfectivity of a preparation containing 8 log10 infectious unitper mL by a factor of 8 log10 leaves zero log10 per mL or one


infectious unit per mL, taking into account the detectionlimit of assay.(6) Variable factor of manufacturing process

Minor variance of a variation factor of a manufacturingprocess, e.g., contact time of a spiked sample to a buffer ora column, will sometimes give influence to viral removal orinactivation effect. In such case, it may be necessary to in-vestigate to what extent such variance of the factor has giveninfluence to the process concerned in aspect of viral inactiva-tion.(7) Existence of anti-viral antiserum

Anti-viral antiserum that exists in the sample preparationused for a test may affect sensitivity of distribution or inacti-vation of a virus, which may result in not only defusing thevirus titer but complicating interpretation of the test result.So, existence of anti-viral antiserum is one of the importantvariable factors.(8) Introduction of a new process for removal/inactivation

Viral clearance is an important factor for securing safetyof drug. In case that an achievement level of infective clear-ance of a process is considered insufficient, a process whichis characterized by inactivation/removal mechanism to meetthe purpose or an inactivation/removal process which canmutually complement to the existence process has to be in-troduced.(9) Limit of viral clearance studies

Viral clearance studies are useful for contributing to theassurance that an acceptable level of safety in the finalproduct is achieved but do not by themselves establish safe-ty. However, a number of factors in the design and execu-tion of viral clearance studies may lead to an incorrect esti-mate of the ability of the process to remove virus infectivity,as described above.

7. StatisticsThe viral clearance studies should include the use of

statistical analysis of the data to evaluate the results. Thestudy results should be statistically valid to support the con-clusions reached.7.1. Statistical considerations for assessing virus assays

Virus titrations suffer the problems of variation commonto all biological assay systems. Assessment of the accuracyof the virus titrations and reduction factors derived fromthem and the validity of the assays should be performed todefine the reliability of a study. The objective of statisticalevaluation is to establish that the study has been carried outto an acceptable level of virological competence.Assay

1. Assay methods may be either semiquantitative orquantitative. Both semiquantitative and quantitative assaysare amenable to statistical evaluation.

2. Variation can arise within an assay as a result of dilu-tion errors, statistical effects and differences within the assaysystem which are either unknown or difficult to control.These effects are likely to be greater when different assayruns are compared (between-assay variation) than whenresults within a single assay run are compared (within-assayvariation).

3. The 95z confidence limits for results of within-assayvariation normally should be on the order of ±0.5 log10 ofthe mean. Within-assay variation can be assessed by stand-ard textbook methods. Between-assay variation can be moni-tored by the inclusion of a reference preparation, the esti-

mate of whose potency should be within approximately 0.5log10 of the mean estimate established in the laboratory forthe assay to be acceptable. Assays with lower precision maybe acceptable with appropriate justification.7.2. Reproducibility and confidence limit of viral clearancestudies

An effective virus inactivation/removal step should givereproducible reduction of virus load shown by at least twoindependent studies. The 95z confidence limits for thereduction factor observed should be calculated whereverpossible in studies of viral clearance. If the 95z confidencelimits for the viral assays of the starting material are±s, andfor the viral assays of the material after the step are±a, the95z confidence limits for the reduction factor are± s2＋ a2.

8. Re-evaluation of viral clearanceWhenever significant changes in the production or purifi-

cation process are made, the effect of that change, bothdirect and indirect, on viral clearance should be re-evaluatedas needed. Changes in process steps may also change theextent of viral clearance.

9. Measurement for viral clearance studies9.1. Measurement of virus infective titer

Assay methods may be either semiquantitative or quan-titative. Semiquantitative methods include infectivity assaysin animals or in cultured cell infections dose (CCID) assays,in which the animal or cell culture is scored as either infectedor not. Infectivity titers are then measured by the proportionof animals or culture infected. In quantitative methods, theinfectivity measured varies continuously with the virus input.Quantitative methods include plaque assays where eachplaque counted corresponds to a single infectious unit. Bothquantal and quantitative assays are amenable to statisticalevaluation.9.2. Testing by nucleic-acid-amplification test (NAT)

NAT can detect individual or pooled raw material/cellsubstrate or virus genome at a high sensitivity even in a stagethat serum test on each virus is negative. Further, it can de-tect HBV or HCV gene, which can not be measured in cul-ture system. Window period can be drastically shortened atthe test on HBV, HCV and HIV, and the method is expectedto contribute as an effective measure for ensuring viral safe-ty. However, depending upon a choice of primer, there maybe a case that not all the subtype of objective virus can be de-tected by this method, and, therefore, it is recommendableto evaluate, in advance, if subtype of a broad range can bedetected.

NAT will be an effective evaluation method for virusremoval capability for viral clearance. However, in case ofviral inactivation process, viral inactivation obtained by thismethod may be underrated, since there is a case that inacti-vated virus still shows positive on nucleic acid. Further, atintroduction of NAT, cautions should be taken onrationality of detection sensitivity, choice of a standardwhich is used as run-control, quality assurance and main-tenance of a reagent used for primer, interpretation of posi-tive and negative results, etc.

10. Reporting and preservationAll the items relating to virus test and viral clearance stu-

dies should be reported and preserved.


11. OthersThe Notice, Iyakushin No. 329, should be used as a refer-

ence at virus test and viral clearance studies.

ConclusionAs mentioned at the Introduction, assurance of quality/

safety etc. of JP listed drugs should be achieved by state-of-the-art methods and concepts reflecting the progress ofscience and accumulation of experiences.

The basis for ensuring viral safety of JP listed biotechno-logical/biological products is detailed in this General Infor-mation. What is discussed here is that an almost equal levelof measures are required for both development of new drugsand for existing products as well, which means that similarlevel of concerns should be paid on both existing and newproducts in aspect of viral safety. This document is intendedto introduce a basic concept that quality and safety assur-ance of JP listed product should be based upon the most ad-vanced methods and concepts. This document has been writ-ten to cover all conceivable cases, which can be applied to allbiotechnological/biological products. Therefore, there maybe cases that it is not so rational to pursue virus tests andviral clearance studies in accordance with this document oneach product, which has been used for a long time withoutany safety issue. So, it will be necessary to elaborate themost rational ways under a case-by-case principle taking intodue consideration source, origin, type, manufacturingprocess, characteristics, usages at clinical stage, accumula-tion of the past usage record, etc. relating to such biotechno-logical/biological products.

Capillary ElectrophoresisThis test is harmonized with the European Pharmacopoeia


Capillary electrophoresis is a physical method of analysisbased on the migration, inside a capillary, of charged ana-lytes dissolved in an electrolyte solution, under the influenceof a direct-current electric field.

The migration velocity of an analyte under an electric fieldof intensity E, is determined by the electrophoretic mobilityof the analyte and the electroosmotic mobility of the bufferinside the capillary. The electrophoretic mobility of a solute(mep) depends on the characteristics of the solute (electriccharge, molecular size and shape) and those of the buffer inwhich the migration takes place (type and ionic strength ofthe electrolyte, pH, viscosity and additives). The elec-trophoretic velocity (nep) of a solute, assuming a sphericalshape, is given by the equation:

nep＝ mepE＝ Ø q6phr»ØVL»

q: effective charge of the solute,h: viscosity of the electrolyte solution,r: Stoke's radius of the solute,V: applied voltage,L: total length of the capillary.

When an electric field is applied through the capillaryfilled with buffer, a flow of solvent is generated inside thecapillary, called electroosmotic flow. The velocity of theelectroosmotic flow depends on the electroosmotic mobility

(meo) which in turn depends on the charge density on thecapillary internal wall and the buffer characteristics. Theelectroosmotic velocity (neo) is given by the equation:

neo＝ meoE＝ Øezh »ØVL»e: dielectric constant of the buffer,z: zeta potential of the capillary surface.

The velocity of the solute (n) is given by:

n＝ nep＋ neo

The electrophoretic mobility of the analyte and the elec-troosmotic mobility may act in the same direction or in op-posite directions, depending on the charge of the solute. Innormal capillary electrophoresis, anions will migrate in theopposite direction to the electroosmotic flow and their veloc-ities will be smaller than the electroosmotic velocity. Cationswill migrate in the same direction as the electroosmotic flowand their velocities will be greater than the electroosmoticvelocity. Under conditions in which there is a fast electroos-motic velocity with respect to the electrophoretic velocity ofthe solutes, both cations and anions can be separated in thesame run.

The time (t) taken by the solute to migrate the distance (l )from the injection end of the capillary to the detection point(capillary effective length) is given by the expression:

t＝l

nep＋ neo＝

l× L(mep＋ meo)V

In general, uncoated fused-silica capillaries above pH 3have negative charge due to ionized silanol groups in theinner wall. Consequently, the electroosmotic flow is fromanode to cathode. The electroosmotic flow must remainconstant from run to run if good reproducibility is to be ob-tained in the migration velocity of the solutes. For some ap-plications, it may be necessary to reduce or suppress the elec-troosmotic flow by modifying the inner wall of the capillaryor by changing the concentration, composition and/or pH ofthe buffer solution.

After the introduction of the sample into the capillary,each analyte ion of the sample migrates within the back-ground electrolyte as an independent zone, according to itselectrophoretic mobility. Zone dispersion, that is the spread-ing of each solute band, results from different phenomena.Under ideal conditions the sole contribution to the solute-zone broadening is molecular diffusion of the solute alongthe capillary (longitudinal diffusion). In this ideal case theefficiency of the zone, expressed as the number of theoreticalplates (N ), is given by:

N＝(mep＋ meo)× V× l

2× D× L

D: molecular diffusion coefficient of the solute in thebuffer.

In practice, other phenomena such as heat dissipation,sample adsorption onto the capillary wall, mismatched con-ductivity between sample and buffer, length of the injectionplug, detector cell size and unlevelled buffer reservoirs canalso significantly contribute to band dispersion.

Separation between two bands (expressed as the resolu-tion, RS) can be obtained by modifying the electrophoreticmobility of the analytes, the electroosmotic mobility induced


in the capillary and by increasing the efficiency for the bandof each analyte, according to the equation:

RS＝N(mepb－ mepa)4( �mep＋ meo)

mepa and mepb: electrophoretic mobilities of the two analytesseparated,

�mep: mean electrophoretic mobility of the two analytes

�mep＝12

(mepb＋ mepa).

ApparatusAn apparatus for capillary electrophoresis is composed of:

(1) a high-voltage, controllable direct-current power supp-ly,(2) two buffer reservoirs, held at the same level, containingthe prescribed anodic and cathodic solutions,(3) two electrode assemblies (the cathode and the anode),immersed in the buffer reservoirs and connected to the pow-er supply,(4) a separation capillary (usually made of fused-silica)which, when used with some specific types of detectors, hasan optical viewing window aligned with the detector. Theends of the capillary are placed in the buffer reservoirs. Thecapillary is filled with the solution prescribed in the mono-graph,(5) a suitable injection system,(6) a detector able to monitor the amount of substances ofinterest passing through a segment of the separation capil-lary at a given time. It is usually based on absorption spec-trophotometry (UV and visible) or fluorometry, but conduc-timetric, amperometric or mass spectrometric detection canbe useful for specific applications. Indirect detection is an al-ternative method used to detect non-UV-absorbing and non-fluorescent compounds,(7) a thermostatic system able to maintain a constant tem-perature inside the capillary is recommended to obtain agood separation reproducibility,(8) a recorder and a suitable integrator or a computer.

The definition of the injection process and its automationare critical for precise quantitative analysis. Modes of injec-tion include gravity, pressure or vacuum injection and elec-trokinetic injection. The amount of each sample componentintroduced electrokinetically depends on its electrophoreticmobility, leading to possible discrimination using this injec-tion mode.

Use the capillary, the buffer solutions, the preconditioningmethod, the sample solution and the migration conditionsprescribed in the monograph of the considered substance.The employed electrolytic solution is filtered to remove par-ticles and degassed to avoid bubble formation that could in-terfere with the detection system or interrupt the electricalcontact in the capillary during the separation run. A rigorousrinsing procedure should be developed for each analyticalmethod to achieve reproducible migration times of the so-lutes.

1. Capillary Zone ElectrophoresisIn capillary zone electrophoresis, analytes are separated in

a capillary containing only buffer without any anticonvectivemedium. With this technique, separation takes place becausethe different components of the sample migrate as discretebands with different velocities. The velocity of each band de-pends on the electrophoretic mobility of the solute and the

electroosmotic flow in the capillary (see General Principles).Coated capillaries can be used to increase the separationcapacity of those substances adsorbing on fused-silica sur-faces.

Using this mode of capillary electrophoresis, the analysisof both small (Mr＜ 2000) and large molecules (2000＜Mr

＜ 100,000) can be accomplished. Due to the high efficiencyachieved in capillary zone electrophoresis, separation ofmolecules having only minute differences in their charge-to-mass ratio can be effected. This separation mode also allowsthe separation of chiral compounds by addition of chiralselectors to the separation buffer.Optimization

Optimization of the separation is a complex process whereseveral separation parameters can play a major role. Themain factors to be considered in the development of separa-tions are instrumental and electrolytic solution parameters.Instrumental parameters(1) Voltage: A Joule heating plot is useful in optimizingthe applied voltage and column temperature. Separationtime is inversely proportional to applied voltage. However,an increase in the voltage used can cause excessive heatproduction, giving rise to temperature and, as a result there-of, viscosity gradients in the buffer inside the capillary. Thiseffect causes band broadening and decreases resolution.(2) Polarity: Electrode polarity can be normal (anode atthe inlet and cathode at the outlet) and the electroosmoticflow will move toward the cathode. If the electrode polarityis reversed, the electroosmotic flow is away from the outletand only charged analytes with electroosmotic mobilitiesgreater than the electroosmotic flow will pass to the outlet.(3) Temperature: The main effect of temperature isobserved on buffer viscosity and electrical conductivity, andtherefore on migration velocity. In some cases, an increase incapillary temperature can cause a conformational change inproteins, modifying their migration time and the efficiencyof the separation.(4) Capillary: The dimensions of the capillary (length andinternal diameter) contribute to analysis time, efficiency ofseparations and load capacity. Increasing total length candecrease the electric fields (working at constant voltage), andincreasing both effective length and total length increasemigration time. For a given buffer and electric field, heatdissipation, and hence sample band-broadening, depend onthe internal diameter of the capillary. The latter also affectsthe detection limit, depending on the sample volume injectedand the detection system employed.

Since the adsorption of the sample components on thecapillary wall limits efficiency, methods to avoid these inter-actions should be considered in the development of a separa-tion method. In the specific case of proteins, several strate-gies have been devised to avoid adsorption on the capillarywall. Some of these strategies (use of extreme pH and ad-sorption of positively charged buffer additives) only requiremodification of the buffer composition to prevent proteinadsorption. In other strategies, the internal wall of the capil-lary is coated with a polymer, covalently bonded to thesilica, that prevents interaction between the proteins and thenegatively charged silica surface. For this purpose, ready-to-use capillaries with coatings consisting of neutral-hydro-philic, cationic and anionic polymers are available.Electrolytic solution parameters(1) Buffer type and concentration: Suitable buffers for


capillary electrophoresis have an appropriate buffer capacityin the pH range of choice and low mobility to minimize cur-rent generation.

Matching buffer-ion mobility to solute mobility, wheneverpossible, is important for minimizing band distortion. Thetype of sample solvent used is also important to achieve on-column sample focusing, which increases separation efficien-cy and improves detection.

An increase in buffer concentration (for a given pH)decreases electroosmotic flow and solute velocity.(2) Buffer pH: The pH of the buffer can affect separa-tion by modifying the charge of the analyte or additives, andby changing the electroosmotic flow. In protein and peptideseparation, changing the pH of the buffer from above tobelow the isoelectric point (pI) changes the net charge of thesolute from negative to positive. An increase in the bufferpH generally increases the electroosmotic flow.(3) Organic solvents: Organic modifiers (methanol,acetonitrile, etc.) may be added to the aqueous buffer to in-crease the solubility of the solute or other additives and/orto affect the degree of ionization of the sample components.The addition of these organic modifiers to the buffer gener-ally causes a decrease in the electroosmotic flow.(4) Additives for chiral separations: For the separation ofoptical isomers, a chiral selector is added to the separationbuffer. The most commonly used chiral selectors are cy-clodextrins, but crown ethers, polysaccharides and proteinsmay also be used. Since chiral recognition is governed by thedifferent interactions between the chiral selector and each ofthe enantiomers, the resolution achieved for the chiral com-pounds depends largely on the type of chiral selector used. Inthis regard, for the development of a given separation it maybe useful to test cyclodextrins having a different cavity size(a-, b-, or g-cyclodextrin) or modified cyclodextrins withneutral (methyl, ethyl, hydroxyalkyl, etc.) or ionizable(aminomethyl, carboxymethyl, sulfobutyl ether, etc.)groups. When using modified cyclodextrins, batch-to-batchvariations in the degree of substitution of the cyclodextrinsmust be taken into account since it will influence the selectiv-ity. Other factors controlling the resolution in chiral separa-tions are concentration of chiral selector, composition andpH of the buffer and temperature. The use of organic addi-tives, such as methanol or urea can also modify the resolu-tion achieved.

2. Capillary Gel ElectrophoresisIn capillary gel electrophoresis, separation takes place in-

side a capillary filled with a gel that acts as a molecular sieve.Molecules with similar charge-to-mass ratios are separatedaccording to molecular size since smaller molecules movemore freely through the network of the gel and therefore mi-grate faster than larger molecules. Different biological mac-romolecules (for example, proteins and DNA fragments),which often have similar charge-to-mass ratios, can thus beseparated according to their molecular mass by capillary gelelectrophoresis.Characteristics of Gels

Two types of gels are used in capillary electrophoresis:permanently coated gels and dynamically coated gels. Per-manently coated gels, such as cross-linked polyacrylamide,are prepared inside the capillary by polymerization of themonomers. They are usually bonded to the fused-silica walland cannot be removed without destroying the capillary. If

the gels are used for protein analysis under reducing condi-tions, the separation buffer usually contains sodium dodecylsulfate and the samples are denatured by heating a mixtureof sodium dodecyl sulfate and 2-mercaptoethanol ordithiothreitol before injection. When non-reducing condi-tions are used (for example, analysis of an intact antibody),2-mercaptoethanol and dithiothreitol are not used. Separa-tion in cross-linked gels can be optimized by modifying theseparation buffer (as indicated in the capillary zone elec-trophoresis section) and controlling the gel porosity duringthe gel preparation. For cross-linked polyacrylamide gels,the porosity can be modified by changing the concentrationof acrylamide and/or the proportion of cross-linker. As arule, a decrease in the porosity of the gel leads to a decreasein the mobility of the solutes. Due to the rigidity of thesegels, only electrokinetic injection can be used.

Dynamically coated gels are hydrophilic polymers, such aslinear polyacrylamide, cellulose derivatives, dextran, etc.,which can be dissolved in aqueous separation buffers givingrise to a separation medium that also acts as a molecularsieve. These separation media are easier to prepare thancross-linked polymers. They can be prepared in a vial andfilled by pressure in a wall-coated capillary (with no electro-osmotic flow). Replacing the gel before every injection gen-erally improves the separation reproducibility. The porosityof the gels can be increased by using polymers of higher mo-lecular mass (at a given polymer concentration) or bydecreasing the polymer concentration (for a given polymermolecular mass). A reduction in the gel porosity leads to adecrease in the mobility of the solute for the same buffer.Since the dissolution of these polymers in the buffer giveslow viscosity solutions, both hydrodynamic and electrokinet-ic injection techniques can be used.

3. Capillary Isoelectric FocusingIn isoelectric focusing, the molecules migrate under the

influence of the electric field, so long as they are charged, ina pH gradient generated by ampholytes having pI values in awide range (poly-aminocarboxylic acids), dissolved in theseparation buffer.

The three basic steps of isoelectric focusing are loading,focusing and mobilization.(1) Loading step: Two methods may be employed:

( i ) loading in one step: the sample is mixed with ampho-lytes and introduced into the capillary either by pres-sure or vacuum;

(ii) sequential loading: a leading buffer, then the ampho-lytes, then the sample mixed with ampholytes, againampholytes alone and finally the terminating bufferare introduced into the capillary. The volume of thesample must be small enough not to modify the pHgradient.

(2) Focusing step: When the voltage is applied, ampho-lytes migrate toward the cathode or the anode, according totheir net charge, thus creating a pH gradient from anode(lower pH) to cathode (higher pH). During this step the com-ponents to be separated migrate until they reach a pH cor-responding to their isoelectric point (pI) and the currentdrops to very low values.(3) Mobilization step: If mobilization is required for de-tection, use one of the following methods. Three methodsare available:

( i ) in the first method, mobilization is accomplished


during the focusing step under the effect of the elec-troosmotic flow; the electroosmotic flow must besmall enough to allow the focusing of the compo-nents;

( ii ) in the second method, mobilization is accomplishedby applying positive pressure after the focusing step;

(iii) in the third method, mobilization is achieved afterthe focusing step by adding salts to the cathodereservoir or the anode reservoir (depending on thedirection chosen for mobilization) in order to alterthe pH in the capillary when the voltage is applied.As the pH is changed, the proteins and ampholytesare mobilized in the direction of the reservoir whichcontains the added salts and pass the detector.

The separation achieved, expressed as DpI, depends on thepH gradient (dpH/dx), the number of ampholytes havingdifferent pI values, the molecular diffusion coefficient (D),the intensity of the electric field (E) and the variation of theelectrophoretic mobility of the analyte with the pH (－dm/dpH ):

DpI＝ 3D(dpH/dx)

E(－dm/dpH )

OptimizationThe main parameters to be considered in the development

of separations are:(1) Voltage: Capillary isoelectric focusing utilises veryhigh electric fields, 300 V/cm to 1000 V/cm in the focusingstep.(2) Capillary: The electroosmotic flow must be reducedor suppressed depending on the mobilization strategy (seeabove). Coated capillaries tend to reduce the electroosmoticflow.(3) Solutions: The anode buffer reservoir is filled with asolution with a pH lower than the pI of the most acidic am-pholyte and the cathode reservoir is filled with a solutionwith a pH higher than the pI of the most basic ampholyte.Phosphoric acid for the anode and sodium hydroxide for thecathode are frequently used.

Addition of a polymer, such as methylcellulose, in the am-pholyte solution tends to suppress convective forces (if any)and electroosmotic flow by increasing the viscosity. Com-mercial ampholytes are available covering many pH rangesand may be mixed if necessary to obtain an expanded pHrange. Broad pH ranges are used to estimate the isoelectricpoint whereas narrower ranges are employed to improve ac-curacy. Calibration can be done by correlating migrationtime with isoelectric point for a series of protein markers.

During the focusing step precipitation of proteins at theirisoelectric point can be prevented, if necessary, using bufferadditives such as glycerol, surfactants, urea or zwitterionicbuffers. However, depending on the concentration, ureadenatures proteins.

4. Micellar Electrokinetic Chromatography (MEKC)In micellar electrokinetic chromatography, separation

takes place in an electrolyte solution which contains a sur-factant at a concentration above the critical micellar concen-tration (cmc). The solute molecules are distributed betweenthe aqueous buffer and the pseudo-stationary phase com-posed of micelles, according to the partition coefficient ofthe solute. The technique can therefore be considered as ahybrid of electrophoresis and chromatography. It is a tech-

nique that can be used for the separation of both neutral andcharged solutes, maintaining the efficiency, speed and in-strumental suitability of capillary electrophoresis. One of themost widely used surfactants in MEKC is the anionic sur-factant sodium dodecyl sulfate, although other surfactants,for example cationic surfactants such as cetyltrimethylam-monium salts, are also used.

The separation mechanism is as follows. At neutral andalkaline pH, a strong electroosmotic flow is generated andmoves the separation buffer ions in the direction of thecathode. If sodium dodecyl sulfate is employed as the sur-factant, the electrophoretic migration of the anionic micelleis in the opposite direction, towards the anode. As a result,the overall micelle migration velocity is slowed down com-pared to the bulk flow of the electrolytic solution. In the caseof neutral solutes, since the analyte can partition between themicelle and the aqueous buffer, and has no electrophoreticmobility, the analyte migration velocity will depend only onthe partition coefficient between the micelle and the aqueousbuffer. In the electropherogram, the peaks corresponding toeach uncharged solute are always between that of the electro-osmotic flow marker and that of the micelle (the timeelapsed between these two peaks is called the separation win-dow). For electrically charged solutes, the migration velocitydepends on both the partition coefficient of the solute be-tween the micelle and the aqueous buffer, and on the elec-trophoretic mobility of the solute in the absence of micelle.

Since the mechanism in MEKC of neutral and weaklyionized solutes is essentially chromatographic, migration ofthe solute and resolution can be rationalized in terms of theretention factor of the solute (k?), also referred to as massdistribution ratio (Dm), which is the ratio of the number ofmoles of solute in the micelle to those in the mobile phase.For a neutral compound, k? is given by:

k?＝tR－ t0

t0 Ø1－ tRtmc»

＝ KVS

VM

tR: migration time of the solute,t0: analysis time of an unretained solute (determined by in-

jecting an electroosmotic flow marker which does notenter the micelle, for instance methanol),

tmc: micelle migration time (measured by injecting amicelle marker, such as Sudan III, which migrateswhile continuously associated in the micelle),

K: partition coefficient of the solute,VS: volume of the micellar phase,VM: volume of the mobile phase.

Likewise, the resolution between two closely-migrating so-lutes (RS) is given by:

RS＝N4×

a－ 1a×

k?bk?b＋ 1

×

1－ Ø t0tmc»

1＋ Ø t0tmc» k?a

N: number of theoretical plates for one of the solutes,a: selectivity,k?a and k?b: retention factors for both solutes, respectively

(k?b＞ k?a).

Similar, but not identical, equations give k? and RS valuesfor electrically charged solutes.


OptimizationThe main parameters to be considered in the development

of separations by MEKC are instrumental and electrolyticsolution parameters.Instrumental parameters(1) Voltage: Separation time is inversely proportional toapplied voltage. However, an increase in voltage can causeexcessive heat production that gives rise to temperaturegradients and viscosity gradients of the buffer in the cross-section of the capillary. This effect can be significant withhigh conductivity buffers such as those containing micelles.Poor heat dissipation causes band broadening and decreasesresolution.(2) Temperature: Variations in capillary temperature af-fect the partition coefficient of the solute between the bufferand the micelles, the critical micellar concentration and theviscosity of the buffer. These parameters contribute to themigration time of the solutes. The use of a good cooling sys-tem improves the reproducibility of the migration time forthe solutes.(3) Capillary: As in capillary zone electrophoresis, thedimensions of the capillary (length and internal diameter)contribute to analysis time and efficiency of separations. In-creasing both effective length and total length can decreasethe electric fields (working at constant voltage), increasemigration time and improve the separation efficiency. Theinternal diameter controls heat dissipation (for a givenbuffer and electric field) and consequently the sample bandbroadening.Electrolytic solution parameters(1) Surfactant type and concentration: The type of sur-factant, in the same way as the stationary phase in chroma-tography, affects the resolution since it modifies separationselectivity. Also, the log k? of a neutral compound increaseslinearly with the concentration of surfactant in the mobilephase. Since resolution in MEKC reaches a maximum whenk? approaches the value of tm/t0, modifying the concentra-tion of surfactant in the mobile phase changes the resolutionobtained.(2) Buffer pH: Although pH does not modify the parti-tion coefficient of non-ionized solutes, it can modify theelectroosmotic flow in uncoated capillaries. A decrease in thebuffer pH decreases the electroosmotic flow and thereforeincreases the resolution of the neutral solutes in MEKC,resulting in a longer analysis time.(3) Organic solvents: To improve MEKC separation ofhydrophobic compounds, organic modifiers (methanol,propanol, acetonitrile, etc.) can be added to the electrolyticsolution. The addition of these modifiers usually decreasesmigration time and the selectivity of the separation. Sincethe addition of organic modifiers affects the critical micellarconcentration, a given surfactant concentration can be usedonly within a certain percentage of organic modifier beforethe micellization is inhibited or adversely affected, resultingin the absence of micelles and, therefore, in the absence ofpartition. The dissociation of micelles in the presence of ahigh content of organic solvent does not always mean thatthe separation will no longer be possible; in some cases thehydrophobic interaction between the ionic surfactantmonomer and the neutral solutes forms solvophobic com-plexes that can be separated electrophoretically.(4) Additives for chiral separations: For the separation ofenantiomers using MEKC, a chiral selector is included in the

micellar system, either covalently bound to the surfactant oradded to the micellar separation electrolyte. Micelles thathave a moiety with chiral discrimination properties includesalts of N-dodecanoyl-L-amino acids, bile salts, etc. Chiralresolution can also be achieved using chiral discriminators,such as cyclodextrins, added to the electrolytic solutionswhich contain micellized achiral surfactants.(5) Other additives: Several strategies can be carried outto modify selectivity, by adding chemicals to the buffer. Theaddition of several types of cyclodextrins to the buffer canalso be used to reduce the interaction of hydrophobic soluteswith the micelle, thus increasing the selectivity for this typeof compound.

The addition of substances able to modify solute-micelleinteractions by adsorption on the latter, is used to improvethe selectivity of the separations in MEKC. These additivesmay be a second surfactant (ionic or non-ionic) which givesrise to mixed micelles or metallic cations which dissolve inthe micelle and form co-ordination complexes with the so-lutes.

QuantificationPeak areas must be divided by the corresponding migra-

tion time to give the corrected area in order to:(1) compensate for the shift in migration time from run to

run, thus reducing the variation of the response,(2) compensate for the different responses of sample con-

stituents with different migration times.Where an internal standard is used, verify that no peak of

the substance to be examined is masked by that of the inter-nal standard.Calculations

From the values obtained, calculate the content of thecomponent or components being examined. Whenprescribed, the percentage content of one or more compo-nents of the sample to be examined is calculated by deter-mining the corrected area(s) of the peak(s) as a percentage ofthe total of the corrected areas of all peaks, excluding thosedue to solvents or any added reagents (normalization proce-dure). The use of an automatic integration system (integratoror data acquisition and processing system) is recommended.

System SuitabilityIn order to check the behavior of the capillary electropho-

resis system, system suitability parameters are used. Thechoice of these parameters depends on the mode of capillaryelectrophoresis used. They are: retention factor (k?) (only formicellar electrokinetic chromatography), apparent numberof theoretical plates (N ), symmetry factor (AS) and resolu-tion (RS). In previous sections, the theoretical expressionsfor N and RS have been described, but more practical equa-tions that allow these parameters to be calculated from theelectropherograms are given below.Apparent Number of Theoretical Plates

The apparent number of theoretical plates (N ) may be cal-culated using the expression:

N＝ 5.54 Ø tRwh»

2

tR: migration time or distance along the baseline from thepoint of injection to the perpendicular dropped fromthe maximum of the peak corresponding to the compo-nent,


wh: width of the peak at half-height.

ResolutionThe resolution (RS) between peaks of similar height of two

components may be calculated using the expression:

RS＝ Ø1.18(tR2－ tR1)wh1＋ wh2 »

tR2＞ tR1

tR1 and tR2: migration times or distances along the baselinefrom the point of injection to the perpendicu-lars dropped from the maxima of two adjacentpeaks,

wh1 and wh2: peak widths at half-height.

When appropriate, the resolution may be calculated bymeasuring the height of the valley (Hv) between two partlyresolved peaks in a standard preparation and the height ofthe smaller peak (Hp) and calculating the peak-to-valleyratio:

p/n＝Hp

Hv

Symmetry FactorThe symmetry factor (AS) of a peak may be calculated

using the expression:

AS＝w0.05

2d

w0.05: width of the peak at one-twentieth of the peakheight,

d: distance between the perpendicular dropped from thepeak maximum and the leading edge of the peak at one-twentieth of the peak height.

Tests for area repeatability (standard deviation of areas orof the area/migration-time ratio) and for migration timerepeatability (standard deviation of migration time) are in-troduced as suitability parameters. Migration time repeata-bility provides a test for the suitability of the capillary wash-ing procedures. An alternative practice to avoid the lack ofrepeatability of the migration time is to use migration timerelative to an internal standard.

A test for the verification of the signal-to-noise ratio for astandard preparation (or the determination of the limit ofquantification) may also be useful for the determination ofrelated substances.Signal-to-noise Ratio

The detection limit and quantification limit correspond tosignal-to-noise ratios of 3 and 10 respectively. The signal-to-noise ratio (S/N) is calculated using the expression:

S/N＝2Hh

H: height of the peak corresponding to the componentconcerned, in the electropherogram obtained with theprescribed reference solution, measured from the maximumof the peak to the extrapolated baseline of the signal ob-served over a distance equal to twenty times the width athalf-height,

h: range of the background in an electropherogram ob-tained after injection of a blank, observed over a distanceequal to twenty times the width at the half-height of the peak

in the electropherogram obtained with the prescribed refer-ence solution and, if possible, situated equally around theplace where this peak would be found.

Isoelectric FocusingThis test is harmonized with the European Pharmacopoeia

and the U.S. Pharmacopeia. The parts of the text that arenot harmonized are marked with symbols ( ).

General PrinciplesIsoelectric focusing (IEF) is a method of electrophoresis

that separates proteins according to their isoelectric point.Separation is carried out in a slab of polyacrylamide oragarose gel that contains a mixture of amphoteric electro-lytes (ampholytes). When subjected to an electric field, theampholytes migrate in the gel to create a pH gradient. Insome cases gels containing an immobilized pH gradient, pre-pared by incorporating weak acids and bases to specificregions of the gel network during the preparation of the gel,are used. When the applied proteins reach the gel fractionthat has a pH that is the same as their isoelectric point (pI),their charge is neutralized and migration ceases. Gradientscan be made over various ranges of pH, according to themixture of ampholytes chosen.

Theoretical AspectsWhen a protein is at the position of its isoelectric point, it

has no net charge and cannot be moved in a gel matrix by theelectric field. It may, however, move from that position bydiffusion. The pH gradient forces a protein to remain in itsisoelectric point position, thus concentrating it; this concen-trating effect is called ``focusing''. Increasing the appliedvoltage or reducing the sample load result in improved sepa-ration of bands. The applied voltage is limited by the heatgenerated, which must be dissipated. The use of thin gels andan efficient cooling plate controlled by a thermostatic circu-lator prevents the burning of the gel whilst allowing sharpfocusing. The separation R is estimated by determining theminimum pI difference (DpI ), which is necessary to separate2 neighboring bands:

R: DpI＝ 3D(dpH/dx)

E(－dm/dpH )

D: Diffusion coefficient of the proteindpH/dx: pH gradientE: Intensity of the electric field, in volts per centimeter－dm/dpH: Variation of the solute mobility with the pH in

the region close to the pI

Since D and －dm/dpH for a given protein cannot be al-tered, the separation can be improved by using a narrowerpH range and by increasing the intensity of the electric field.Resolution between protein bands on an IEF gel preparedwith carrier ampholytes can be quite good. Improvements inresolution may be achieved by using immobilized pHgradients where the buffering species, which are analogousto carrier ampholytes, are copolymerized within the gelmatrix. Proteins exhibiting pIs differing by as little as 0.02pH units may be resolved using a gel prepared with carrierampholytes while immobilized pH gradients can resolve pro-teins differing by approximately 0.001 pH units.

2185

Figure. Mould


Practical AspectsSpecial attention must be paid to sample characteristics

and/or preparation. Having salt in the sample can beproblematic and it is best to prepare the sample, if possible,in deionized water or 2 per cent ampholytes, using dialysis orgel filtration if necessary.

The time required for completion of focusing in thin-layerpolyacrylamide gels is determined by placing a colored pro-tein (e.g. hemoglobin) at different positions on the gel sur-face and by applying the electric field: the steady state isreached when all applications give an identical band pattern.In some protocols the completion of the focusing is indicatedby the time elapsed after the sample application.

The IEF gel can be used as an identity test when the migra-tion pattern on the gel is compared to a suitable standardpreparation and IEF calibration proteins, the IEF gel can beused as a limit test when the density of a band on IEF is com-pared subjectively with the density of bands appearing in astandard preparation, or it can be used as a quantitative testwhen the density is measured using a densitometer or similarinstrumentation to determine the relative concentration ofprotein in the bands subject to validation.

ApparatusAn apparatus for IEF consists of:�a controllable generator for constant potential, current

and power. Potentials of 2500 V have been used and areconsidered optimal under a given set of operating condi-tions. Supply of up to 30 W of constant power is recom-mended,

�a rigid plastic IEF chamber that contains a cooled plate,of suitable material, to support the gel,

�a plastic cover with platinum electrodes that are con-nected to the gel by means of paper wicks of suitablewidth, length and thickness, impregnated with solutionsof anodic and cathodic electrolytes.

Isoelectric Focusing in Polyacrylamide Gels: Detailed Proce-dure

The following method is a detailed description of an IEFprocedure in thick polyacrylamide slab gels, which is usedunless otherwise stated in the monograph.

Preparation of the GelsMould The mould (see Figure) is composed of a glass

plate (A) on which a polyester film (B) is placed to facilitatehandling of the gel, one or more spacers (C), a second glassplate (D) and clamps to hold the structure together.

7.5z Polyacrylamide gel Dissolve 29.1 g of acrylamideand 0.9 g of N,N?-methylenebisacrylamide in 100 mL ofwater. To 2.5 volumes of this solution, add the mixture ofampholytes specified in the monograph and dilute to 10volumes with water. Mix carefully and degas the solution.

Preparation of the mould Place the polyester film on thelower glass plate, apply the spacer, place the second glassplate and fit the clamps. Place 7.5z polyacrylamide gel pre-pared before use on a magnetic stirrer, and add 0.25 volumesof a solution of ammonium persulfate (1 in 10) and 0.25volumes of N,N,N?,N?-tetramethylethylenediamine. Imme-diately fill the space between the glass plates of the mouldwith the solution.

MethodDismantle the mould and, making use of the polyester

film, transfer the gel onto the cooled support, wetted with a

few millilitres of a suitable liquid, taking care to avoid form-ing air bubbles. Prepare the test solutions and reference solu-tions as specified in the monograph. Place strips of paper forsample application, about 10 mm× 5 mm in size, on the geland impregnate each with the prescribed amount of the testand reference solutions. Also apply the prescribed quantityof a solution of proteins with known isoelectric points as pHmarkers to calibrate the gel. In some protocols the gel haspre-cast slots where a solution of the sample is applied in-stead of using impregnated paper strips. Cut 2 strips ofpaper to the length of the gel and impregnate them with theelectrolyte solutions: acid for the anode and alkaline for thecathode. The compositions of the anode and cathode solu-tions are given in the monograph. Apply these paper wicksto each side of the gel several millimetres from the edge. Fitthe cover so that the electrodes are in contact with the wicks(respecting the anodic and cathodic poles). Proceed with theisoelectric focusing by applying the electrical parametersdescribed in the monograph. Switch off the current when themigration of the mixture of standard proteins has stabilized.Using forceps, remove the sample application strips and the2 electrode wicks. Immerse the gel in fixing solution for isoe-lectric focusing in polyacrylamide gel. Incubate with gentleshaking at room temperature for 30 minutes. Drain off thesolution and add 200 mL of destaining solution. Incubatewith shaking for 1 hour. Drain the gel, add coomassie stain-ing TS. Incubate for 30 minutes. Destain the gel by passivediffusion with destaining solution until the bands are wellvisualized against a clear background. Locate the positionand intensity of the bands in the electropherogram asprescribed in the monograph.

Variations to the Detailed Procedure (Subject to Validation)Where reference to the general method on isoelectric

focusing is made, variations in methodology or proceduremay be made subject to validation. These include:(1) the use of commercially available pre-cast gels and of

commercial staining and destaining kits,(2) the use of immobilized pH gradients,(3) the use of rod gels,(4) the use of gel cassettes of different dimensions, includ-

ing ultra-thin (0.2 mm) gels,(5) variations in the sample application procedure, includ-

ing different sample volumes or the use of sample appli-cation masks or wicks other than paper,

(6) the use of alternate running conditions, including varia-tions in the electric field depending on gel dimensionsand equipment, and the use of fixed migration times


rather than subjective interpretation of band stability,(7) the inclusion of a pre-focusing step,(8) the use of automated instrumentation,(9) the use of agarose gels.

Validation of Iso-Electric Focusing ProceduresWhere alternative methods to the detailed procedure are

employed they must be validated. The following criteria maybe used to validate the separation:(1) formation of a stable pH gradient of desired character-

istics, assessed for example using colored pH markersof known isoelectric points,

(2) comparison with the electropherogram provided withthe chemical reference substance for the preparation tobe examined,

(3) any other validation criteria as prescribed in the mono-graph.

Specified Variations to the General MethodVariations to the general method required for the analysis

of specific substances may be specified in detail in mono-graphs. These include:(1) the addition of urea in the gel (3 mol/L concentration is

often satisfactory to keep protein in solution but up to 8mol/L can be used): some proteins precipitate at theirisoelectric point. In this case, urea is included in the gelformulation to keep the protein in solution. If urea isused, only fresh solutions should be used to preventcarbamylation of the protein,

(2) the use of alternative staining methods,(3) the use of gel additives such as non-ionic detergents

(e.g. octylglucoside) or zwitterionic detergents (e.g., 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesul-fonate (CHAPS) or 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO)),and the addition of ampholyte to the sample, to preventproteins from aggregating or precipitating.

Points to ConsiderSamples can be applied to any area on the gel, but to pro-

tect the proteins from extreme pH environments samplesshould not be applied close to either electrode. Duringmethod development the analyst can try applying the proteinin 3 positions on the gel (i.e. middle and both ends); the pat-tern of a protein applied at opposite ends of the gel may notbe identical.

A phenomenon known as cathodic drift, where the pHgradient decays over time, may occur if a gel is focused toolong. Although not well understood, electroendoosmosis andabsorption of carbon dioxide may be factors that lead tocathodic drift. Cathodic drift is observed as focused proteinmigrating off the cathode end of the gel. Immobilized pHgradients may be used to address this problem.

Efficient cooling (approximately 49C) of the bed that thegel lies on during focusing is important. High field strengthsused during isoelectric focusing can lead to overheating andaffect the quality of the focused gel.

Reagents and Solutions�Fixing solution for isoelectric focusing in polyacrylamide

gel Dissolve 35 g of 5-sulfosalicylic acid dihydrate and100 g of trichloroacetic acid in water to make 1000 mL.Coomassie staining TS Dissolve 125 mg of coomassie

brilliant blue R-250 in 100 mL of a mixture of water, metha-nol and acetic acid (100) (5:4:1), and filter.

Destaining solution A mixture of water, methanol andacetic acid (100) (5:4:1).

Mass Spectrometry of Peptidesand Proteins

Mass spectrometry (MS) is based on the ionization ofmolecules and separation of the electrically charged ionsaccording to the m/z. The results are expressed as a massspectrum with m/z values of the ions on the x-axis and signalintensity of the ions on the y-axis. The mass of the moleculecalculated from the m/z values is expressed in unified atomicmass units (u) or daltons (Da). Tandem mass spectrometry(MS/MS) is based on the selection of the precursor ion in thefirst stage analyzer, fragmentation of the precursor ion andmeasurement of the product ions in the second stage massanalyzer. This technique provides useful information forstructural analysis of the molecule. Information obtained inMS is qualitative and is sometimes used for qualification.MS and MS/MS are useful for measuring masses of peptidesand proteins and for confirming amino acid sequences andpost-translational modifications. Both methods are thereforeused for identification of pharmaceutical peptides and pro-teins.

1. InstrumentA mass spectrometer is composed of an ion source, an

analyzer, an ion detector, and a data system (Figure 1). Apeptide or protein sample introduced into the ion source isionized by soft-ionization methods, such as matrix-assistedlaser desorption/ionization (MALDI) and electrospray ioni-zation (ESI). The charged and gas-phased ions are sorted ac-cording to the m/z under a vacuum in the analyzer, whichmay be a quadrupole, time-of-flight, ion trap or Fouriertransform ion cyclotron resonance analyzer. The ion fluxcollected in the detector is converted to an electric signal.Then the signal is recorded as a mass spectrum. MS/MS iscarried out by using two mass spectrometers connected inseries, an ion-trap mass spectrometer and Fourier transformion cyclotron resonance mass spectrometer. The precursorions are generally fragmented by collision-induced dissocia-tion, post-source decay, electron capture dissociation, etc.

2. Analytical mode2.1. MS

There are two useful modes for MS:(1) Full scan mode

The signals of the entire ion are acquired over the chosenrange of m/z. This mode provides information on themasses of the molecule of interest and different species.(2) Selected ion monitoring

The signals of the ion at chosen m/z are acquired. Thismode is useful for the sensitive measurement of the chosenmolecule.2.2. MS/MS

There are four essential modes for MS/MS:(1) Product ion analysis

The signals of all the product ions produced from theprecursor at chosen m/z are acquired. This mode providesstructural information on the analytes and various co-existing species.

2187

Fig� 1 Concept of MS and MS/MS


(2) Precursor ion scan modeThe precursor that yields the product ion at chosen m/z

are monitored. This mode is used for sorting the moleculescontaining a component of interest.(3) Constant neutral loss scan mode

The precursor that loses the fragment at chosen m/z aremonitored. This mode is useful to sort the molecules con-taining a component of interest.(4) Selected reaction monitoring

The product ions at chosen m/z that are produced fromthe precursor at chosen m/z are monitored. This modeallows for sensitive and selective measurement and is usedfor quantification of a molecule in a complex mixture.

3. Analytical procedure3.1. MS

System suitability is tested by measuring the mass of a testsample specified in the monograph. The system performanceshould be confirmed based on the difference between the cal-culated mass and observed mass of the test sample. If the de-tectability and system performance do not meet the criteria,the system should be optimized by adjustment of the ionsource, analyzer and detector, as well as by calibration usingappropriate molecules. MS is performed according to thesample preparation and operating conditions indicated in themonograph. The general procedure is described as follows.(1) Matrix-assisted laser desorption/ionization (MALDI)

A desalted peptide or protein sample is dissolved in anappropriate solvent, e.g., an aqueous solution of trifluoroa-cetic acid. A suitable matrix, such as a-ciano-4-hydroxycin-namic acid, 2,5-dihydroxybenzoic acid, or sinapic acid, isdissolved in an aqueous solution containing acetonitrile andtrifluoroacetic acid. A mixture of sample solution andmatrix solution is deposited on a sample plate and dried. Thesample on the plate is set in the ion source, and ionized by alaser beam at suitable intensity.

(2) Electrospray ionization (ESI)A desalted peptide or protein sample is dissolved in a suit-

able solvent, such as an aqueous solution containing aceticacid and methanol or acetonitrile. The sample solution issprayed through a capillary and held at a potential of severalkilovolts. The sample is introduced by using a syringe orHPLC.3.2. MS/MS

System suitability is tested by MS/MS of the test samplespecified in the monograph. The detectability and systemperformance should be confirmed based on the detection ofthe product ions specified in the monograph. The sample isionized in the same way as for MS, and the chosen precursoris fragmented by the suitable conditions specified in themonograph. The signals are recorded as a mass spectrum. Apeptide containing disulfide bonds is generally reduced bydithiothreitol, 2-mercaptethanol and tris (2-carboxyethyl)phosphine. The reduced peptides are derivatized withmonoiodoacetic acid, iodoacetamide, and 4-binylpyridine.

4. Identification test4.1. Mass of the molecule

The monoisotopic mass of the peptide is preferably ac-quired. If the monoisotopic peak is not detectable, theaverage mass is calculated using the weighed average ofisotopic masses. Deconvolution is effective for calculatingthe average mass of multiply-charged proteins. The massshould meet the criteria specified in the monograph.4.2. Amino acid sequence

After measuring the mass of the sample peptide, thepresence of the specified product ions that arise from theselected precursor is confirmed according to the conditionsindicated in the monograph. Digestion of sample proteinswith a suitable enzyme followed by MS/MS is sometimes ef-fective for sequencing of the high-molecular weight proteinswhich provide insufficient product ions. Details of the diges-


tion procedure are provided in the section on peptide map-ping.

5. GlossaryIon-trap (IT)

Ion-trap refers to the quadrupole ion trap mass analyzer ina restricted sense. Ions stored in the analyzer by applying ra-dio frequency voltage to ring electrodes are separated bysubsequent ejection of the ions from the analyzer by varyingthe voltage on the ring electrodes. This allows multiple stageMS in which a selected ion is repeatedly trapped, fragmentedand ejected.

Electrospray ionization (ESI)The sample in solution is sprayed through a capillary and

held at high-voltage at atmospheric pressure. The sample isionized by a formation of charged liquid droplets. High-molecular weight proteins are detected as multiply-chargedions. The analyzer can be connected with HPLC.

Quadrupole (Q)The analyzer is composed of four parallel electrodes which

have a hyperboloidal or cylindrical cross-section. The ionstransmitted to the analyzer are separated by varying thepotential of direct and radio frequency components appliedto the rods so that the filter for sorting the m/z values ofions is changed.

Collision-induced dissociation (CID)When an ion collides with a neutral atom or molecule (He,

Ar, N2 and so on), some of the translational energy of thecollision is converted into internal energy, thereby causingdissociation. The terms low-energy CID and high-energyCID refer to those CIDs for which the translational energyof the precursor ions is lower than 1000 eV and higher than1000 eV, respectively.

Electron capture dissociation (ECD)Multiply-charged positive ions interact with low energy

electrons producing charge-reduced radical ions, which rea-dily dissociate. This method is primarily used for MS/MS inFT-ICR MS or IT MS.

Time-of-flight (TOF)The ionized sample is accelerated at high-voltage and sepa-

rated based on the time required for an ion to travel to thedetector. There are two types of analyzer, a linear type inwhich ions travel linearly from the ion source to the detector,and a reflectron type where ions are inverted by a reflectron.The latter type allows high-resolution measurement by cor-rection of the variation in the initial energy of ions.

Fourier transform ion cyclotron resonance (FT-ICR)The analyzer is based on the principle that the cyclotron

frequency of the ions in a magnetic field is inversely propor-tional to its m/z value. Ions are excited to a larger radius or-bit using radio frequency energy and their image current isdetected on receiver plates. The resulting data are devolvedby applying a Fourier transform to give a mass spectrum.

Post-source decay (PSD)Metastable ion decay occurs by excess internal energy and

collision with residual gas during ion acceleration out of theMALDI ion source and prior to reaching the detector. Thismethod is used for MS/MS by using MALDI-TOF MS witha reflectron mode.

Matrix-assisted laser desorption/ionization (MALDI)The sample, which is mixed with a suitable matrix and

deposited on a target plate, is ionized by irradiation withnanosecond laser pulses. Proteins, carbohydrates,oligonucleotides, and lipids can be ionized without any dis-sociation. Singly-charged ions are mainly detected.

Mycoplasma Testing for CellSubstrates used for the Production

of Biotechnological/BiologicalProducts

This document describes the currently available methodsof mycoplasma testing that should be performed for cell sub-strates that are used in the manufacture of biotechnological/biological products.

Methods suggested for detection of mycoplasma are, A.culture method, B. indicator cell culture method, and C.polymerase chain reaction (PCR) method.

Mycoplasma testing should be performed on the mastercell bank (MCB) and the working cell bank (WCB), as wellas on the cell cultures used during the manufacturing processof the product. For the assessment of these cells, mycoplas-ma testing should be performed using both methods A andB. Method B, however, does not detect only DNA derivedfrom mycoplasma. Therefore, if a positive result is obtainedonly from method B, method C can be used to determinewhether mycoplasma is actually present. When method C isused, it is necessary to demonstrate the rationale for deter-mining a negative result. In such a case, the sensitivity andspecificity of the method, the appropriateness of the samplepreparation, and the suitability of the selection of the testmethod, including selection of reagents, reaction conditionsand primers should be taken into account.

Prior to mycoplasma testing, the sample should be testedto detect the presence of any factors inhibiting the growth ofmycoplasma. If such growth-inhibiting factors are detectedthey should be neutralized or eliminated by an appropriatemethod, such as centrifugation or cell passage.

If the test will be performed within 24 hours of obtainingthe sample, the sample should be stored at a temperature be-tween 29C and 89C. If more than 24 hours will elapse beforethe test is performed, the sample should be stored at－609Cor lower.

If mycoplasma is detected, additional testing to identifythe species may be helpful in determining the source of con-tamination.

A. Culture Method1. Culture Medium

Both agar plates and broth are used. Each lot of agar andbroth medium should be free of antibiotics except forpenicillin. Refer to the Minimum Requirements for Biologi-cal Products regarding selection of the culture media. Otherculture media may be used if they fulfill the requirementsdescribed in the following section 2.2. Suitability of Culture Medium

Each lot of medium should be examined for mycoplasmagrowth-promoting properties. To demonstrate the capacityof the media to detect known mycoplasma, each test should


include control cultures of at least two known species orstrains of mycoplasma, one of which should be a dextrosefermenter (i.e., M. pneumoniae ATCC15531 or equivalentspecies or strains) and one of which should be an argininehydrolyser (i.e., M. orale ATCC23714 or equivalent speciesor strains). The mycoplasma strains used for the positivecontrol tests should be those with a low number of passagesobtained from an official or suitably accredited agency, andhandled appropriately. Inoculate the culture medium with100 colony-forming units (CFU) or 100 color-changing units(CCU) or less.3. Culture and Observation

1) Inoculate no less than 0.2 mL of test sample (cell sus-pension) in evenly distributed amounts over the surface ofeach of two or more agar plates. After the surfaces of the in-oculated plates are dried, the plates should be incubatedunder microaerophilic conditions in an atmosphere of nitro-gen containing 5 to 10 percent carbon dioxide and adequatehumidity at 36± 19C for no less than 14 days.

2) Inoculate no less than 10 mL of the test sample (cellsuspension) into each of one or more vessels containing 100mL of broth medium, and incubate at 36± 19C.

If the culture medium for the sample cells contains anygrowth-inhibiting factors, such as antibiotics, these factorsmust be removed. A method such as centrifugation is recom-mended for this purpose. Refer to the Validation tests forgrowth-inhibiting factors described in the Minimum Re-quirements for Biological Products for the detection ofgrowth-inhibiting factors.

3) Subculture 0.2 mL of broth culture from each vesselon the 3rd, 7th, and 14th days of incubation onto two or moreagar plates. The plates should be inoculated microaerophili-cally at 36± 19C for no less than 14 days.

4) Examination of all plates for mycoplasma coloniesshould be done microscopically on the 7th and 14th day at 100times magnification or greater.

B. Indicator Cell Culture MethodUsing Vero cell culture substrate, pretest the suitability of

the method using an inoculum of 100 CFU or 100 CCU orless of M. hyorhinis (ATCC29052, ATCC17981 or equiva-lent species or strains) and M. orale (ATCC23714 or equiva-lent species or strains).

An equivalent indicator cell substrate and suitablemycoplasma strains may be acceptable if data demonstrateat least equal sensitivity for the detection of knownmycoplasma contaminants. The mycoplasma strains shouldbe those with a low number of passages obtained from anofficial or suitably accredited agency, and handled appropri-ately, and the unit of inoculation should be determined be-fore use. The cell substrate used should be obtained from aqualified cell bank and certified to be mycoplasma free. Theacquired cells should be carefully cultured and propagated,and sufficient volumes of seed stock should be prepared withthe proper precautions to avoid mycoplasma contamination.The stock should be tested for mycoplasma contaminationusing at least one of the methods described in this document,then frozen for storage. For each test a new container fromthe stock should be thawed and used within 6 passages.

Indicator cell cultures should be grown on cover slips sub-merged in culture dishes or equivalent containers for oneday. Inoculate no less than 1 mL of the test sample (cell cul-ture supernatant) into two or more of the culture dishes.

The test should include a negative (non-infected) controland two positive mycoplasma controls, such as M. hyorhinis(ATCC29052, ATCC17981 or equivalent species or strains)and M. orale (ATCC23714 or equivalent species or strains).Use an inoculum of 100 CFU or 100 CCU or less for thepositive controls.

Incubate the cell cultures for 3 to 6 days at 36± 19C in anatmosphere of air containing 5 percent carbon dioxide.

Examine the cell cultures after fixation for the presence ofmycoplasma by epifluorescence microscopy (400 to 600times magnification or greater) using a DNA-bindingfluorochrome, such as bisbenzimidazole or an equivalentstain. Compare the microscopical appearance of the test cul-tures with that of the negative and positive controls.Procedure

1) Aseptically place a sterilized glass cover slip into eachcell culture dish (35 mm diameter).

2) Prepare Vero cell suspension in Eagle's minimumessential medium containing 10 percent bovine calf serum ata concentration of 1× 104 cells per 1 mL. The bovine calfserum should be tested and confirmed to be free frommycoplasma prior to use.

3) Inoculate aliquots of 2 mL of the Vero cell suspensioninto each culture dish. Ensure that the cover slips are com-pletely submerged, and not floating on the surface of the cul-ture medium. Incubate the cultures at 36± 19C in an at-mosphere of air containing 5 percent carbon dioxide for oneday, so that the cells are attached to the glass cover slip.

4) Replace 2 mL of the culture medium with fresh me-dium, then add 0.5 mL of the test sample (cell culture super-natant) to each of two or more culture dishes. Perform thesame procedure for the positive (2 types of mycoplasma,such as M. hyorhinis (ATCC29052, ATCC17981 or equiva-lent species or strains) and M. orale (ATCC23714 or equiva-lent species or strains) and negative controls.

5) Incubate the cultures for 3 to 6 days at 36± 19C in anatmosphere of air containing 5 percent carbon dioxide.

6) Remove the culture medium from the culture dishes,and add 2 mL of a mixture of acetic acid (100) and methanol(1:3) (fixative) to each dish; then, allow them to stand for 5minutes.

7) Remove the fixative from each dish, then add thesame amount of fixative again, and leave the dishes to standfor 10 minutes.

8) Remove the fixative and then completely air-dry allthe dishes.

9) Add 2 mL of bisbenzamide fluorochrome staining so-lution to each culture dish. Cover the dishes and let themstand at room temperature for 30 minutes.

10) Aspirate the staining solution and rinse each dishwith 2 mL of distilled water 3 times. Take out the glass coverslips and dry them.

11) Mount each cover slip with a drop of a mountingfluid. Blot off surplus mounting fluid from the edges of thecover slips.

12) Examine by epifluorescence microscopy at 400 to 600times magnification or greater.

13) Compare the microscopic appearance of the testsample with that of the negative and positive controls.

14) The test result is judged to be positive if there aremore than 5 cells per 1000 (0.5z) that have minute fluores-cent spots that appear to surround, but are outside, the cellnucleus.


C. Polymerase Chain Reaction (PCR) Detection MethodThe PCR method is a highly specific method that enables

the detection of trace amounts of mycoplasma DNA, andhas come to be widely used in recent years as a means of de-tecting mycoplasma contamination. However, the sensitivityand specificity depend on the procedure employed, and apositive result from PCR does not always indicate thepresence of viable mycoplasma.

The PCR method is based on amplifying DNA extractedfrom the cell culture with specific primers so that thepresence of the target DNA is detected. A two-step PCR(nested PCR) is recommended in order to increase sensitivityand specificity. The tests should include both a positivecontrol (such as M. hyorhinis (ATCC29052, ATCC17981 orequivalent species or strains) of 100 CFU or 100 CCU orless) and a negative control.

Mycoplasma DNA from the sample of cells or cell culturesis amplified using primers which should be able to amplifysome common conserved mycoplasma DNA sequence. Theamplification should be performed using an appropriateheat-resistant DNA polymerase, and suitable conditions.The amplified DNA can be identified after agarose gel elec-trophoresis, followed by ethidium bromide staining and UVirradiation of the gel.

For this method, it is important to use primers that arespecific to mycoplasma by choosing base sequences that arewell-conserved for a wide range of mycoplasma species, forexample, the spacer region between the 16S-23S ribosomegenes.

It is recommended that a two-step PCR using nestedprimers should be performed to increase the sensitivity andspecificity, if the one-step PCR is negative.

The primers to be selected for the second stage of a two-step PCR are nested primers from the inner portion of thesequence. The outer and inner primers should have proveneffectiveness and specificity as described in publications orbe validated experimentally.

It is possible to increase the accuracy of the detection ofmycoplasma DNA by performing PCR tests after cultivationof mycoplasma that may be present in samples using Verocells.

The following is an example of a two-step PCR procedure.The reagents and reaction conditions in this example are notexclusive. If the suitability of other reagents and conditionsis verified, they may be used. If another procedure is used,the procedure should be justified and documented in detail,and the information provided should include the sensitivityand specificity of the method.Example Procedure1. Preparation of template

1) Place 600 mL of the test cell suspension (if necessary,subcultured with Vero cells) in a tube and dissolve the cellswith 0.1z SDS or an equivalent. Add an equal volume (600mL) of TE (10 mmol/L tris-hydrochloric acid (pH 8.0), 1mmol/L EDTA) buffer-saturated phenol, and mix.

2) Centrifuge at 15,000 min－1 for 5 minutes at roomtemperature.

3) Transfer 400 mL of the supernatant to another tube,and add 10 mL of 3 mol/L sodium acetate.

4) Add 1 mL (2.5 volumes) of ethanol (95) and stir thor-oughly. Ice the mixture for 15 minutes, then centrifuge at15,000 min－1 for 10 minutes at 49C.

5) Discard the supernatant and rinse the precipitate once

or twice with 200 to 300 mL of 80z ethanol. Remove therinse solution using a pipette. Centrifuge at 15,000 min－1 for10 minutes at 49C, then remove the supernatant thoroughlyand dry up the precipitate.

6) Dissolve the precipitate in 40 mL of distilled water.2. Perform the same procedure for the positive and nega-tive controls3. First stage of a two-step PCR

1) Make a mixture of the heat-resistant DNA poly-merase, dNTP solution, outer primer, and reaction buffersolution (including Mg ions), and place 90 mL in each tube.

2) Add 10 mL of the template prepared as above to eachtube containing the first stage PCR solution (90 mL).

3) Perform the DNA amplification by repeating 30 cy-cles of denaturation at 949C for 30 seconds, annealing at anappropriate temperature for the primer (559C for the primerin this example), and elongation at 729C for 2 minutes.4. Second stage of a two-step PCR

1) Make a mixture of the heat-resistant DNA poly-merase, dNTP solution, inner primer, and reaction buffersolution (including Mg ions), and place 99 mL in each tube.

2) Add 1 mL of the first stage PCR product from eachtube to a tube containing the second stage PCR solution (99mL).

3) Perform the DNA amplification by repeating 30 cy-cles of denaturation at 949C for 30 seconds, annealing at anappropriate temperature for the primer (559C for the primerin this example), and elongation at 729C for 2 minutes.5. Agarose gel electrophoresis

1) Mix 10 mL of each of the first stage and second stagePCR products with 2 mL of an appropriate dye as a migra-tion marker, and perform 1z agarose gel electrophoresis.

2) Stain the gel with ethidium bromide and take a photo-graph under UV irradiation.

3) The test is judged to be positive if a DNA band is de-tected.

[An Example of Primer]For mycoplasma detectionOuter primerF1 : 5?-ACACCATGGGAG(C/T)TGGTAAT-3?R1 : 5?-CTTC(A/T)TCGACTT(C/T)CAGACCCAAGG-

CAT-3?Inner primerF2 : 5?-GTG(G/C)GG(A/C)TGGATCACCTCCT-3?R2 : 5?-GCATCCACCA(A/T)A(A/T)AC(C/T)CTT-3?

( ) indicates a mixture.

[PCR reaction solution][First stage] [Second stage]

dNTP solution (1.25 mmol/L) 16 mL 16 mLPrimer (10 pmol/mL) F1 2 mL F2 2 mLPrimer (10 pmol/mL) R1 2 mL R2 2 mLHeat-resistant DNA poly-

merase (1 U/mL) 2 mL 2 mL

Reaction buffer solution25 mmol/L magnesium

chloride hexahydrate 8 mL 8 mL

10-fold buffer solution* 10 mL 10 mLSterile distilled water 50 mL 59 mL

*Composition of 10-fold buffer solution2-amino-2-hydroxymethyl-1,3-


propanediol-hydrochloric acid(pH 8.4) 100 mmol/L

Potassium chloride 500 mmol/LGelatin 0.1 g/L

[Method of cultivating mycoplasma within Vero cells]1) Use at least two cell culture dishes for each of the test

sample, positive control and negative control.2) Into each cell culture dish (35 mm diameter), inoculate

2 mL of the Vero cell suspension (1× 104 cells per 1 mL) inEagle's minimum essential medium containing 10 percentbovine calf serum (tested in advance using the PCR methodto verify that it does not contain any detectable mycoplasmaDNA). Incubate the cultures at 36± 19C in an atmosphereof air containing 5 percent carbon dioxide for one day.

3) Replace the culture media with fresh media, and add0.5 mL of the test sample (cell culture supernatant) to eachof two or more Vero cell culture dishes. Perform the sameprocedure for the positive (such as 100 CFU or 100 CCU orless of M. hyorhinis (ATCC29052, ATCC17981 or equiva-lent species or strains)) and negative controls.

4) Incubate the Vero cell culture dishes for the testsample, positive and negative controls for 3 to 6 days at36± 19C in an atmosphere of air containing 5 percentcarbon dioxide.

Peptide MappingThis test is harmonized with the European Pharmacopoeia


Peptide mapping is an identity test for proteins, especiallythose obtained by r-DNA technology. It involves the chemi-cal or enzymatic treatment of a protein, resulting in the for-mation of peptide fragments, followed by separation andidentification of the fragments in a reproducible manner. Itis a powerful test that is capable of identifying single aminoacid changes resulting from events such as errors in the read-ing of complementary DNA (cDNA) sequences or point mu-tations. Peptide mapping is a comparative procedure be-cause the information obtained, compared to a referencestandard or reference material similarly treated, confirms theprimary structure of the protein, is capable of detectingwhether alterations in structure have occurred, and demon-strates process consistency and genetic stability. Each pro-tein presents unique characteristics which must be wellunderstood so that the scientific and analytical approachespermit validated development of a peptide map that providessufficient specificity.

This chapter provides detailed assistance in the applicationof peptide mapping and its validation to characterize thedesired protein product, to evaluate the stability of theexpression construct of cells used for recombinant DNAproducts, to evaluate the consistency of the overall processand to assess product stability as well as to ensure the identi-ty of the protein product, or to detect the presence of proteinvariant.

1. The Peptide MapPeptide mapping is not a general method, but involves de-

veloping specific maps for each unique protein. Althoughthe technology is evolving rapidly, there are certain methods

that are generally accepted. Variations of these methods willbe indicated, when appropriate, in specific monographs.

A peptide map may be viewed as a fingerprint of a proteinand is the end product of several chemical processes thatprovide a comprehensive understanding of the protein beinganalyzed. Four major steps are necessary for the develop-ment of the procedure: isolation and purification of the pro-tein, if the protein is part of a formulation; selective cleavageof the peptide bonds; chromatographic separation of thepeptides; and analysis and identification of the peptides. Atest sample is digested and assayed in parallel with a refer-ence standard or a reference material. Complete cleavage ofpeptide bonds is more likely to occur when enzymes such asendoproteases (e.g., trypsin) are used, instead of chemicalcleavage reagents. A map should contain enough peptides tobe meaningful. On the other hand, if there are too manyfragments, the map might lose its specificity because manyproteins will then have the same profiles.

2. Isolation and PurificationIsolation and purification are necessary for analysis of

bulk drugs or dosage forms containing interfering excipientsand carrier proteins and, when required, will be specified inthe monograph. Quantitative recovery of protein from thedosage form should be validated.

3. Selective Cleavage of Peptide BondsThe selection of the approach used for the cleavage of

peptide bonds will depend on the protein under test. Thisselection process involves determination of the type ofcleavage to be employed �enzymatic or chemical� and thetype of cleavage agent within the chosen category. Severalcleavage agents and their specificity are shown in Table 1.This list is not all-inclusive and will be expanded as othercleavage agents are identified.3.1. Pretreatment of Sample

Depending on the size or the configuration of the protein,different approaches in the pretreatment of samples can beused. For monoclonal antibodies, the heavy and light chainswill need to be separated before mapping. If trypsin is usedas a cleavage agent for proteins with a molecular mass great-er than 100,000 Da, lysine residues must be protected bycitraconylation or maleylation; otherwise, too many peptideswill be generated.3.2. Pretreatment of the Cleavage Agent

Pretreatment of cleavage agents �especially enzymaticagents� might be necessary for purification purposes toensure reproducibility of the map. For example, trypsin usedas a cleavage agent will have to be treated with tosyl-L-phenylalanine chloromethyl ketone to inactivate chymotryp-sin. Other methods, such as purification of trypsin by HPLCor immobilization of enzyme on a gel support, have beensuccessfully used when only a small amount of protein isavailable.3.3. Pretreatment of the Protein

Under certain conditions, it might be necessary to concen-trate the sample or to separate the protein from added sub-stances and stabilizers used in formulation of the product, ifthese interfere with the mapping procedure. Physical proce-dures used for pretreatment can include ultrafiltration,column chromatography, and lyophilization. Other pretreat-ments, such as the addition of chaotropic agents (e.g., urea)can be used to unfold the protein prior to mapping. To allowthe enzyme to have full access to cleavage sites and permit

2192

Table 1 Examples of Cleavage Agents

Type Agent Specificity

Enzymatic Trypsin (EC 3.4.21.4) C-terminal side ofArg and Lys

Chymotrypsin(EC 3.4.21.1)

C-terminal side ofhydrophobicresidues (e.g.,Leu, Met, Ala,aromatics)

Pepsin (EC 3.4.23.1 & 2) Nonspecific digestLysyl endopeptidase(Lys-C Endopeptidase)(EC 3.4.21.50)

C-terminal side ofLys

Glutamyl endopeptidase(from S. aureus strain V8)(EC 3.4.21.19)

C-terminal side ofGlu and Asp

Peptidyl-Asp metalloendopeptidase(Endoproteinase Asp-N)(EC 3.24.33)

N-terminal side ofAsp

Clostripain (EC 3.4.22.8) C-terminal side ofArg

Chemical Cyanogen bromide C-terminal side ofMet

2-Nitro-5-thio-cyanobenzoicacid N-terminal side of

Cyso-Iodosobenzoic acid C-terminal side of

Trp and TyrDilute acid Asp and ProBNPS-skatole Trp


some unfolding of the protein, it is often necessary to reduceand alkylate the disulfide bonds prior to digestion.

Digestion with trypsin can introduce ambiguities in thetryptic map due to side reactions occurring during the diges-tion reaction, such as nonspecific cleavage, deamidation,disulfide isomerization, oxidation of methionine residues, orformation of pyroglutamic groups created from the deami-dation of glutamine at the N-terminal side of a peptide.Furthermore, peaks may be produced by autohydrolysis oftrypsin. Their intensities depend on the ratio of trypsin toprotein. To avoid autohydrolysis, solutions of proteases maybe prepared at a pH that is not optimal (e.g., at pH 5 fortrypsin), which would mean that the enzyme would notbecome active until diluted with the digest buffer.3.4. Establishment of Optimal Digestion Conditions

Factors that affect the completeness and effectiveness ofdigestion of proteins are those that could affect any chemicalor enzymatic reactions.

(i) pH: The pH of the digestion mixture is empiricallydetermined to ensure the optimal performance of the givencleavage agent. For example, when using cyanogen bromideas a cleavage agent, a highly acidic environment (e.g., pH 2,formic acid) is necessary; however, when using trypsin as acleavage agent, a slightly alkaline environment (pH 8) is op-timal. As a general rule, the pH of the reaction milieu shouldnot alter the chemical integrity of the protein during thedigestion and should not change during the course of the

fragmentation reaction.(ii) Temperature: A temperature between 259C and

379C is adequate for most digestions. The temperature usedis intended to minimize chemical side reactions. The type ofprotein under test will dictate the temperature of the reactionmilieu, because some proteins are more susceptible todenaturation as the temperature of the reaction increases.For example, digestion of recombinant bovine somatropin isconducted at 49C, because at higher temperatures it will pre-cipitate during digestion.

(iii) Time: If sufficient sample is available, a timecourse study is considered in order to determine the opti-mum time to obtain a reproducible map and avoid incom-plete digestion. Time of digestion varies from 2 to 30 hours.The reaction is stopped by the addition of an acid whichdoes not interfere in the tryptic map or by freezing.

(iv) Amount of Cleavage Agent: Although excessiveamounts of cleavage agent are used to accomplish a reasona-bly rapid digestion time (i.e., 6 to 20 hours), the amount ofcleavage agent is minimized to avoid its contribution to thechromatographic map pattern. A protein to protease ratiobetween 20:1 and 200:1 is generally used. It is recommendedthat the cleavage agent can be added in two or more stages tooptimize cleavage. Nonetheless, the final reaction volumeremains small enough to facilitate the next step in peptidemapping �the separation step. To sort out digestion ar-tifacts that might be interfering with the subsequent analysis,a blank determination is performed, using a digestion con-trol with all the reagents, except the test protein.

4. Chromatographic SeparationMany techniques are used to separate peptides for map-

ping. The selection of a technique depends on the proteinbeing mapped. Techniques that have been successfully usedfor separation of peptides are shown in Table 2. In this sec-tion, a most widely used reverse-phase High PerformanceLiquid Chromatographic (RP-HPLC) method is described asone of the procedures of chromatographic separation.

The purity of solvents and mobile phases is a critical fac-tor in HPLC separation. HPLC-grade solvents and waterthat are commercially available are recommended for RP-HPLC. Dissolved gases present a problem in gradient sys-tems where the solubility of the gas in a solvent may be lessin a mixture than in a single solvent. Vacuum degassing andagitation by sonication are often used as useful degassingprocedures. When solid particles in the solvents are drawninto the HPLC system, they can damage the sealing of pumpvalves or clog the top of the chromatographic column. Bothpre- and post-pump filtration is also recommended.4.1. Chromatographic Column

The selection of a chromatographic column is empiricallydetermined for each protein. Columns with 100 Å or 300 Åpore size with silica support can give optimal separation. Forsmaller peptides, octylsilane chemically bonded to totallyporous silica articles, 3 to 10 mm in diameter (L7) and oc-tadecylsilane chemically bonded to porous silica or ceramicmicro-particles, 3 to 10 mm in diameter (L1) column pack-ings are more efficient than the butyl silane chemically bond-ed to totally porous silica particles, 5 to 10 mm in diameter(L26) packing.4.2. Solvent

The most commonly used solvent is water with acetonitrileas the organic modifier to which less than 0.1z trifluoroa-

2193

Table 2 Techniques Used for the Separation of Peptides

Reverse-Phase High Performance Liquid Chromatography(RP-HPLC)

Ion-Exchange Chromatography (IEC)Hydrophobic Interaction Chromatography (HIC)Polyacrylamide Gel Electrophoresis (PAGE), nondenaturat-

ingSDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)Capillary Electrophoresis (CE)Paper Chromatography-High Voltage (PCHV)High-Voltage Paper Electrophoresis (HVPE)


cetic acid is added. If necessary, add isopropyl alcohol or n-propyl alcohol to solubilize the digest components, providedthat the addition does not unduly increase the viscosity ofthe components.4.3. Mobile Phase

Buffered mobile phases containing phosphate are used toprovide some flexibility in the selection of pH conditions,since shifts of pH in the 3.0 to 5.0 range enhance the separa-tion of peptides containing acidic residues (e.g., glutamicand aspartic acids). Sodium or potassium phosphates, am-monium acetate, phosphoric acid, and a pH between 2 and 7(or higher for polymer-based supports) have also been usedwith acetonitrile gradients. Acetonitrile containingtrifluoroacetic acid is used quite often.4.4. Gradient Selection

Gradients can be linear, nonlinear, or include step func-tions. A shallow gradient is recommended in order to sepa-rate complex mixtures. Gradients are optimized to provideclear resolution of one or two peaks that will become ``mar-ker'' peaks for the test.4.5. Isocratic Selection

Isocratic HPLC systems using a single mobile phase areused on the basis of their convenience of use and improveddetector responses. Optimal composition of a mobile phaseto obtain clear resolution of each peak is sometimes difficultto establish. Mobile phases for which slight changes in com-ponent ratios or in pH significantly affect retention times ofpeaks in peptide maps should not be used in isocratic HPLCsystems.4.6. Other Parameters

Temperature control of the column is usually necessary toachieve good reproducibility. The flow rates for the mobilephases range from 0.1 to 2.0 mL per minute, and the detec-tion of peptides is performed with a UV detector at 200 to230 nm. Other methods of detection have been used (e.g.,postcolumn derivatization), but they are not as robust orversatile as UV detection.4.7 Validation

This section provides an experimental means for measur-ing the overall performance of the test method. The accep-tance criteria for system suitability depend on the identifica-tion of critical test parameters that affect data interpretationand acceptance. These critical parameters are also criteriathat monitor peptide digestion and peptide analysis. An indi-cator that the desired digestion endpoint was achieved is bythe comparison with a reference standard or referencematerial, which is treated exactly as the article under test.The use of a reference standard or reference material in

parallel with the protein under test is critical in the develop-ment and establishment of system suitability limits. In addi-tion a specimen chromatogram should be included with thereference standard or reference material for additional com-parison purposes. Other indicators may include visual in-spection of protein or peptide solubility, the absence of in-tact protein, or measurement of responses of a digestion-dependent peptide. The critical system suitability parametersfor peptide analysis will depend on the particular mode ofpeptide separation and detection and on the data analysisrequirements.

When peptide mapping is used as an identification test,the system suitability requirements for the identified peptidescovers selectivity and precision. In this case, as well as whenidentification of variant protein is done, the identification ofthe primary structure of the peptide fragments in the peptidemap provides both a verification of the known primarystructure and the identification of protein variants by com-parison with the peptide map of the reference standard/reference material for the specified protein. The use of adigested reference standard or reference material for a givenprotein in the determination of peptide resolution is themethod of choice. For an analysis of a variant protein, acharacterized mixture of a variant and a reference standardor reference material can be used, especially if the variantpeptide is located in a less-resolved region of the map. Theindex of pattern consistency can be simply the number ofmajor peptides detected. Peptide pattern consistency can bebest defined by the resolution of peptide peaks. Chromato-graphic parameters �such as peak-to-peak resolution, maxi-mum peak width, peak area, peak tailing factors, andcolumn efficiency� may be used to define peptide resolu-tion. Depending on the protein under test and the method ofseparation used, single peptide or multiple peptide resolutionrequirements may be necessary.

The replicate analysis of the digest of the reference stand-ard or reference material for the protein under test yieldsmeasures of precision and quantitative recovery. Recoveryof the identified peptides is generally ascertained by the useof internal or external peptide standards. The precision is ex-pressed as the relative standard deviation (RSD). Differencesin the recovery and precision of the identified peptides areexpected; therefore, the system suitability limits will have tobe established for both the recovery and the precision of theidentified peptides. These limits are unique for a given pro-tein and will be specified in the individual monograph.

Visual comparison of the relative retention times, the peakresponses (the peak area or the peak height), the number ofpeaks, and the overall elution pattern is completed initially.It is then complemented and supported by mathematicalanalysis of the peak response ratios and by the chromato-graphic profile of a 1:1 (v/v) mixture of sample and refer-ence standard or reference material digest. If all peaks in thesample digest and in the reference standard or reference ma-terial digest have the same relative retention times and peaksresponse ratios, then the identity of the sample under test isconfirmed.

If peaks that initially eluted with significantly differentrelative retention times are then observed as single peaks inthe 1:1 mixture, the initial difference would be an indicationof system variability. However, if separate peaks are ob-served in the 1:1 mixture, this would be evidence of thenonequivalence of the peptides in each peak. If a peak in the


1:1 mixture is significantly broader than the correspondingpeak in the sample and reference standard or reference mate-rial digest, it may indicate the presence of different peptides.The use of computer-aided pattern recognition software forthe analysis of peptide mapping data has been proposed andapplied, but issues related to the validation of the computersoftware preclude its use in a compendial test in the nearfuture. Other automated approaches have been used thatemploy mathematical formulas, models, and pattern recog-nition. Such approaches are, for example, the automatedidentification of compounds by IR spectroscopy and theapplication of diode-array UV spectral analysis for identifi-cation of peptides. These methods have limitations due toinadequate resolutions, co-elution of fragments, or absolutepeak response differences between reference standard orreference material and sample fragments.

The numerical comparison of the retention times and peakareas or peak heights can be done for a selected group ofrelevant peaks that have been correctly identified in thepeptide maps. Peak areas can be calculated using one peakshowing relatively small variation as an internal reference,keeping in mind that peak area integration is sensitive tobaseline variation and likely to introduce error in the analy-sis. Alternatively, the percentage of each peptide peak heightrelative to the sum of all peak heights can be calculated forthe sample under test. The percentage is then compared tothat of the corresponding peak of the reference standard/reference material. The possibility of auto-hydrolysis oftrypsin is monitored by producing a blank peptide map, thatis, the peptide map obtained when a blank solution is treatedwith trypsin.

The minimum requirement for the qualification of peptidemapping is an approved test procedure that includes systemsuitability as a test control. In general, early in the regulatoryprocess, qualification of peptide mapping for a protein issufficient. As the regulatory approval process for the proteinprogresses, additional qualifications of the test can include apartial validation of the analytical procedure to provideassurance that the method will perform as intended in thedevelopment of a peptide map for the specified protein.

5. Analysis and Identification of PeptidesThis section gives guidance on the use of peptide mapping

during development in support of regulatory applications.The use of a peptide map as a qualitative tool does not re-

quire the complete characterization of the individual peptidepeaks. However, validation of peptide mapping in supportof regulatory applications requires rigorous characterizationof each of the individual peaks in the peptide map. Methodsto characterize peaks range from N-terminal sequencing ofeach peak followed by amino acid analysis to the use of massspectroscopy (MS).

For characterization purposes, when N-terminal sequenc-ing and amino acids analysis are used, the analytical separa-tion is scaled up. Since scale-up might affect the resolutionof peptide peaks, it is necessary, using empirical data, toassure that there is no loss of resolution due to scale-up.Eluates corresponding to specific peptide peaks are collect-ed, vacuum-concentrated, and chromatographed again, ifnecessary. Amino acid analysis of fragments may be limitedby the peptide size. If the N-terminus is blocked, it mayneed to be cleared before sequencing. C-terminal sequencingof proteins in combination with carboxypeptidase digestion

and MALDI-TOF MS can also be used for characterizationpurposes.

The use of MS for characterization of peptide fragments isby direct infusion of isolated peptides or by the use of on-line LC-MS for structure analysis. In general, it includeselectrospray and MALDI-TOF MS analyzer as well as fastatom bombardment (FAB). Tandem MS has also been usedto sequence a modified protein and to determine the type ofamino acid modification that has occurred. The comparisonof mass spectra of the digests before and after reduction pro-vides a method to assign the disulfide bonds to the varioussulfhydryl-containing peptides.

If regions of the primary structure are not clearly demon-strated by the peptide map, it might be necessary to developa secondary peptide map. The goal of a validated method ofcharacterization of a protein through peptide mapping is toreconcile and account for at least 95z of the theoreticalcomposition of the protein structure.

Qualification of Animals as Originof Animal-derived Medicinal

Products provided in the GeneralNotices of Japanese Pharmacopoeia

and Other StandardsIntroduction

The Official Gazette issued on March 29, 2002 announcedthat General Notices of the Japanese Pharmacopoeia andother standards were amended to add a provision that``When a drug product or a drug substance which is used tomanufacture a drug product, is manufactured from a rawmaterial of animal origin, the animal in question should bein principle a healthy subject, if not otherwise provided.''.

The Notice Iyaku-hatsu No. 0329001, which was issued onthe same date, provided that ``Healthy subject herein provid-ed is the animal which does not cause any disease or any in-fection to human being at an appropriate production processand use of the drug product, and as for the oral or externaldrug for example, the animal, as its raw material of animalorigin, should be confirmed at this stage to meet the FoodStandard. It has to be noted that this standard of healthysubject has to be revised timely taking into account the up-to-date information with respect to the amphixenosis infec-tions common between human beings and animals.''.

This General Information describes safety assuranceagainst infection associated with the use of drugs, which aremanufactured from raw materials of animal origin, to followup the Notice as mentioned above.

1. Basic conceptWhen drugs derived from raw materials of animal origin

including human are used, it is important to take into ac-count any possibility that communicable disease agents suchas virus may cause infectious disease or any possible hazardsto patients. In such case, it goes without saying that the pri-mary subject that has to be considered is the absence of anyinfectious agents such as virus in the raw materials of animalorigin including human as the source of the drug. More im-portant points are whether the drugs derived from such raw


materials are free of such infectious agents and whetherthere is any possibility of transmission of infectious agentswhen the drugs are administered to patient. The eligibility ofanimals including human, as the source of raw materials ofdrugs, in other words ``the subject which is free from anydisease or transmission of infectious agents that is infectiousto human being at an appropriate production process anduse of the drug product'' is that ``The drug should beentirely free from any risk of infections by means of wholeprocedures which include evaluation of appropriateness ofthe animals including human as the source of their raw mate-rials, establishment of appropriate production processes andtheir appropriate control, and strict adherence to the clinicalindications of the final product.''

2. Animals including human as the source of raw materialsof drugs

What is the most clear and appropriate preventive meas-ures against infection to human being due to administrationof drugs which are derived from animals including human isto assure the absence of any infectious agents such as virus inits raw materials or an appropriate critical raw material byeach of the followings: (1) the use of raw materials ofhealthy animal origin, which are proved to be free fromcommunicable disease agents to human, or (2) the use of ap-propriate critical raw materials (e.g., cell substrate, bloodplasma, pooled urine after some treatments) for drugproduction, which are proved to be free from communicabledisease agents after certain appropriate processing on rawmaterials of animal origin.

As for raw materials of drugs of human origin, cell, tissue,blood, placenta, urine, etc. are used. Whenever it issufficient and possible each donor, as the origin of such rawmaterials, should be asked his (her) health condition andundergoes his (her) medical examination at this stage, so thatthe appropriateness as a donor can be confirmed from thestandpoint of safety concerning communicable diseaseagents such as virus.

For example, ``Basic concept on handling and use of adrug product, etc. which is derived from cell/tissue''(Attachment 1 of the Notice Iyaku-Hatsu No. 1314 datedDecember 26, 2000) issued by the Director-General of theMedicinal Safety Bureau, Ministry of Health and Welfare,states that since the cell/tissue supplied by a human donorcomes to be applied to patients without processing throughany sufficient inactivation or removal of communicable dis-ease agents, the selection and qualification criteria on suchdonor has to be established. These criteria are to be com-posed with the respect to the check items on the case historyand the physical conditions as well as the test items on thevarious transmission of infectious agents through cell/tissue,and that the appropriateness of these criteria has to be clari-fied. Hepatitis Type-B (HBV), Hepatitis Type-C (HCV),Human Immune Deficiency Viral infections (HIV), AdultT-Cell Leukemia and Parvovirus B19 Infections should bedenied through the interview to the donor and the tests(serologic test, nucleic-acid amplification test, etc.). Further,if necessary, Cytomegalovirus infection and EB Virus infec-tion should be denied by tests. ``Infections caused by bacter-ia such as Treponema pallidum, Chlamydia, Gonococci,Tubercule bacillus, etc.'', ``septicemia and its suspiciouscase'', ``vicious tumor'', ``serious metabolic or endocrine-related disorders'', ``collagenosis and haematological disord-

er'', ``hepatic disease'' and ``dementia (transmissible spon-giform encephalopathies and its suspicious case)'' should bechecked on the case history or by the interview, etc. and theexperience of being transfused or/and transplanted shouldbe checked to confirm eligibility as a donor. The most ap-propriate check items and test methods then available are tobe used, which need to be reconsidered at appropriate timingtaking into account the updated knowledge and the progressof the science and the technologies. At screening of a donor,reexaminations has to be made at appropriate timing usingthe eligible check items and the test methods taking intoaccount the window period (Initial period after infection,in which antibody against bacteria, fungi or virus is notdetected.)

In the case of plasma derivatives produced from the do-nated blood in Japan, the donor should be checked by meansof self-assessed report about health conditions, and a sero-logic check and a nucleic acid amplification test (NAT) onmini pooled plasma should be performed at the stage of do-nated blood. Further, the plasma material (i.e., critical rawmaterial ) for fractionation should be stored 4 months inminimum so that the arrangement could be taken based onthe information available after collection of the blood andthe blood infusion to exclude the possibility of using anycritical raw material which might cause infection to patients.

On the other hand, as for the materials such as urinewhich are taken from the unspecified number of the donorsand come to be critical raw materials for drug productionafter some treatments, it is unrealistic and not practical toconduct the tests of virus infection, etc. on the individualdonor. Consequently, appropriate tests such as virus test hasto be performed on such critical raw materials for drugproduction.

In the case of the animals besides human, the wild onesshould be excluded. Only the animals, which are raisedunder well sanitarily controlled conditions taken to preventbacterial contamination or under the effective bacterial pol-lution monitoring systems, have to be used, and it is recom-mended that the animals from a colony appropriately con-trolled under specific pathogen-free (SPF) environment areto be used as far as possible. Further, for the animals regu-lated under the Food Standard, only the animals that metthis standard should be used. It should be confirmed by ap-propriate tests that the animals were free from pathogen, ifnecessary.

The concrete measures to avoid transmittance or spread ofinfectivity of prion, which is considered to be the pathogenof transmissible spongiform encephalopathies (TSEs), as faras possible are the followings: avoidance of use ofanimals, which are raised in the areas where high incidenceor high risk of TSEs (Scrapie in sheep and goat, bovinespongiform encephalopathies (BSE) in cattle, chronic wast-ing disease (CWD) in deer, new type of Creutzfeldt-Jacob-Disease (CJD) in human, etc.) is reported, and humans, whohave stayed long time (more than 6 months) in such areas, asraw materials or related substances of drugs; avoidance ofuse of any substances that are derived from the individual in-fected with scrapie, BSE, CJD, etc.; avoidance of using amaterial derived from organ, tissue and cell, etc. of high riskof TSEs; and taking appropriate measures basing on theinformation collected, which includes incidence of TSEs, theresults of epidemiological investigation and the experimentalresearch on prion, and incidence of tardive infection on


donors after collecting raw materials, etc.

3. Human or animal cells which are used as critical rawmaterials for drug production

Cell substrates derived from humans or animals are usedfor drug production. In such case, it is desirable that thehumans or the animals, which are the origins of the cell sub-strates, are healthy subjects. However, it is considered prac-tical that viral safety of the drugs derived from the cell sub-strates are evaluated on the cells, which are so called criticalraw materials for production of such drugs. In such case, thesafety should be confirmed through the test and analysis onestablished cell bank thoroughly with respect to virus etc., asfar as possible. The items and the methods of the tests thathave been followed in this case are described in detail in theNotice of Japanese version on the internationally acceptedICH Guideline entitled ``Viral safety evaluation ofbiotechnology products derived from cell lines of human oranimal origin'' (Iyakushin No. 329 issued on February 22,2000 by Director, Evaluation and Licensing Division, Phar-maceutical and Medical Safety Bureau, Ministry of Healthand Welfare). In the meantime, it is important how to han-dle the cell in case that any virus has been detected under thecell level tests. This Notice describes how to cope with thissituation as follows: ``It is recognised that some cell linesused for the manufacture of product will contain en-dogenous retroviruses, other viruses or viral sequences. Insuch circumstances, the action plan recommended for manu-facturer is described in Section V (Rationale and action planfor viral clearance studies and virus tests on purified bulk) ofthe Notice. The acceptability of cell lines containing virusesother than endogenous retroviruses will be considered on anindividual basis by the regulatory authorities, by taking intoaccount a risk/benefit analysis based on the benefit of theproduct and its intended clinical use, the nature of the con-taminating viruses, their potential for infecting humans orfor causing disease in humans, the purification process forthe product (e.g., viral clearance evaluation data), and theextent of the virus tests conducted on the purified bulk.'' Forexample, it is well known that Type A-, R- and C-endogenous particles like retrovirus are observed in the cellsof the rodents used most often for drug production. It is alsoknown that they are not infectious to human and is not dan-gerous, and CHO cells are generally used for drug produc-tion. The established cell lines (e.g., NAMALWA Cell,BALL-1 Cell, etc.) derived from cancer patients are some-times used, but through the thorough virus tests, etc., theirsafety are confirmed. The established cell lines are assumedto be safer than the primary cultured cells which are hard toconduct the thorough virus test.

4. Establishment and control of appropriate productionprocess and adherence to the clinical indication of finalproduct for safety assurance

Safety assurance against potential infections at only thelevel of animals that are source of raw materials of drugs islimited. Further, ``health of animal'' can not be definedunivocally, and the various factors have to be taken intoaccount. The final goal of this subject is to protect humanfrom any infectious disease caused by drugs. Achieving thisgoal, the establishment and control of appropriate produc-tion processes of each drug and the adherence to the clinicalindications of the final product are important.

As mentioned above, the rodent cells used most often for

the production of the drugs are known to have endogenousretrovirus-like particles sometimes. The reason why suchcells can be used for the production of the drugs is that mul-tiple measures are applied for safety in the purificationstages which include appropriate inactivation or removalprocesses. There are cases in which the production procedureinvolves intentional use of a virus or a microorganism. Inthis case, relevant measures capable of removing or inac-tivating of such virus or microorganism are appropriatelyincorporated in the purification process, so that the risk ofinfection to human can be fully denied and its safety can beassured when it is used as a drug. Further, even in the casethat it is difficult to clarify the risk of contamination of theinfectious agents or that the raw material are contaminatedby viruses etc., the raw material in question may be used forthe production of drugs so long as appropriate inactivationor removal processes are introduced, their effectiveness canbe confirmed and the safety can be assured by appropriatecontrol of the manufacturing processes under GMP, etc.

5. ConclusionThe qualification of animals including human, as the

source of raw materials of drugs, in other words ``the subjectwhich does not cause any infectious diseases to human beingat an appropriate production process and use of the drugproduct'' is that ``the drug has to be entirely free from anyrisk of infections by means of whole procedures which in-clude evaluation of appropriateness of the animal includinghuman as the source of their raw materials, establishment ofappropriate production processes and their appropriatecontrol, and strict adherence to the clinical indication of thefinal product.''

To cope with this subject, the advanced scientific meas-ures, which actually reflect the updated knowledge andprogress of the science and the technology about infectiousdiseases in human and infection of animal origin, have to betaken into account timely.

SDS-Polyacrylamide GelElectrophoresis

This test is harmonized with the European Pharmacopoeiaand the U.S. Pharmacopeia.

The SDS-Polyacrylamide Gel Electrophoresis is used forthe characterization of proteins in biotechnological and bio-logical products and for control of purity and quantitativedeterminations.

This technique is a suitable analytical method with whichto identify and to assess the homogeneity of proteins inbiotechnological and biological products. The method is alsoroutinely used for the estimation of protein subunit molecu-lar masses and for determining the subunit compositions ofpurified proteins.

Ready-to-use gels and reagents are widely available on themarket and can be used instead of those described in thistext, provided that they give equivalent results and that theymeet the validity requirements given below under Validationof the Test.

1. Characteristics of Polyacrylamide GelsThe sieving properties of polyacrylamide gels are afforded


by the three-dimensional network of fibers and pores whichis formed as the bifunctional bisacrylamide cross-linksadjacent polyacrylamide chains. Polymerization is catalyzedby a free radical-generating system composed of ammoniumpersulfate and N,N,N?,N?-tetramethylethylenediamine(TEMED).

As the acrylamide concentration of a gel increases, itseffective pore size decreases. The effective pore size of a gelis operationally defined by its sieving properties; that is, bythe resistance it imparts to the migration of macromolecules.There are limits on the acrylamide concentration that can beused. At high acrylamide concentrations, gels break muchmore easily and are difficult to handle. As the pore size of agel decreases, the migration rate of a protein through the geldecreases. By adjusting the pore size of a gel, throughmanipulating the acrylamide concentration, the resolution ofthe method can be optimized for a given protein product.Thus, the physical characteristics of a given gel are deter-mined by the relative concentrations of acrylamide andbisacrylamide, used in its preparation.

In addition to the composition of the gel, the state of theprotein is an important determinant of the electrophoreticmobility. In the case of proteins, the electrophoretic mobilityis dependent on the pK values of the charged groups and thesize of the molecule. It is also influenced by the type, con-centration and pH of the buffer, the temperature and thefield strength, as well as by the nature of the support mate-rial.

2. Polyacrylamide Gel Electrophoresis under DenaturingConditions

The method cited as an example is limited to the analysisof monomeric polypeptides with a mass range of 14,000 to100,000 daltons. It is possible to extend this mass range byvarious techniques (e.g., by using gradient gels, particularbuffer systems, etc.), but those techniques are not discussedin this chapter.

Analysis by electrophoresis on sodium dodecyl sulfate(SDS) polyacrylamide gel (SDS-Polyacrylamide Gel Elec-trophoresis) under denaturing conditions is the most com-mon mode of electrophoresis used in assessing the quality ofproteins in biotechnological and biological products, andwill be the focus of the example described here. Typically,analytical electrophoresis of proteins is carried out in poly-acrylamide gels under conditions that ensure dissociation ofthe proteins into their individual polypeptide subunits andthat minimize aggregation. Most commonly, the stronglyanionic detergent SDS is used in combination with heat todissociate the proteins before they are loaded on the gel. Thedenatured polypeptides bind to SDS, become negativelycharged and exhibit a consistent charge-to-mass ratio regard-less of protein type. Because the amount of SDS bound isalmost always proportional to the molecular mass of thepolypeptide and is independent of its amino acid sequence,SDS-polypeptide complexes migrate through polyacrylamidegels with mobilities that are dependent on the size of thepolypeptides.

The electrophoretic mobilities of the resultant SDS-polypeptide complexes all assume the same functionalrelationship to their molecular masses. Migration of SDS-complexes occurs toward the anode in a predictable manner,with low-molecular-mass complexes migrating faster thanlarger ones. The molecular mass of a protein can therefore

be estimated from its relative mobility calibrated in SDS-Polyacrylamide Gel Electrophoresis and the occurrence of asingle band in such a gel is a criterion of purity.

However, modifications to the polypeptide backbone,such as N- or O-1inked glycosylation, have a significant im-pact on the apparent molecular mass of a protein, since SDSdoes not bind to a carbohydrate moiety in a manner similarto a polypeptide. Thus, a consistent charge-to-mass ratio isnot maintained. The apparent molecular masses of proteinsthat have undergone post-translational modifications do nottruly reflect the masses of the polypeptides.2.1. Reducing conditions

Polypeptide subunits and three-dimensional structure ofproteins are often fixed, at least in part, by the presence ofdisulfide bonds. A goal of SDS-Polyacrylamide Gel Elec-trophoresis under reducing conditions is to disrupt this struc-ture by reducing the disulfide bonds. Complete denaturationand dissociation of proteins by treatment with 2-mercap-toethanol or dithiothreitol (DTT) will result in unfolding ofthe polypeptide backbone and subsequent complexation withSDS. Under these conditions, the molecular masses of thepolypeptide subunits can be calculated by linear regression inthe presence of suitable molecular-mass standards.2.2. Non-reducing conditions

For some analyses, complete dissociation of the protein ofinterest into subunit peptides is not desirable. In the absenceof treatment with reducing agents such as 2-mercaptoethanolor DTT, disulfide covalent bonds remain intact, preservingthe oligomeric form of the protein. Oligomeric SDS-proteincomplexes migrate more slowly than their SDS-polypeptidesubunits. In addition, non-reduced proteins may not be com-pletely saturated with SDS and, hence, may not bind thedetergent in the expected mass ratio. This makes molecular-mass determinations of these molecules by SDS-Polyacrylamide Gel Electrophoresis less straightforwardthan analyses of fully denatured polypeptides, since it isnecessary that both standards and unknown proteins be insimilar configurations for valid comparisons. However, thestaining of a single band in such a gel is a criterion of purity.

3. Characteristics of Discontinuous Buffer System GelElectrophoresis

The most widely used electrophoretic method for theanalysis of complex mixtures of proteins involves the use ofa discontinuous buffer system consisting of two contiguous,but distinct gels: a resolving (lower) gel and a stacking(upper) gel. The two gels are cast with different porosities,pH, and ionic strengths. In addition, different mobile ionsare used in the gel and electrode buffers. The buffer discon-tinuity acts to concentrate large-volume samples in the stack-ing gel, resulting in improved resolution. When power is ap-plied, a voltage drop develops across the sample solutionwhich drives the proteins into the stacking gel. Glycinateions from the electrode buffer follow the proteins into thestacking gel. A moving boundary region is rapidly formedwith the highly mobile chloride ions in the front and the rela-tively slow glycinate ions in the rear. A localized high-vol-tage gradient is formed between the leading and trailing ionfronts, causing the SDS-protein complex to form into a verythin zone (called the stack) and to migrate between the chlo-ride and glycinate phases. Regardless of the height of the ap-plied sample solution in the wells, all SDS-protein complexescondense within a very narrow range and enter the resolving


gel as a well-defined, thin zone of high protein density. Thelarge-pore stacking gel does not retard the migration of mostproteins and serves mainly as an anticonvective medium. Atthe interface of the stacking and resolving gels, the proteinsexperience a sharp increase in retardation due to the smallerpore size of the resolving gel. Once the proteins are in theresolving gel, their mobility continues to be slowed down bythe molecular sieving effect of the matrix. The glycinateions overtake the proteins, which then move in a spaceof uniform pH formed by the tris(hydroxy-methyl)aminomethane and glycine. Molecular sieving causesthe SDS-polypeptide complexes to separate on the basis oftheir molecular masses.

4. Preparing Vertical Discontinuous Buffer SDS-Polyacrylamide Gels4.1. Assembling of the gel moulding cassette

Clean the two glass plates (size: e.g. 10 cm× 8 cm), thesample comb made of polytetrafluoroethylene, the twospacers and the silicone rubber tubing (diameter, e.g. 0.6mm× 35 cm) with mild detergent and rinse extensively withwater. Dry all the items with a paper towel or tissue. Lubri-cate the spacers and the silicone rubber tubing with non-silicone grease. Apply the spacers along each of the twoshort sides of the glass plate 2 mm away from the edges and2 mm away from the long side corresponding to the bottomof the gel. Begin to lay the silicone rubber tubing on the glassplate by using one spacer as a guide. Carefully twist the sili-cone rubber tubing at the bottom of the spacer and followthe long side of the glass plate. While holding the siliconerubber tubing with one finger along the long side again twistthe tubing and lay it on the second short side of the glassplate, using the spacer as a guide. Place the second glassplate in perfect alignment and hold the mould together byhand pressure. Apply two clamps on each of the two shortsides of the mould. Carefully apply four clamps on the lon-ger side of the gel mould, thus forming the bottom of the gelmould. Verify that the silicone rubber tubing is runningalong the edge of the glass plates and has not been extrudedwhile placing the clamps.4.2. Preparation of the gel

In a discontinuous buffer SDS polyacrylamide gel, it isrecommended to pour the resolving gel, let the gel set, andthen pour the stacking gel, since the compositions of the twogels in acrylamide-bisacrylamide, buffer and pH are differ-ent.4.2.1. Preparation of the resolving gel

In a conical flask, prepare the appropriate volume of solu-tion containing the desired concentration of acrylamide forthe resolving gel, using the values given in Table 1. Mix thecomponents in the order shown. Where appropriate, beforeadding the ammonium persulfate solution and thetetramethylethylenediamine (TEMED), filter the solution ifnecessary under vacuum through a cellulose acetate mem-brane (pore size: 0.45 mm); keep the solution under vacuumby swirling the filtration unit until no more bubbles areformed in the solution. Add appropriate amounts of ammo-nium persulfate solution and TEMED as indicated in Table1, swirl and pour immediately into the gap between the twoglass plates of the mould. Leave sufficient space for thestacking gel (the length of the teeth of the sample comb plus1 cm). Using a pipette, carefully overlay the solution withwater-saturated isobutanol. Leave the gel in a vertical posi-

tion at room temperature to allow polymerization to occur.4.2.2. Preparation of the stacking gel

After polymerization is complete (about 30 minutes), pouroff the isobutanol and wash the top of the gel several timeswith water to remove the isobutanol overlay and any un-polymerized acrylamide. Drain as much fluid as possiblefrom the top of the gel, and then remove any remainingwater with the edge of a paper towel.

In a conical flask, prepare the appropriate volume of solu-tion containing the desired concentration of acrylamide,using the values given in Table 2. Mix the components in theorder shown. Where appropriate, before adding the ammo-nium persulfate solution and the TEMED, filter the solutionif necessary under vacuum through a cellulose acetate mem-brane (pore size: 0.45 mm); keep the solution under vacuumby swirling the filtration unit until no more bubbles areformed in the solution. Add appropriate amounts of ammo-nium persulfate solution and TEMED as indicated in Table2, swirl and pour immediately into the gap between the twoglass plates of the mould directly onto the surface of thepolymerized resolving gel. Immediately insert a clean samplecomb into the stacking gel solution, taking care to avoidtrapping air bubbles. Add more stacking gel solution to fillcompletely the spaces of the sample comb. Leave the gel in avertical position and allow to polymerize at room tempera-ture.4.3. Mounting the gel in the electrophoresis apparatus andelectrophoretic separation

After polymerization is complete (about 30 minutes), re-move the sample comb carefully. Rinse the wells immedi-ately with water or with the running buffer for SDS-Polyacrylamide Gel Electrophoresis to remove any un-polymerized acrylamide. If necessary, straighten the teeth ofthe sample comb of the stacking gel with a blunt hypodermicneedle attached to a syringe. Remove the clamps on oneshort side, carefully pull out the silicone rubber tubing andreplace the clamps. Proceed similarly on the other short side.Remove the silicone rubber tubing from the bottom part ofthe gel. Mount the gel in the electrophoresis apparatus. Addthe electrophoresis buffers to the top and bottom reservoirs.Remove any bubbles that become trapped at the bottom ofthe gel between the glass plates. This is best done with a benthypodermic needle attached to a syringe. Never pre-run thegel before loading solutions, such as samples, since this willdestroy the discontinuity of the buffer systems. Before load-ing solutions, such as samples, carefully rinse the stackinggel wells with the running buffer for SDS-PolyacrylamideGel Electrophoresis. Prepare the test and reference solutionsin the recommended sample buffer and treat as specified inthe individual monograph. Apply the appropriate volume ofeach solution to the stacking gel wells. Start the electropho-resis using suitable operating conditions for the electropho-resis equipment to be used. There are commercially availablegels of different surface area and thickness that are ap-propriate for various types of electrophoresis equipment.Electrophoresis running time and current/voltage may needto be altered depending on the type of apparatus used, inorder to achieve optimum separation. Check that the dyefront is moving into the resolving gel. When the dye is reach-ing the bottom of the gel, stop the electrophoresis. Removethe gel assembly from the apparatus and separate the glassplates. Remove the spacers, cut off and discard the stackinggel and immediately proceed with staining.


5. Detection of Proteins in GelsCoomassie staining is the most common protein staining

method, with a detection level of the order of 1 mg to 10 mgof protein per band. Silver staining is the most sensitivemethod for staining proteins in gels and a band containing l0ng to 100 ng can be detected. All of the steps in gel stainingare done at room temperature with gentle shaking in anyconvenient container. Gloves must be worn when staininggels, since fingerprints will stain.5.1. Coomassie staining

Immerse the gel in a large excess of Coomassie staining TSand allow to stand for at least 1 hour. Remove the stainingsolution.

Destain the gel with a large excess of destaining TS.Change the destaining solution several times, until thestained protein bands are clearly distinguishable on a clearbackground. The more thoroughly the gel is destained, thesmaller is the amount of protein that can be detected by themethod. Destaining can be speeded up by including 2 to 3 gof anion-exchange resin or a small sponge in the destainingTS.

NOTE: the acid-alcohol solutions used in this proceduredo not completely fix proteins in the gel. This can lead tolosses of some low-molecular-mass proteins during the stain-ing and destaining of the gel. Permanent fixation is ob-tainable by allowing the gel to stand in trichloroacetic acidTS for fixing for 1 hour before it is immersed in Coomassiestaining TS.5.2. Silver staining

Immerse the gel in a large excess of fixing TS and allow tostand for 1 hour. Remove the fixing solution, add fresh fix-ing solution and incubate either for at least 1 hour or over-night, if convenient. Discard the fixing solution and washthe gel in water for 1 hour. Soak the gel for 15 minutes in a 1volz glutaraldehyde solution. Wash the gel twice for 15minutes in water. Soak the gel in fresh silver nitrate TS forsilver staining for 15 minutes, in darkness. Wash the gelthree times for 5 minutes in water. Immerse the gel for about1 minute in developer TS until satisfactory staining has beenobtained. Stop the development by incubation in blockingTS for 15 minutes. Rinse the gel with water.

6. Drying of Stained SDS-Polyacrylamide GelsDepending on the staining method used, gels are pretreat-

ed in a slightly different way. For Coomassie staining, afterthe destaining step, allow the gel to stand in a diluted solu-tion of concentrated glycerin (1 in 10) for at least 2 hours(overnight incubation is possible). For silver staining, afterthe final rinse, allow the gel to stand in a diluted solution ofconcentrated glycerin (1 in 50) for 5 minutes.

Immerse two sheets of porous cellulose film in water andincubate for 5 to 10 minutes. Place one of the sheets on adrying frame. Carefully lift the gel and place it on the cellu-lose film. Remove any trapped air bubbles and pour 2 to 3mL of water around the edges of the gel. Place the secondsheet on top and remove any trapped air bubbles. Completethe assembly of the drying frame. Place in an oven or leaveat room temperature until dry.

7. Molecular-Mass DeterminationMolecular masses of proteins are determined by compari-

son of their mobilities with those of several marker proteinsof known molecular mass. Mixtures of proteins with precise-ly known molecular masses blended for uniform staining are

commercially available for calibrating gels. They are ob-tainable in various molecular mass ranges. Concentratedstock solutions of proteins of known molecular mass arediluted in the appropriate sample buffer and loaded on thesame gel as the protein sample to be studied.

Immediately after the gel has been run, the position of thebromophenol blue tracking dye is marked to identify theleading edge of the electrophoretic ion front. This can bedone by cutting notches in the edges of the gel or by insertinga needle soaked in India ink into the gel at the dye front.After staining, measure the migration distances of each pro-tein band (markers and unknowns) from the top of theresolving gel. Divide the migration distance of each proteinby the distance traveled by the tracking dye. The normalizedmigration distances so obtained are called the relative mobil-ities of the proteins (relative to the dye front) and conven-tionally denoted as Rf. Construct a plot of the logarithm ofthe relative molecular masses (Mr) of the protein standardsas a function of the Rf values. Note that the graphs areslightly sigmoid. Unknown molecular masses can be estimat-ed by linear regression analysis or interpolation from thecurves of log Mr against Rf as long as the values obtained forthe unknown samples are positioned along the linear part ofthe graph.

8. Suitability of the Test (Validation)The test is not valid unless the proteins of the molecular

mass marker are distributed along 80z of the length of thegel and over the required separation range (e.g., the rangecovering the product and its dimer or the product and itsrelated impurities) the separation obtained for the relevantprotein bands shows a linear relationship between thelogarithm of the molecular mass and the Rf as described in7. Additional requirements with respect to the solution undertest may be specified in individual monographs.

9. Quantification of ImpuritiesWhere the impurity limit is specified in the individual

monograph, a reference solution corresponding to that levelof impurity should be prepared by diluting the test solution.For example, where the limit is 5z, a reference solutionwould be a 1:20 dilution of the test solution. No impurity(any band other than the main band) in the electrophoreto-gram obtained with the test solution may be more intensethan the main band obtained with the reference solution.

Under validated conditions, impurities may be quantifiedby normalization to the main band, using an integrating den-sitometer. In this case, the responses must be validated forlinearity.

10. Test solutions:(i) Coomassie staining TS: Dissolve 125 mg of coomassiebrilliant blue R-250 in 100 mL of a mixture of water, metha-nol and acetic acid (100) (5:4:1), and filter.(ii) Developer TS: Dissolve 2 g of citric acid monohydratein water to make 100 mL. To 2.5 mL of this solution add0.27 mL of formaldehyde solution and water to make 500mL.(iii) Fixing TS: To 250 mL of methanol add 0.27 mL offormaldehyde solution and water to make 500 mL.(iv) Silver nitrate TS for silver staining: To 40 mL of so-dium hydroxide TS add 3 mL of ammonia solution (28),then add dropwise 8 mL of a solution of silver nitrate (1 in 5)while stirring, and add water to make 200 mL.

2200

Table 1 Preparation of resolving gel

Solution componentsComponent volumes (mL) per gel mould volume of

5 mL 10 mL 15 mL 20 mL 25 mL 30 mL 40 mL 50 mL

6z AcrylamideWater 2.6 5.3 7.9 10.6 13.2 15.9 21.2 26.5Acrylamide solution(1) 1.0 2.0 3.0 4.0 5.0 6.0 8.0 10.01.5 mol/L Tris solution (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5100 g/L SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5100 g/L APS(4) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5TEMED(5) 0.004 0.008 0.012 0.016 0.02 0.024 0.032 0.04

8z AcrylamideWater 2.3 4.6 6.9 9.3 11.5 13.9 18.5 23.2Acrylamide solution(1) 1.3 2.7 4.0 5.3 6.7 8.0 10.7 13.31.5 mol/L Tris solution (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5100 g/L SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5100 gL APS(4) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5TEMED(5) 0.003 0.006 0.009 0.012 0.015 0.018 0.024 0.03





(1) Acrylamide solution: 30z acrylamide/bisacrylamide (29:1) solution(2) 1.5 mol/L Tris solution (pH 8.8): 1.5 mol/L tris-hydrochloride buffer solution, pH 8.8(3) 100 g/L SDS: 100 g/L solution of sodium dodecyl sulfate(4) 100 g/L APS: 100 g/L solution of ammonium persulfate. Ammonium persulfate provides the free radicals that drive

polymerization of acrylamide and bisacrylamide. Since ammonium persulfate solution decomposes slowly, fresh solu-tions must be prepared before use.

(5) TEMED: N,N,N?,N?-tetramethylethylenediamine


(v) Destaining TS: A mixture of water, methanol andacetic acid (100) (5:4:1).(vi) Blocking TS: To 10 mL of acetic acid (100) add waterto make 100 mL.(vii) Trichloroacetic acid TS for fixing: Dissolve 10 g of

trichloroacetic acid in a mixture of water and methanol (5:4)to make 100 mL.

2201

Table 2 Preparation of stacking gel

Solution componentsComponent volumes (mL) per gel mould volume of

1 mL 2 mL 3 mL 4 mL 5 mL 6 mL 8 mL 10 mL

Water 0.68 1.4 2.1 2.7 3.4 4.1 5.5 6.8Acrylamide solution(1) 0.17 0.33 0.5 0.67 0.83 1.0 1.3 1.71.0 mol/L Tris solution (pH 6.8)(2) 0.13 0.25 0.38 0.5 0.63 0.75 1.0 1.25100 g/L SDS(3) 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1100 g/L APS(4) 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1TEMED(5) 0.001 0.002 0.003 0.004 0.005 0.006 0.008 0.01

(1) Acrylamide solution: 30z acrylamide/bisacrylamide (29:1) solution(2) 1.0 mol/L Tris solution (pH 6.8): 1 mol/L tris-hydrochloride buffer solution, pH 6.8(3) 100 g/L SDS: 100 g/L solution of sodium dodecyl sulfate(4) 100 g/L APS: 100 g/L solution of ammonium persulfate. Ammonium persulfate provides the free radicals that drive

polymerization of acrylamide and bisacrylamide. Since ammonium persulfate solution decomposes slowly, fresh solu-tions must be prepared before use.

(5) TEMED: N,N,N?,N?-tetramethylethylenediamine


Total Protein AssayThis test is harminized with the European Pharmacopoeia

and the U.S. Pharmacopeia. The parts of the text that arenot harmonized are marked with symbols ( ).

The following procedures are provided as illustrations ofthe determination of total protein content in pharmacopoeialpreparations. Other techniques, such as HPLC, are also ac-ceptable if total protein recovery is demonstrated. Many ofthe total protein assay methods described below can be per-formed successfully using kits from commercial sources.

Note: Where water is required, use distilled water.

Method 1 (UV method)Protein in solution absorbs UV light at a wavelength of

280 nm, due to the presence of aromatic amino acids, mainlytyrosine and tryptophan. This property is the basis of thismethod. Protein determination at 280 nm is mainly a func-tion of the tyrosine and tryptophan content of the protein. Ifthe buffer used to dissolve the protein has a high absorbancerelative to that of water, there is an interfering substance inthe buffer. This interference can be compensated for whenthe spectrophotometer is adjusted to zero buffer absorbance.If the interference results in a large absorbance thatchallenges the limit of sensitivity of the spectrophotometer,the results may be compromised. Furthermore, at low con-centrations protein can be absorbed onto the cuvette, there-by reducing the content in solution. This can be prevented bypreparing samples at higher concentrations or by using anonionic detergent in the preparation.

Note: Keep the Test Solution, the Standard Solution, andthe buffer at the same temperature during testing.Standard Solution Unless otherwise specified in the in-dividual monograph, prepare a solution of the referencestandard or reference material for the protein under test inthe same buffer and at the same concentration as the TestSolution.Test Solution Dissolve a suitable quantity of the proteinunder test in the appropriate buffer to obtain a solution hav-ing a concentration of 0.2 to 2 mg per mL.Procedure Concomitantly determine the absorbances of

the Standard Solution and the Test Solution in quartz cells ata wavelength of 280 nm, with a suitable spectrophotometer,using the buffer as the blank. To obtain accurate results, theresponse should be linear in the range of protein concentra-tions to be assayed.Light-Scattering The accuracy of the UV spectroscopic de-termination of protein can be decreased by the scattering oflight by the test specimen. If the proteins in solution exist asparticles comparable in size to the wavelength of the measur-ing light (250 to 300 nm), scattering of the light beam resultsin an apparent increase in absorbance of the test specimen.To calculate the absorbance at 280 nm due to light-scatter-ing, determine the absorbances of the Test Solution at wave-lengths of 320, 325, 330, 335, 340, 345, and 350 nm. Usingthe linear regression method, plot the log of the observed ab-sorbance versus the log of the wavelength, and determine thestandard curve best fitting the plotted points. From thegraph so obtained, extrapolate the absorbance value due tolight-scattering at 280 nm. Subtract the absorbance fromlight-scattering from the total absorbance at 280 nm toobtain the absorbance value of the protein in solution.Filtration with a filter having a 0.2-mm porosity or clarifica-tion by centrifugation may be performed to reduce the effectof light-scattering, especially if the solution is noticeablyturbid.Calculations Calculate the concentration, CU, of protein inthe test specimen by the formula:

CU＝ CS (AU/AS),

in which CS is the concentration of the Standard Solution;and AU and AS are the corrected absorbances of the TestSolution and the Standard Solution, respectively.

Method 2 (Lowry method)This method, commonly referred to as the Lowry assay, is

based on the reduction by protein of the phosphomolybdic-tungstic mixed acid chromogen in the Folin-Ciocalteu'sphenol reagent, resulting in an absorbance maximum at 750nm. The Folin-Ciocalteu's phenol reagent (Folin's TS) reactsprimarily with tyrosine residues in the protein, which canlead to variation in the response of the assay to differentproteins. Because the method is sensitive to interfering sub-


stances, a procedure for precipitation of the protein from thetest specimen may be used. Where separation of interferingsubstances from the protein in the test specimen is necessary,proceed as directed below for Interfering Substances prior topreparation of the Test Solution. The effect of interferingsubstances can be minimized by dilution provided the con-centration of the protein under test remains sufficient for ac-curate measurement. Variations of the Lowry test that areindicated in national regulatory documents1) can be substit-uted for the method described below.Standard Solutions Unless otherwise specified in the in-dividual monograph, dissolve the reference standard or ref-erence material for the protein under test in the buffer usedto prepare the Test Solution. Dilute portions of this solutionwith the same buffer to obtain not fewer than five StandardSolutions having concentrations between 5 and 100 mg ofprotein per mL, the concentrations being evenly spaced.Test Solution Dissolve a suitable quantity of the proteinunder test in the appropriate buffer to obtain a solution hav-ing a concentration within the range of the concentrations ofthe Standard Solutions. An appropriate buffer will producea pH in the range of 10 to 10.5.Blank Use the buffer used for the Test Solution and theStandard Solutions.Reagents and Solutions�

Copper Sulfate Reagent Dissolve 100 mg of copper (II)sulfate pentahydrate and 200 mg of sodium tartrate dihy-drate in water, dilute with water to 50 mL, and mix. Dissolve10 g of anhydrous sodium carbonate in water to a finalvolume of 50 mL, and mix. Slowly pour the sodium carbon-ate solution into the copper sulfate solution with mixing.Prepare this solution fresh daily.

5z SDS TS Dissolve 5 g of sodium dodecyl sulfate inwater, and dilute with water to 100 mL.

Alkaline Copper Reagent Prepare a mixture of 5z SDSTS, Copper Sulfate Reagent, and Sodium Hydroxide Solu-tion (4 in 125) (2:1:1). This reagent may be stored at roomtemperature for up to 2 weeks.

Diluted Folin's TS Mix 10 mL of Folin's TS with 50 mLof water. Store in an amber bottle, at room temperature.Procedure To 1 mL of each Standard Solution, the TestSolution, and the Blank, add 1 mL of Alkaline Copper Rea-gent, and mix. Allow to stand at room temperature for 10minutes. Add 0.5 mL of the Diluted Folin's TS to each solu-tion, and mix each tube immediately after the addition, andallow to stand at room temperature for 30 minutes. Deter-mine the absorbances of the solutions from the Standard So-lutions and the Test Solution at the wavelength of maximumabsorbance at 750 nm, with a suitable spectrophotometer,using the solution from the Blank to set the instrument tozero.Calculations [Note: The relationship of absorbance to pro-tein concentration is nonlinear; however, if the standardcurve concentration range is sufficiently small, it will ap-proach linearity.] Using the linear regression method, plotthe absorbances of the solutions from the Standard Solu-tions versus the protein concentrations, and determine thestandard curve best fitting the plotted points. From thestandard curve so obtained and the absorbance of the TestSolution, determine the concentration of protein in the TestSolution.Interfering Substances In the following procedure, deoxy-cholate-trichloroacetic acid is added to a test specimen to re-

move interfering substances by precipitation of proteins be-fore testing. This technique also can be used to concentrateproteins from a dilute solution.

Sodium Deoxycholate Reagent Prepare a solution of so-dium deoxycholate in water having a concentration of 150mg in 100 mL.

Trichloroacetic Acid Reagent Prepare a solution oftrichloroacetic acid in water having a concentration of 72 gin 100 mL.Procedure Add 0.1 mL of Sodium Deoxycholate Reagentto 1 mL of a solution of the protein under test. Mix on a vor-tex mixer, and allow to stand at room temperature for 10minutes. Add 0.1 mL of Trichloroacetic Acid Reagent, andmix on a vortex mixer. Centrifuge at 3000× g for 30minutes, decant the liquid, and remove any residual liquidwith a pipet. Redissolve the protein pellet in 1 mL of Alka-line Copper Reagent. Proceed as directed for the Test Solu-tion. [Note: Color development reaches a maximum in 20 to30 minutes during incubation at room temperature, afterwhich there is a gradual loss of color. Most interfering sub-stances cause a lower color yield; however, some detergentscause a slight increase in color. A high salt concentrationmay cause a precipitate to form. Because different proteinspecies may give different color response intensities, thestandard protein and test protein should be the same.]

Method 3 (Bradford method)This method, commonly referred to as the Bradford assay,

is based on the absorption shift from 470 nm to 595 nm ob-served when Coomassie brilliant blue G-250 binds to protein.The Coomassie brilliant blue G-250 binds most readily toarginyl and lysyl residues in the protein, which can lead tovariation in the response of the assay to different proteins.Standard Solutions Unless otherwise specified in the in-dividual monograph, dissolve the reference standard or thereference material for the protein under test in the bufferused to prepare the Test Solution. Dilute portions of this so-lution with the same buffer to obtain not fewer than fiveStandard Solutions having concentrations between 100 mgand 1 mg of protein per mL, the concentrations being evenlyspaced.Test Solution Dissolve a suitable quantity of the proteinunder test in the appropriate buffer to obtain a solution hav-ing a concentration within the range of the concentrations ofthe Standard Solutions.Blank Use the buffer used to prepare the Test Solution andthe Standard Solutions.Coomassie Reagent Dissolve 100 mg of Coomassie brilliantblue G-2502) in 50 mL of ethanol (95). [Note: Not all dyeshave the same brilliant blue G content, and different prod-ucts may give different results.] Add 100 mL of phosphoricacid, dilute with water to 1000 mL, and mix. Filter the solu-tion through filter paper (Whatman No.1 or equivalent), andstore the filtered reagent in an amber bottle at room temper-ature. [Note: Slow precipitation of the dye will occur duringstorage of the reagent. Filter the reagent before use.]Procedure Add 5 mL of the Coomassie Reagent to 100 mLof each Standard Solution, the Test Solution, and the Blank,and mix by inversion. Avoid foaming, which will lead topoor reproducibility. Determine the absorbances of the solu-tions from the Standard Solutions and the Test Solution at595 nm, with a suitable spectrophotometer, using the Blankto set the instrument to zero.


[Note: Do not use quartz (silica) spectrophotometer cells:the dye binds to this material. Because different protein spe-cies may give different color response intensities, the stand-ard protein and test protein should be the same.] There arerelatively few interfering substances, but detergents and am-pholytes in the test specimen should be avoided. Highly alka-line specimens may interfere with the acidic reagent.Calculations [Note: The relationship of absorbance to pro-tein concentration is nonlinear; however, if the standardcurve concentration range is sufficiently small, it will ap-proach linearity.] Using the linear regression method, plotthe absorbances of the solutions from the Standard Solu-tions versus the protein concentrations, and determine thestandard curve best fitting the plotted points. From thestandard curve so obtained and the absorbance of the TestSolution, determine the concentration of protein in the TestSolution.

Method 4 (Bicinchoninic acid method)This method, commonly referred to as the bicinchoninic

acid or BCA assay, is based on reduction of the cupric(Cu2＋) ion to cuprous (Cu＋) ion by protein. The bicincho-ninic acid reagent is used to detect the cuprous ion. Themethod has few interfering substances. When interferingsubstances are present, their effect may be minimized by di-lution, provided that the concentration of the protein undertest remains sufficient for accurate measurement.Standard Solutions Unless otherwise specified in the in-dividual monograph, dissolve the reference standard or thereference material for the protein under test in the bufferused to prepare the Test Solution. Dilute portions of this so-lution with the same buffer to obtain not fewer than fiveStandard Solutions having concentrations between 10 and1200 mg of protein per mL, the concentrations being evenlyspaced.Test Solution Dissolve a suitable quantity of the proteinunder test in the appropriate buffer to obtain a solution hav-ing a concentration within the range of the concentrations ofthe Standard Solutions.Blank Use the buffer used to prepare the Test Solution andthe Standard Solutions.Reagents and Solutions�

BCA Reagent Dissolve about 10 g of bicinchoninic acid,20 g of sodium carbonate monohydrate, 1.6 g of sodiumtartrate dihydrate, 4 g of sodium hydroxide, and 9.5 g ofsodium hydrogen carbonate in water. Adjust, if necessary,with sodium hydroxide or sodium hydrogen carbonate to apH of 11.25. Dilute with water to 1000 mL, and mix.

Copper Sulfate Reagent Dissolve about 2 g of copper (II)sulfate pentahydrate in water to a final volume of 50 mL.

Copper-BCA Reagent Mix 1 mL of Copper Sulfate Rea-gent and 50 mL of BCA Reagent.Procedure Mix 0.1 mL of each Standard Solution, the TestSolution, and the Blank with 2 mL of the Copper-BCA Rea-gent. Incubate the solutions at 379C for 30 minutes, note thetime, and allow to come to room temperature. Within 60minutes following the incubation time, determine the absor-bances of the solutions from the Standard Solutions and theTest Solution in quartz cells at 562 nm, with a suitable spec-trophotometer, using the Blank to set the instrument to zero.After the solutions are cooled to room temperature, thecolor intensity continues to increase gradually. If substancesthat will cause interference in the test are present, proceed as

directed for Interfering Substances under Method 2. Becausedifferent protein species may give different color responseintensities, the standard protein and test protein should bethe same.Calculations [Note: The relationship of absorbance to pro-tein concentration is nonlinear; however, if the standardcurve concentration range is sufficiently small, it will ap-proach linearity.] Using the linear regression method, plotthe absorbances of the solutions from the Standard Solu-tions versus the protein concentrations, and determine thestandard curve best fitting the plotted points. From thestandard curve so obtained and the absorbance of the TestSolution, determine the concentration of protein in the TestSolution.

Method 5 (Biuret method)This method, commonly referred to as the Biuret assay, is

based on the interaction of cupric (Cu2＋ ) ion with protein inan alkaline solution and the resultant development of absor-bance at 545 nm.Standard Solutions Unless otherwise specified in the in-dividual monograph, prepare a solution of Albumin Humanfor which the protein content has been previously deter-mined by nitrogen analysis (using the nitrogen-to-proteinconversion factor of 6.25) or of the reference standard orreference material for the protein under test in sodium chlo-ride solution (9 in 1000). Dilute portions of this solution withsodium chloride solution (9 in 1000) to obtain not fewer thanthree Standard Solutions having concentrations between 0.5and 10 mg per mL, the concentrations being evenly spaced.[Note: Low responses may be observed if the sample undertest has significantly different level of proline than that ofAlbumin Human. A different standard protein may beemployed in such cases.]Test Solution Prepare a solution of the test protein in so-dium chloride solution (9 in 1000) having a concentrationwithin the range of the concentrations of the Standard Solu-tions.Blank Use sodium chloride solution (9 in 1000).Biuret Reagent Dissolve about 3.46 g of copper (II) sulfatepentahydrate in 10 mL of water, with heating if necessary,and allow to cool (Solution A). Dissolve about 34.6 g of so-dium citrate dihydrate and 20.0 g of anhydrous sodium car-bonate in 80 mL of water, with heating if necessary, andallow to cool (Solution B). Mix Solutions A and B, anddilute with water to 200 mL. This Biuret Reagent is stable atroom temperature for 6 months. Do not use the reagent if itdevelops turbidity or contains any precipitate.Procedure To one volume of the Standard Solutions and asolution of the Test Solution add an equal volume of sodiumhydroxide solution (6 in 100), and mix. Immediately add avolume of Biuret Reagent equivalent to 0.4 volume of theTest Solution, and mix. Allow to stand at a temperature be-tween 159C and 259C for not less than 15 minutes. Within90 minutes after the addition of the Biuret Reagent, deter-mine the absorbances of the Standard Solutions and the so-lution from the Test Solution at the wavelength of maximumabsorbance at 545 nm, with a suitable spectrophotometer,using the Blank to set the instrument to zero. [Note: Any so-lution that develops turbidity or a precipitate is not accepta-ble for calculation of protein concentration.]Calculations Using the least-squares linear regressionmethod, plot the absorbances of the Standard Solutions ver-


sus the protein concentrations, and determine the standardcurve best fitting the plotted points, and calculate the corre-lation coefficient for the line. [Note: Within the given rangeof the standards, the relationship of absorbance to proteinconcentration is approximately linear.] A suitable system isone that yields a line having a correlation coefficient of notless than 0.99. From the standard curve and the absorbanceof the Test Solution, determine the concentration of proteinin the test specimen, making any necessary correction.Interfering Substances To minimize the effect of interfer-ing substances, the protein can be precipitated from the ini-tial test specimen as follows. Add 0.1 volume of 50ztrichloroacetic acid to 1 volume of a solution of the testspecimen, withdraw the supernatant layer, and dissolve theprecipitate in a small volume of 0.5 mol/L sodium hydroxideTS. Use the solution so obtained to prepare the Test Solu-tion.Comments This test shows minimal difference betweenequivalent IgG and albumin samples. Addition of the so-dium hydroxide and the Biuret Reagent as a combined rea-gent, insufficient mixing after the addition of the sodium hy-droxide, or an extended time between the addition of the so-dium hydroxide solution and the addition of the Biuret Rea-gent will give IgG samples a higher response than albuminsamples. The trichloroacetic acid method used to minimizethe effects of interfering substances also can be used to de-termine the protein content in test specimens at concentra-tions below 500 mg per mL.

Method 6 (Fluorometric method)This fluorometric method is based on the derivatization of

the protein with o-phthalaldehyde (OPA), which reacts withthe primary amines of the protein (i.e., NH2-terminal aminoacid and the e-amino group of the lysine residues). The sensi-tivity of the test can be increased by hydrolyzing the proteinbefore testing. Hydrolysis makes the a-amino group of theconstituent amino acids of the protein available for reactionwith the OPA reagent. The method requires very small quan-tities of the protein.

Primary amines, such as tris(hydroxymethyl)amino-methane and amino acid buffers, react with OPA and mustbe avoided or removed. Ammonia at high concentrationswill react with OPA as well. The fluorescence obtained whenamine reacts with OPA can be unstable. The use of automat-ed procedures to standardize this procedure may improve theaccuracy and precision of the test.Standard Solutions Unless otherwise specified in the in-dividual monograph, dissolve the reference standard or thereference material for the protein under test in the bufferused to prepare the Test Solution. Dilute portions of this so-lution with the same buffer to obtain not fewer than fiveStandard Solutions having concentrations between 10 and200 mg of protein per mL, the concentrations being evenlyspaced.Test Solution Dissolve a suitable quantity of the proteinunder test in the appropriate buffer to obtain a solution hav-ing a concentration within the range of the concentrations ofthe Standard Solutions.Blank Use the buffer used to prepare the Test Solution andthe Standard Solutions.Reagents and Solutions�

Borate Buffer Dissolve about 61.83 g of boric acid inwater, and adjust with potassium hydroxide to a pH of 10.4.

Dilute with water to 1000 mL, and mix.Stock OPA Reagent Dissolve about 120 mg of o-

phthalaldehyde in 1.5 mL of methanol, add 100 mL ofBorate Buffer, and mix. Add 0.6 mL of polyoxyethylene (23)lauryl ether, and mix. This solution is stable at room temper-ature for at least 3 weeks.

OPA Reagent To 5 mL of Stock OPA Reagent add 15mL of 2-mercaptoethanol. Prepare at least 30 minutes priorto use. This reagent is stable for one day.Procedure Adjust each of the Standard Solutions and theTest Solution to a pH between 8.0 and 10.5. Mix 10 mL ofthe Test Solution and each of the Standard Solutions with100 mL of OPA Reagent, and allow to stand at room tem-perature for 15 minutes. Add 3 mL of 0.5 mol/L sodiumhydroxide TS, and mix. Using a suitable fluorometer,determine the fluorescent intensities of solutions from theStandard Solutions and the Test Solution at an excitationwavelength of 340 nm and an emission wavelength between440 and 455 nm. [Note: The fluorescence of an individualspecimen is read only once because irradiation decreases thefluorescent intensity.]Calculations The relationship of fluorescence to proteinconcentration is linear. Using the linear regression method,plot the fluorescent intensities of the solutions from theStandard Solutions versus the protein concentrations, anddetermine the standard curve best fitting the plotted points.From the standard curve so obtained and the fluorescentintensity of the Test Solution, determine the concentrationof protein in the test specimen.

Method 7 (Nitrogen method)This method is based on nitrogen analysis as a means of

protein determination. Interference caused by the presenceof other nitrogen-containing substances in the test proteincan affect the determination of protein by this method.Nitrogen analysis techniques destroy the protein under testbut are not limited to protein presentation in an aqueousenvironment.Procedure A Determine the nitrogen content of the proteinunder test as directed elsewhere in the Pharmacopoeia. Com-mercial instrumentation is available for the Kjeldahl nitro-gen assay.Procedure B Commercial instrumentation is available fornitrogen analysis. Most nitrogen analysis instruments usepyrolysis (i.e., combustion of the sample in oxygen at tem-peratures approaching 10009C), which produces nitric oxide(NO) and other oxides of nitrogen (NOx) from the nitrogenpresent in the test protein. Some instruments convert thenitric oxides to nitrogen gas, which is quantified with athermal conductivity detector. Other instruments mix nitricoxide (NO) with ozone (O3) to produce excited nitrogendioxide (NO2

.) which emits light when it decays and can bequantified with a chemiluminescence detector. A protein ref-erence standard or reference material that is relatively pureand is similar in composition to the test proteins is used tooptimize the injection and pyrolysis parameters and to evalu-ate consistency in the analysis.Calculations The protein concentration is calculated bydividing the nitrogen content of the sample by the knownnitrogen content of the protein. The known nitrogen contentof the protein can be determined from the chemical composi-tion of the protein or by comparison with the nitrogen con-tent of the appropriate reference standard or reference mate-

22052205JP XVI General Information / Microorganisms

rial.

1) Example: Minimum Requirements for BiologicalProducts and individual monograph in JP.2) Purity of the reagent is important.

G4 Microorganisms

Decision of Limit for BacterialEndotoxins

The endotoxin limit for injections is to be decided asfollows:

Endotoxin limit＝ K/M

where K is a threshold pyrogenic dose of endotoxin per kgbody mass (EU/kg), and M is equal to the maximum bolusdose of product per kg body mass. When the product is to beinjected at frequent intervals or infused continuously, M isthe maximum total dose administered in a single hourperiod.

M is expressed in mL/kg for products to be administeredby volume, in mg/kg or mEq/kg for products to be ad-ministered by mass, and in Unit/kg for products to be ad-ministered by biological units. Depending on the administra-tion route, values for K are set as in the following table.

Intended route of administration K (EU/kg)

Intravenous 5.0

Intravenous, forradiopharmaceuticals 2.5

Intraspinal 0.2

Notes:1) For products to be administered by mass or by units,

the endotoxin limit should be decided based on the labeledamount of the principal drug.

2) Sixty kg should be used as the average body mass ofan adult when calculating the maximum adult dose per kg.

3) The pediatric dose per kg body mass should be usedwhen this is higher than the adult dose.

4) The K values for the intravenous route are applicableto drugs to be administered by any route other than thoseshown in the table.

Disinfection and SterilizationMethods

Disinfection and Sterilization Methods are applied to killmicroorganisms in processing equipment/utensils and areasused for drug manufacturing, as well as to performmicrobiological tests specified in the monographs, and sodiffer from ``Terminal Sterilization'' and ``FiltrationMethod'' described in ``Terminal Sterilization and Steriliza-tion Indicators''. The killing effect on microorganisms or the

estimated level of sterility assurance is greatly variable, sothe conditions for disinfection and sterilization treatmentmust be chosen appropriately for each application. General-ly, the following methods are to be used singly or in combi-nation after appropriate optimization of operation proce-dures and conditions, in accordance with the kind and thedegree of the contaminating microorganisms and the natureof the item to which the methods are applied.

The validation of sterilization in accordance with Termi-nal Sterilization and Sterilization Indicators is required whenthe methods are applied to the manufacturing processes ofdrug products.

1. Disinfection methodsThese methods are used to reduce the number of living

microorganisms, but do not always remove or kill allmicroorganisms present. Generally, disinfection is classifiedinto chemical disinfection with the use of chemical drugs(disinfectants) and physical disinfection with the use of moistheat, ultraviolet light, and other agents.1.1. Chemical disinfection

Microorganisms are killed with chemical drugs. The kill-ing effect and mechanisms of a chemical drug differ depend-ing on the type, applied concentration, action temperature,and action time of the chemical drug used, the degree of con-tamination on the object to be disinfected, and the speciesand state (e.g., vegetative bacteria or spore bacteria) ofmicroorganisms.

Therefore, in applying the method, full consideration isrequired of the sterility and permissible storage period of theprepared chemical drug, the possibility of resistance ofmicroorganisms at the site of application, and the effect ofresidual chemical drug on the product. In selecting a suitablechemical drug, the following items should be considered inrelation to the intended use.

(i) The antimicrobial spectrum(ii) Action time for killing microorganisms(iii) Action durability(iv) Effect of the presence of proteins(v) Influence on the human body(vi) Solubility in water(vii) Influence on the object to be disinfected(viii) Odor(ix) Convenience of use(x) Easy disposability(xi) Influence on the environment at disposal(xii) Frequency of occurrence of resistance

1.2. Physical disinfectionMicroorganisms are killed without a chemical drug.(i) Steam flow methodMicroorganisms are killed by direct application of steam.

This method is used for a product which may be denaturedby the moist heat method. As a rule, the product is kept inflowing steam at 1009C for 30 � 60 minutes.

(ii) Boiling methodMicroorganisms are killed by putting the object in boiling

water. This method is used for a product which may bedenatured by the moist heat method. As a rule, the productis put in boiling water for 15 minutes or more.

(iii) Intermittent methodMicroorganisms are killed by heating for 30 � 60 minutes

repeatedly, three to five times, once a day in water at 80 �1009C or in steam. This method is used for a product which

22062206 JP XVIMicroorganisms / General Information

may be denatured by the moist heat method. There isanother method called the low temperature intermittentmethod with repeated heating at 60 � 809C. During the inter-mission periods between heating or warming, a suitabletemperature for the growth of microorganisms of 209C orhigher, must be maintained.

(iv) Ultraviolet methodAs a rule, microorganisms are killed by irradiation with

ultraviolet rays at a wavelength of around 254 nm. Thismethod is used for products which are resistant to ultravioletrays, such as smooth-surfaced articles, facilities, and equip-ment, or water and air. This method does not suffer fromthe occurrence of resistance, which is observed in chemicaldisinfection, and shows a killing effect on bacteria, fungi,and viruses. It must be taken into consideration that directultraviolet irradiation of the human body can injure the eyesand skin.

2. Sterilization methods2.1. Heating methods

In these methods, the heating time before the temperatureor pressure reaches the prescribed value differs according tothe properties of the product, the size of the container, andthe conditions. The duration of heating in conducting thesemethods is counted from the time when all the parts of theproduct have reached the prescribed temperature.

(i) Moist heat methodMicroorganisms are killed in saturated steam at a suitable

temperature and pressure. This method is generally used forheat-stable substances, such as glass, porcelain, metal, rub-ber, plastics, paper, and fiber, as well as heat-stable liquids,such as water, culture media, reagents, test solutions, liquidsamples, etc. As a rule, one of the following conditions isused.

115 � 1189C for 30 minutes121 � 1249C for 15 minutes126 � 1299C for 10 minutes

(ii) Dry-heat methodMicroorganisms are killed in dry-heated air. This method

is generally used for heat-stable substances, such as glass,porcelain, and metal, as well as heat-stable products, such asmineral oils, fats and oils, powder samples, etc. This methodis generally conducted in the way of direct heating by gas orelectricity or circulating heated air. As a rule, one of the fol-lowing conditions is used.

160 � 1709C for 120 minutes170 � 1809C for 60 minutes180 � 1909C for 30 minutes

2.2. Irradiation methods(i) Radiation methodMicroorganisms are killed by gamma-rays emitted from a

radioisotope or electron beam and bremsstrahlung (X-ray)generated from an electron accelerator. This method is gen-erally used for radiation-resistant substances such as glass,porcelain, metal, rubber, plastics, fiber, etc. The dose isdecided according to the material properties, and the degreeof contamination of the product to be sterilized. Special con-sideration is necessary of the possibility of qualitative changeof the product after the application of the method.

(ii) Microwave methodMicroorganisms are killed by the heat generated by direct

microwave irradiation. This method is generally used formicrowave-resistant products such as water, culture media,

test solutions, etc. As a rule, microwave radiation with awavelength of around 2450± 50 MHz is used.2.3. Gas methods

Microorganisms are killed by a sterilizing gas. Suitablegases for killing microorganisms include ethylene oxide gas,formaldehyde gas, hydrogen peroxide gas, chlorine dioxidegas, etc. Temperature, humidity, the concentration of gas,and the exposure time differ in accordance with the speciesof gas used. As sterilizing gases are generally toxic to hu-mans, full consideration is required of the environmentalcontrol for the use of gases and the concentration of residualgas. In some of the gas methods, it may be difficult to meas-ure or estimate quantitatively the killing of microorganisms.2.4. Filtration method

Microorganisms are removed by filtration with a suitablefiltering device. This method is generally used for gas, water,or culture media and test solutions containing a substancethat is water-soluble and unstable to heat. As a rule, a filterhaving a pore size of 0.22 mm or smaller is used for thesterilization. However, in this method, a filter with a poresize of 0.45 mm or smaller is permitted to be used.

Media Fill Test (Process Simulation)The media fill test is one of the process validations em-

ployed to evaluate the propriety of the aseptic processing ofpharmaceutical products using sterile media, etc. instead ofactual products. Therefore, media fill should be conductedunder conditions that simulate routine manufacturingprocedures, e.g. filling and sealing, operating environment,processing operation, number of personnel involved, etc.,and include permissible worst case conditions. Process simu-lation can be applied to the other aseptic manufacturingprocesses in addition to aseptic manufacturing processes forfinished drug products such as ``filling'' and ``sealing''.

1. Frequency of media fills1.1. Initial performance qualification

Initial performance qualification should be conducted foreach new facility, item of equipment, filling line, and con-tainer design (except for multiple sizes of the same containerdesign), etc. As referring to Table 1, a sufficient number ofunits should be used to simulate aseptic manufacturingprocess. A minimum of three consecutive separate successfulruns should be performed on each separate day.1.2. Periodic performance requalification

1) As referring to Table 2, a sufficient number of unitsshould be used to simulate aseptic manufacturing process.Media fill run should be conducted at least on semi-annualbase for each shift and processing line. All personnel work-ing in the critical processing area should be trained aboutaseptic processing and participate in a media fill run at leastonce a year.

2) When filling lines have not been used for over sixmonths, conduct appropriate numbers of media fill runs inthe same way as for the initial performance qualificationprior to the use of the filling lines.

3) In cases of facility and equipment modification (inter-changing parts may not require requalification), majorchanges in personnel working in critical aseptic processing,anomalies in environmental monitoring results, or a productsterility test showing contaminated products, conduct ap-

2207

Table 1 Initial performance qualification

Minimumnumber ofsimulations

Numberof unitsfilled persimulation

Contaminatedunits in any of

the threesimulations

Action

3 ＜5000 ≧1Investigation, cor-rective measures,restart validation

35000 �10000

1Investigation, con-sideration of repeatof one media fill

＞1Investigation, cor-rective measures,restart validation

3 ＞10000

1 Investigation

＞1Investigation, cor-rective measures,restart validation

Table 2 Periodic performance requalification

Minimumnumber ofsimulations

Numberof unitsfilled persimulation

Contaminatedunits

Action

Every halfyear

＜5000 1Investigation, reval-idation

5000 �10000

1Investigation, con-sideration of repeatmedia fill

＞1Investigation, cor-rective measures,revalidation

＞10000

1 Investigation

＞1Investigation, cor-rective measures,revalidation


propriate numbers of media fill runs in the same way as forthe initial performance qualification prior to the scheduledmedia fills.

2. Acceptance criteria of media fillsBoth in initial performance qualification and periodic per-

formance qualification, the target should be zero growthregardless of number of units filled per simulation. Wherecontaminated units are found, action shown in Tables 1 and2 should be taken.2.1. Investigation of positive units

Where contaminated units are found in media fill, an in-vestigation should be conducted regarding the cause, takinginto consideration the following points:

1) Microbial monitoring data

2) Particulate monitoring data3) Personnel monitoring data (microbial monitoring

data on gloves, gowns, etc. at the end of work)4) Sterilization cycle data for media, commodities, eq-

uipment, etc.5) Calibration data of sterilization equipment6) Storage conditions of sterile commodities7) HEPA filter evaluation (integrity tests, velocity, etc.)8) Pre and post filter integrity test data (including filter

housing assembly)9) Air flow patterns and pressures

10) Unusual events that occurred during the media fill run11) Characterization of contaminants12) Hygienic control and training programs13) Gowning procedures and training programs14) Aseptic processing technique and training programs15) Operator's health status (especially coughing, sneez-

ing, etc., due to respiratory diseases)16) Other factors that affect sterility

3. Data guidance for media fillsEach media fill run should be fully documented and the

following information recorded:1) Data and time of media fill2) Identification of filling room and filling line used3) Container/closure type and size4) Volume filled per container5) Filling speed6) Filter type and integrity test result (in case of filtra-

tion)7) Type of media filled8) Number of units filled9) Number of units not incubated and reason

10) Number of units incubated11) Number of units positive12) Incubation time and temperature13) Procedures used to simulate any step of a normal

production fill (e.g., mock lyophilization or substitution ofvial headspace gas)14) Microbiological monitoring data obtained during the

media fill set-up and run15) List of personnel who took part in the media fill16) Growth promotion results of the media (in case of

powder fill, an antimicrobial activity test for the powder isnecessary)17) Identification and characterization of the microorgan-

isms from any positive units18) Product(s) covered by the media fill19) Investigation of runs with a positive unit or failed

runs20) Management review

4. Media fill proceduresMethods to validate aseptic processing of liquid, powder

and freeze-dried products are described. Basically, it is possi-ble to apply media fill procedures for liquid products toother dosage forms.4.1. Media selection and growth promotion

Soybean-casein digest medium or other suitable media areused. When strains listed in the Microbial Limit Test <4.05>and, if necessary, one or two representative microorganismswhich are frequently isolated in environmental monitoringare inoculated under the specified conditions, each strainshould show obvious growth.


4.2. Sterile medium preparationThe medium is sterilized according to the pre-validated

method.4.3. Incubation and inspection of media filled units

Leaking or damaged units should be removed and record-ed prior to incubation of media filled units. Units should beincubated at 20 � 359C for not less than 14 days. The use ofanother temperature range should be justified. If two tem-peratures are used for incubation, the units are typically in-cubated for at least 7 days at each temperature starting withthe lower temperature. Established temperature should bekept within ±2.59C. Observe the media filled units forgrowth of microorganisms on the last day of the incubation.Microorganisms in contaminated units should be followed toidentification and characterization. For the identification ofcontaminants, ``Rapid Identification of MicroorganismsBased on Molecular Biological Method'' shown in GeneralInformation or appropriate commercial kit for identificationof microorganisms may be applicable.

A. Liquid productsMedia fill procedureMedia fill should include normal facility/equipment oper-

ations and clean-up routines. Containers, closures, parts ofthe filling machine, trays, etc. are washed and sterilized ac-cording to the standard operating procedures. Media fillsshould be conducted under processing conditions that in-clude ``worst case'' conditions, e.g., correction of line stop-page, repair or replacement of filling needles/tubes, replace-ment of on-line filters, permitted interventions, duration andsize of run, number of personnel involved, etc.

It is not necessary to put all worst-case scenarios in asingle media fill run, but all worst-case scenarios should beevaluated intentionally. While advanced processing lines arehighly automated, often operated at relatively high speeds,and are designed to limit operator intervention, there areprocessing lines showing frequent human interventions.Although the most accurate simulation model would be thefull batch size and duration because it most closely simulatesthe actual production run, other appropriate models can bejustified.

A predetermined volume of medium is filled into sterilizedcontainers at a predetermined filling speed and the contain-ers are sealed. The media are contacted with all interior sur-faces in the containers by an appropriate method, and thenincubated at the predetermined temperature.

B. Powder productsB.1 Powder selection and antimicrobial activity test

Actual products or placebo powder are used. In general,lactose monahydrate, D-mannitol, polyethylene glycol 6,000,carboxymethyl cellulose salts or media powder, etc. are usedas placebo powders. Prior to employing any of the powders,evaluate whether the powder has antimicrobial activity.Media powders are dissolved in water and other powders inliquid medium, and the solutions are inoculated with lessthan 100 CFU microorganisms of each kind, shown in 4.1,for the growth promotion test. If obvious growth appears inthe medium incubated at the predetermined temperature for5 days, the powder has no antimicrobial activity and is avail-able for the media fill test.B.2 Sterilization of powders

Powders are bagged in suitable containers (e.g. doubleheat-sealed polyethylene bags), and are subjected to radia-

tion sterilization.B.3 Sterility of filling powders

The powders must pass the Sterility Test. However, if thesterilization is fully validated, sterility testing of the powderscan be omitted.B.4 Media fill procedures

Chose a suitable procedure from among the followingprocedures.

1) Fill sterilized liquid media into containers by suitablemethods, and then fill actual products or sterilized placebopowder with the powder filling machine. If sterilized powdermedia are used as a placebo powder, fill sterilized water in-stead of sterilized liquid media.

2) Distribute liquid media into containers, and then steri-lize them in an autoclave. Remove the containers to the fill-ing area, and then fill actual products or sterilized placebopowder into the containers with the powder filling machine.

3) Fill actual products or sterilized placebo powder intocontainers with the powder filling machine, and then fillsterilized liquid media into the containers by appropriatemethods. If sterilized powder media are used as a placebopowder, fill sterilized water instead of sterilized liquid me-dia.

C. Lyophilized productsIn the case of lyophilized products, it may be impossible to

conduct a media fill run in the same way as used for actualprocessing of lyophilized products. The process of freezingand lyophilization of the solution may kill contaminant or-ganisms and change the characteristics of the media too. Theuse of inert gas as a blanket gas may inhibit the growth ofaerobic bacteria and fungi. Therefore, in general, the actualfreezing and lyophilization process should be avoided and airis used as the blanket gas. For products manufactured underan anaerobic atmosphere, process simulation should be per-formed with the use of anaerobic growth media and the inertgas such as nitrogen gas.

Media fill proceduresUse the following method or other methods considered to

be equivalent to these methods.1) After filling of the media into containers by the filling

machine, cap the containers loosely and collect them in pre-sterilized trays.

2) After placing the trays in the lyophilizer, close thechamber door, and conduct lyophilization according to theprocedures for production operation. Hold them withoutfreezing and boiling-over under weak vacuum for thepredetermined time.

3) After the vacuum process, break the vacuum, and sealthe stoppers.

4) Contact the media with all product contact surfaces inthe containers by appropriate methods, and then cultivatethem at the predetermined temperature.

ReferencesISO 13408-1 (2008): Aseptic processing of health care prod-ucts: General requirements.


Microbial Attributes of Non-sterilePharmaceutical Products

This chapter is harmonized with the European Phar-macopoeia and the U.S. Pharmacopeia. The parts of the textthat are not harmonized are marked with symbols ( ).

The presence of certain micro-organisms in non-sterilepreparations may have the potential to reduce or even inacti-vate the therapeutic activity of the product and has a poten-tial to adversely affect the health of the patient. Manufac-turers have therefore to ensure a low bioburden of finisheddosage forms by implementing current guidelines on GoodManufacturing Practice during the manufacture, storage anddistribution of pharmaceutical preparations. This chapterprovides guidelines for acceptable limits of viable micro-organisms (bacteria and fungi) existing in raw materials andnon-sterile pharmaceutical products. Microbial examina-tion of non-sterile products is performed according to themethods given in the Microbial Limit Test <4.05> onMicrobiological Examination of Non-sterile Products: I.Microbial Enumeration Tests and II. Tests for SpecifiedMicro-organisms. When these tests are carried out, amicrobial control program must be established as an im-portant part of the quality management system of theproduct. Personnel responsible for conducting the testsshould have specialized training in microbiology, biosafetymeasures and in the interpretation of the testing results.1. Definitions

(i) Non-sterile pharmaceutical products: Non-steriledrugs shown in monographs of the JP and non-sterilefinished dosage forms.

(ii) Raw materials: All materials, including raw ingredi-ents and excipients, used for the preparation of drugs, exceptfor water and gases.

(iii) Bioburden: Number and type of viable micro-organ-isms existing in non-sterile pharmaceutical products.

(iv) Action levels: Established bioburden levels that re-quire immediate follow-up and corrective action if they areexceeded.

(v) Alert levels: Established bioburden levels that giveearly warning of a potential drift from normal bioburdenlevel, but which are not necessary grounds for definitive cor-rective action, though they may require follow-up investiga-tion.

(vi) Quality management system: The procedures, opera-tion methods and organizational structure of a manufacturer(including responsibilities, authorities and relationships be-tween these) needed to implement quality management.

2. ScopeIn general, the test for total viable aerobic count is not ap-

plied to drugs containing viable micro-organisms as an activeingredient.

3. Sampling plan and frequency of testing3.1. Sampling methods

Microbial contaminants are usually not uniformly dis-tributed throughout the batches of non-sterile pharmaceuti-cal products or raw materials. A biased sampling plan, there-fore, cannot be used to estimate the real bioburden in theproduct. A sampling plan which can properly reflect the sta-

tus of the product batch should be established on the basis ofthe bioburden data obtained by retrospective validation and/or concurrent validation. In general, a mixture of samplesrandomly taken from at least different three portions,almost the same amount for each portion, is used for thetests of the product. When the sampling is difficult in a cleanarea, special care is required during sampling to avoid in-troducing microbial contamination into the product or af-fecting the nature of the product bioburden. If it is con-firmed that the product bioburden is stable for a certainperiod, as in the case of non-aqueous or dried products, it isnot necessary to do the tests, immediately after the sampling.3.2. Testing frequency

The frequency of the tests should be established on thebasis of a variety of factors unless otherwise specified. Thesefactors include:

(i) Dosage forms of non-sterile pharmaceutical products(usage);

(ii) Manufacturing processes;(iii) Manufacturing frequency;(iv) Characteristics of raw materials (natural raw mate-

rial, synthetic compound, etc.);(v) Batch sizes;(vi) Variations in bioburden estimates (changes in batch-

es, seasonal variations, etc.);(vii) Changes affecting the product bioburden (changes

in manufacturing process, supplier of raw materials, batchnumber of raw materials, etc.);

(viii) Others.In general, the tests may be performed at a high frequency

during the initial production of a drug to get information onthe microbiological attributes of the product or raw mate-rials used for the production. However, this frequency maybe reduced as bioburden data are accumulated throughretrospective validation and/or concurrent validation. Forexample, the tests may be performed at a frequency based ontime (e.g., weekly, monthly or seasonally), or on alternatebatches.

4. Microbial control programWhen the ``Microbial Limit Test <4.05>'' is applied to a

non-sterile pharmaceutical product, the methods for therecovery, cultivation and estimation of the bioburden fromthe product must be validated and a ``Microbial control pro-gram'' covering the items listed below must be prepared.

(i) Subject pharmaceutical name (product name);(ii) Frequency of sampling and testing;(iii) Sampling methods (including responsible person,

quantity, environment, etc. for sampling);(iv) Transfer methods of the samples to the testing area

(including storage condition until the tests);(v) Treatment of the samples (recovery methods of

microbial contaminants);(vi) Enumeration of viable micro-organisms (including

testing quantity, culture media, growth-supporting test ofthe media, culturing methods, etc.);

(vii) Detection of specified micro-organisms (includingtesting quantity, culture media, growth-supporting test ofthe media, culturing methods, etc.);

(viii) Estimation of the number of and characterizationof microbial contaminants;

(ix) Establishment of ``Microbial acceptance criteria''(including alert level and action level);

2210

Table 2 Acceptance criteria for microbiological quality of non-sterile dosage forms

Route of administrationTotal AerobicMicrobial Count(CFU/g or CFU/mL)

Total CombinedYeasts/Moulds Count(CFU/g or CFU/mL)

Specified Micro-organism

Non-aqueous preparationsfor oral use

103 102 Absence of Escherichia coli(1 g or 1 mL)

Aqueous preparationsfor oral use

102 101 Absence of Escherichia coli(1 g or 1 mL)

Rectal use 103 102 �

Oromucosal useGingival useCutaneous useNasal useAuricular use

102 101 Absence of Staphylococcus aureus(1 g or 1 mL)Absence of Pseudomonas aeruginosa(1 g or 1 mL)

Vaginal use 102 101 Absence of Pseudomonas aeruginosa(1 g or 1 mL)Absence of Staphylococcus aureus(1 g or 1 mL)Absence of Candida albicans(1 g or 1 mL)

Transdermal patches (limitsfor one patch including ad-hesive layer and backing)

102 101 Absence of Staphylococcus aureus(1 patch)Absence of Pseudomonas aeruginosa(1 patch)

Inhalation use (more rigo-rous requirements apply toliquid preparations fornebulization)

102 101 Absence of Staphylococcus aureus(1 g or 1 mL)Absence of Pseudomonas aeruginosa(1 g or 1 mL)Absence of bile-tolerant gram-negativebacteria(1g or 1 mL)

Table 1 Acceptance criteria for microbiological quality ofnon-sterile substances for pharmaceutical use

Total AerobicMicrobial Count(CFU/g or CFU/mL)

Total CombinedYeasts/MouldsCount(CFU/g or CFU/mL)

Substances forpharmaceuticaluse

103 102


(x) Actions to be taken when the levels exceed ``Microbi-al acceptance criteria'';

(xi) Persons responsible for the testing and evaluation,etc.;

(xii) Other necessary items.

5. Microbial acceptance criteria for non-sterile pharmaceu-tical products

By establishing ``Microbial acceptance criteria'' for non-sterile pharmaceutical products based upon the total aerobicmicrobial count (TAMC) and the total combined yeasts/moulds count (TYMC), it is possible to evaluate at the ini-tial processing stage of the product whether the microbiolog-ical quality of the raw materials is adequate or not. Further-more, it is then possible to implement appropriate correctiveaction as needed to maintain or improve the microbiologicalquality of the product. The target limits of microbial levelsfor raw materials (synthetic compounds and minerals) areshown in Table 1.In general, synthetic compounds have low bioburden lev-

els due to the high temperatures, organic solvents, etc., usedin their manufacturing processes. Raw materials originatedfrom plants and animals in general have higher bioburdensthan synthetic compounds.

The microbial quality of the water used in the processing

of active ingredients or non-sterile pharmaceuticals mayhave a direct effect on the quality of the finished dosageform. This means it is necessary to keep the level of microbi-al contaminants in the water as low as possible.

Acceptance criteria for microbiological quality for non-sterile finished dosage forms are shown in Table 2. Thesemicrobial limits are based primarily on the type of dosageform, water activity, and so on. For oral liquids and phar-maceutical products having a high water activity, in general,low microbial acceptance criteria are given.

Table 2 includes a list of specified micro-organisms forwhich acceptance criteria are set. The list is not necessarily

2211

Table 3 Acceptance criteria for crude drugs and crudedrug preparations

Micro-organismsCategory 1

(CFU/g or CFU/mL)Category 2

(CFU/g or CFU/mL)

Aerobic bacteria 107 105

Molds and yeasts 104 103

Enterobacteriaand othergram-negativebacteria

※ 103

Escherichia coli 102 Not detectedSalmonella Not detected Not detectedStaphylococcusaureus

※ ※

※ : The limits are not specified.


exhaustive and for a given preparation it may be necessary totest for other micro-organisms depending on the nature ofthe starting materials and the manufacturing process.

If it has been shown that none of the prescribed tests willallow valid enumeration of micro-organisms at the levelprescribed, a validated method with a limit of detection asclose as possible to the indicated acceptance criterion is used.

In addition to the micro-organisms listed in Table 2, thesignificance of other micro-organisms recovered should beevaluated in terms of:

( i ) the use of the product: hazard varies according tothe route of administration (eye, nose, respiratorytract);

( ii ) the nature of the product: does the product supportgrowth, does it have adequate antimicrobial preser-vation?

(iii) the method of application;(iv) the intended recipient: risk may differ for neonates,

infants, the debilitated;( v ) use of immunosuppressive agents, corticosteroids;(vi) presence of disease, wounds, organ damage.Where warranted, a risk-based assessment of the relevant

factors is conducted by personnel with specialized training inmicrobiology and the interpretation of microbiological data.

For raw materials, the assessment takes account ofprocessing to which the product is subjected, the currenttechnology of testing and the availability of materials of thedesired quality. Acceptance criteria are based on individualresults or on the average of replicate counts when replicatecounts are performed (e.g. direct plating methods).

When an acceptance criterion for microbiological qualityis prescribed it is interpreted as follows:�101 CFU: maximum acceptable count＝ 20,�102 CFU: maximum acceptable count＝ 200,�103 CFU: maximum acceptable count＝ 2000, and soforth.

6. Acceptance criteria for crude drugsTarget limits of microbial contamination for crude drugs

and crude drug preparations are shown in Table 3. Category1 includes crude drugs and crude drug preparations whichare used for extraction by boiling water or to which boilingwater is added before use. Category 2 includes crude drugswhich are taken directly without extraction process and

directly consumed crude drug preparations containing pow-dered crude drugs. In this guideline, enterobacteria andother gram-negative bacteria, Escherichia coli, Salmonella,and Staphylococcus aureus are mentioned as specifiedmicro-organisms, but other micro-organisms such as certainspecies of Bacillus cereus, Clostridia, Pseudomonas, Bur-kholderia, Asperigillus and Enterobacter species are alsonecessary to be tested depending on the origin of raw mate-rials for crude drugs or the preparation method of crudedrug preparations.

Microbiological Evaluation ofProcessing Areas for SterilePharmaceutical Products

This chapter describes the methods for the control andevaluation of microbial contamination in areas used for theprocessing of sterile pharmaceutical products. Such process-ing areas are classified into critical areas and clean areas ac-cording to the required levels of air-cleanliness. A criticalarea is a defined space in which the airborne particulate andmicroorganism levels are controlled to meet grade A. Thecleanliness requirements for such a space extend to the sur-faces of the facilities and equipment which form or are locat-ed within the space, as well as to the supplied raw materials,chemicals, water, etc. Environmental conditions, such astemperature, humidity, and air pressure, are also controlledin this space when required. A clean area is a controlledspace such that the levels of contaminants (particulates andmicroorganisms) in air, gases and liquids are maintainedwithin specified limits, which are less stringent than those ofgrade A. When sterile pharmaceutical products are manufac-tured, the environment, facilities/equipment, and personnelshould be routinely monitored to ensure appropriatemicrobiological control in the processing areas. The detec-tion of microorganisms should be performed under normaloperational conditions, using an appropriate samplingdevice, according to an environmental control program es-tablished previously. The sampling, cultivation, counting,and evaluation methods for airborne microorganisms, aswell as those found on surfaces, should also be chosen ap-propriately, depending on the monitoring purpose, monitor-ing items, and microorganisms being detected. Samplingdevices, measurement methods, media, culture conditions,frequency of monitoring, and recommended limits for en-vironmental microorganisms shown in this chapter are forinformation only, and are not requirements.

1. DefinitionsFor the purposes of this chapter, the following definitions

apply.(i) Processing areas: Areas in which actions such as culti-

vation, extraction/purification, weighing of raw materials,washing and drying of containers and stoppers, preparationof solutions, filling, sealing and packaging are performed,including the gowning area.

(ii) Action levels: Established microbial levels (and typeof microorganisms, if appropriate) that require immediatefollow-up and corrective action if they are exceeded.

(iii) Alert levels: Established microbial levels (and type

2212

Table 1 Air-cleanliness requirements for processing of sterile pharmaceutical products

Air cleanliness Maximum number of airborne particulates per m3

Grade*1at rest

≧0.5 mm

in operation

≧0.5 mm

A (Laminar-airflow zone) 3530 3530B (Non laminar-airflow zone) 3530 353000C 353000 3530000D 3530000 �*2

*1 The maximum permitted number of particles in the ``in operation'' condition corresponds to the standards described under USP <1116> asfollows.

Grade A: Class 100 (M3.5); Grade B: Class 10,000 (M5.5); Grade C: Class 100,000 (M6.5);Grade D: no corresponding standard.

*2 The limit for this area will depend on the nature of the operation carried out there.


of microorganisms if appropriate) that give early warning ofa potential drift from normal operating conditions, butwhich are not necessarily grounds for definitive correctiveaction, though they may require follow-up investigation.

(iv) Contaminants: Particulates and microorganismscausing contamination by adhering to surfaces or by beingincorporated into materials.

(v) Cleanliness: A quantity which indicates the conditionof cleanliness of a monitored item, expressed as mass ornumber of contaminants contained in a certain volume orarea.

(vi) Contamination control: The planning, establishmentof systems and implementation activities performed in orderto maintain the required cleanliness of a specified space orsurface.

(vii) Shift: Scheduled period of work or production, usu-ally less than 12 hours in length, during which operations areconducted by a single defined group of workers.

(viii) Characterization of contaminants: Procedures forclassifying contaminants so that they can be differentiated.In routine control, classification to the genus level issufficient; as required, identification to the species level isperformed.

2. Air-cleanliness of processing areas for sterile phar-maceutical products

Airborne particulates in areas used for the processing ofpharmaceutical products may act physically as a source ofinsoluble particles in the products, and biologically as a car-rier of microorganisms. So, it is necessary to control strictlythe number of particles in the air. The air-cleanliness criteriaare shown in Table 1.2.1. Terminally sterilized products

Solutions should generally be prepared in a grade C en-vironment. Solution preparation may be permitted in a gradeD environment if additional measures are taken to minimizecontamination. For parenterals, filling should be done in agrade A workstation in a grade B or C environment. The re-quirements during the preparation and filling of other sterileproducts are generally similar to those for parenterals.2.2. Sterile products prepared aseptically after filtration

The handling of starting materials and the preparation ofsolutions should be done in a grade C environment. Theseactivities may be permitted in a grade D environment if addi-tional measures are taken to minimize contamination, suchas the use of closed vessels prior to filtration. After sterile

filtration, the product must be handled and filled into con-tainers under grade A aseptic conditions.2.3. Sterile products prepared aseptically from sterile start-ing materials

The handling of starting materials and all further process-ing should be done under grade A conditions.

3. Microbiological environmental monitoring programEnvironmental monitoring is especially important in steril-

ity assurance for sterile pharmaceutical products that aremanufactured by aseptic processing. The major purpose ofenvironmental monitoring is to predict potential deteriora-tion of the processing environment before it occurs, and toproduce high-quality, sterile pharmaceutical products underappropriate contamination control.3.1. Monitoring of environmental microorganisms

(i) An environmental control program document is pre-pared for each area used for the processing of sterile phar-maceutical products. The procedures in the document in-clude: 1) items to be monitored, 2) type of microorganismsto be monitored, 3) frequency of monitoring, 4) methods ofmonitoring, 5) alert and action levels, and 6) actions to betaken when specified levels are exceeded.

(ii) The aseptic processing areas and other processingareas maintained under controlled conditions are monitoredon a routine basis. The critical processing areas where sterileproducts are in contact with environmental air are monitoredduring every operational shift. The items to be monitoredinclude the air, floor, walls, equipment surfaces, and thegowns and gloves of the personnel. Table 2 shows suggestedfrequencies of the environmental monitoring.

(iii) The sampling devices used for monitoring environ-mental microorganisms, as well as the methods and culturemedia, should be suitable to detect microorganisms that maybe present (aerobic bacteria, anaerobic bacteria, molds,yeast, etc.). The cultivation conditions, such as incubationtemperature and time, are selected to be appropriate for thespecific growth requirements of microorganisms to be de-tected. Table 3 shows the culture media and cultivation con-ditions that are generally used in testing for environmentalmicroorganisms.

(iv) The number of microorganisms in the samples isestimated by using the Membrane Filtration, Pour Plating,Spread Plating, or Serial Dilution (Most Probable Number)Methods described in the Microbial Limit Test.

(v) Table 4 shows recommended limits for environmental

2213

Table 2 Suggested frequency of environmental monitoring

Processing area Frequency of monitoring

Critical area (Grade A) Each shiftClean area adjacent to critical area (Grade B) Each shiftOther clean areas (Grade C, D)

Potential product/container contact areas Twice a weekNon-product/container contact areas Once a week

Table 3 Media and culture conditions

Microorganismsto be detected Media*1 Culture conditions

AerobesSoybean-casein digest agar (or fluid) mediumBrain-heart infusion agar (or fluid) mediumNutrient agar (or fluid) medium

30 � 359C*2

More than 5 days*3

Yeast and fungi

Soybean-casein digest agar (or fluid) mediumSabouraud dextrose agar (or fluid) mediumPotato-dextrose agar (or fluid) mediumGlucose peptone agar (or fluid) medium

20 � 259C*2

More than 5 days*3

Anaerobes*4

Soybean-casein digest agar mediumFluid cooked meat mediumReinforced clostridial agar (or fluid) mediumThioglycolate medium I (or thioglycolate agar

medium) for sterility test

30 � 359CMore than 5 days*3

*1 If necessary, antibiotics may be added to media in an appropriate concentration (see Microbial Limit Test). If the existence of disin-fectants that may interfere with the test on the surface of the specimen is suspected, add a substance to inactivate them.

*2 When soybean-casein digest agar medium is used for the detection of aerobes, yeast and fungi, incubation at 25 to 309C for more than 5days is acceptable.

*3 If a reliable count is obtained in a shorter incubation time than 5 days, this may be adopted.*4 Generally, anaerobes are not targets for the monitoring. For the detection of anaerobes, agar medium is incubated in an appropriate

anaerobic jar.

Table 4 Recommended limits for environmental microorganisms*1

GradeAirborne

microorganisms*2

(CFU/m3)Minimum air sample

(m3)

CFU on a surface

instruments/facilities gloves

(CFU/24�30 cm2)*3

A ＜1 0.5 ＜1 ＜1B 10 0.5 5 5C 100 0.2 25 �D 200 0.2 50 �

*1 Maximum acceptable average numbers of microorganisms under each condition.*2 These values are by using a slit sampler or equivalent.*3 Viable microbe cell number per contact plate (5.4 � 6.2 cm in diameter). When swabbing is used in sampling, the number of microorgan-

isms is calculated per 25 cm2. For gloves, usually, put their all fingers on the plate.


microorganisms. The alert and action levels may be adjustedif necessary after sufficient data have been accumulated. Themost important point in environmental monitoring is to con-firm that an acceptable value of each monitoring item ismaintained consistently.

(vi) Microorganisms isolated are characterized if neces-sary. In addition, analysis of hourly or daily variation of air-borne particulate numbers will provide data to assist in thecontrol of the cleanliness of processing areas.3.2. Evaluation of environmental monitoring data

(i) The data from the environmental monitoring areevaluated on a routine basis for each area and location. The

source of any discrepancy should be investigated immedi-ately and the investigation should be documented in areport. After corrective action has been taken, follow-upmonitoring should be done to demonstrate that the affectedarea is once again within specification.

(ii) The report is reviewed and approved by personnelresponsible for quality control and distributed to all key per-sonnel associated with the aseptic processing operation.

4. Sampling devices and measuring methodologyVarious types of sampling devices and measurement

methods are available for the sampling and measurement of


microorganisms in the air and on surfaces, and appropriatesamplers and measuring methodology are selected accordingto the purpose of monitoring and the items to be monitored.4.1. Evaluation of airborne microorganisms4.1.1. Settle plates

Petri dishes of a specified diameter containing a suitableculture medium are placed at the measurement location andthe cover is removed there. The plates are exposed for agiven time and the microorganisms deposited from the aironto the agar surface are enumerated after incubation. Thismethod is not effective for quantitative monitoring of totalairborne microorganisms because it does not detect microor-ganisms that do not settle onto the surface of the culturemedia, and the settling velocity of aggregates of microorgan-isms is affected by air currents and disturbances in airflow.Although the results obtained by the settle plate method areonly qualitative or semi-quantitative, this method is suitablefor long-term evaluation of possible contamination of prod-ucts or devices by airborne microorganisms.4.1.2. Active microbial sampling methods4.1.2.1. Measuring methods

Methods in which a fixed volume of air is aspirated in-clude filtration-type sampling devices and impact-typesampling devices. With the filter-type sampling devices thedesired volume of air can be collected by appropriatelychanging the air intake rate or the filter size. However, caremust be taken to ensure that sterility is maintained while thefilter is placed in and removed from the holder. When airsampling devices are used in critical areas, care must betaken to avoid disturbance of the airflow around the prod-ucts. There are two types of filters; wet-type used gelatinfilters and dry-type used membrane filters. With the dry-typefilters, static electricity effects can make it impossible to col-lect quantitatively microorganisms on the filter. When animpact-type sampling device is used, the following points areimportant: 1) The speed at which the collected air strikes theculture medium surface must be sufficient to capture themicroorganisms, but must not have an adverse effect on thecollected microorganisms. 2) A sufficient volume of air mustbe sampled so that even extremely low levels of microbiolog-ical contaminants are detected, but the procedure must notcause a significant change in the physical or chemical proper-ties of the culture medium. 3) When the device is used in crit-ical areas, care must be exercised to ensure that the process-ing of the sterile pharmaceutical products is not adversely af-fected by the air disturbance.4.1.2.2. Sampling devices

The most commonly used samplers are as follows: Slitsampler, Andersen sampler, pinhole sampler, centrifugalsampler and filtration-type sampler. Each sampler has spe-cific characteristics. The slit sampler is a device to trapmicroorganisms in a known volume of air passed through astandardized slit. The air is impacted on a slowly revolvingPetri dish containing a nutrient agar. The rotation rate of thePetri dish and the distance from the slit to the agar surfaceare adjustable and it is possible to estimate the number ofmicroorganisms in the air passed through the device for aperiod of up to 1 hr. The Andersen sampler consists of a per-forated cover and several pieces of Petri dishes containing anutrient agar, and a known volume of air passed through theperforated cover impacts on the agar medium in the Petridishes. The sampler is suitable for the determination of thedistribution of size ranges of microorganism particulates in

the air. The pinhole sampler resembles the slit of the slitsampler, but has pinholes in place of the slit. A knownvolume of air passed through several pinholes impacts onagar medium in a slowly revolving Petri dish. The cen-trifugal sampler consists of a propeller that pulls a knownvolume of air into the device and then propels the air out-ward to impact on a tangentially placed nutrient agar strip.The sampler is portable and can be used anywhere, but thesampling volume of air is limited.

See above the Measuring methods <4.1.2.1> on the charac-teristics of the filtration-type sampler.4.2. Measurement methods for microorganisms on sur-faces4.2.1. Contact plates

Use a contact plate with an appropriate contact surface.The culture medium surface should be brought into contactwith the sampling site for several seconds by applyinguniform pressure without circular or linear movement. Aftercontact and removal, the plates are covered and, as soon aspossible, incubated using appropriate culture conditions.After a contact plate has been used, the site to which theplate was applied must be wiped aseptically to remove anyadherent culture medium.4.2.2. Swabs

A piece of sterilized gauze, absorbent cotton, cotton swab,or other suitable material premoistened with an appropriaterinse fluid is stroked in closely parallel sweeps or slowly ro-tated over the defined sampling area. After sampling, theswab is agitated in a specified amount of an appropriatesterilized rinse fluid, and the rinse fluid is assayed for viableorganisms.

5. Test methods for collection performance of a samplingdevice for airborne microorganisms

The testing of the collection performance of samplingdevices for airborne microorganisms is performed in accor-dance with JIS K 3836 (Testing methods for collection ef-ficiency of airborne microbe samplers) or ISO 14698 � 1(Cleanrooms and associated controlled environments.Biocontamination control. General principles).

6. Growth-promotion test of media and confirmation ofantimicrobial substances

This test and confirmation are performed according to``Effectiveness of culture media and confirmation of antimi-crobial substances'' in the Microbial Limit Test <4.05>.

7. Media(i) Soybean-casein digest agar medium or Fluid soybean-

casein digest mediumSee Microbial Limit Test.

(ii) Sabouraud dextrose agar medium or Fluid sabourauddextrose medium

See Microbial Limit Test. Antibiotic is added if necessary.

(iii) Potato-dextrose agar medium or Fluid potato-dex-trose medium

See Microbial Limit Test. Antibiotic is added if necessary.

(iv) Glucose peptone agar medium or Fluid glucose pep-tone medium

Glucose 20.0 gYeast extract 2.0 g


Magnesium sulfate heptahydrate 0.5 gPeptone 5.0 gPotassium dihydrogen phosphate 1.0 gAgar 15.0 gWater 1000 mL

Mix all the components, and sterilize by heating in anautoclave at 1219C for 15 to 20 minutes. pH aftersterilization: 5.6 � 5.8. Just prior to use, add 0.10 g ofbenzylpenicillin potassium and 0.10 g of tetracyclin perliter of medium as sterile solutions or, alternatively, add50 mg of chloramphenicol per liter of medium. Anti-biotics are added, as appropriate.

(v) Thioglycolate agar medium or thioglycolate mediumI for sterility test

See Sterility Test. The agar concentration of Thioglycolateagar medium is about 1.5z.

(vi) Brain-heart infusion agar medium or Fluid brain-heart infusion medium

Bovine brain extract powder*1

An amount equivalent to 200 g of calf brainBovine heart extract powder*2

An amount equivalent to 250 g of the materialPeptone 10.0 gGlucose 2.0 gSodium chloride 5.0 gDisodium hydrogenphosphate

dodecahydrate 2.5 gAgar 15.0 gWater 1000 mL

Sterilize by heating in an autoclave at 1219C for 15 to 20min. pH after sterilization: 7.2 � 7.6.

(vii) Nutrient agar medium or Fluid nutrient mediumMeat extract 3.0 gPeptone 5.0 gAgar 15.0 gWater 1000 mL


(viii) Fluid cooked meat mediumBovine heart extract powder*2

An amount equivalent to 450 g of the materialPeptone 20.0 gGlucose 2.0 gSodium chloride 5.0 gWater 1000 mL


(ix) Reinforced clostridial agar medium or Fluid rein-forced clostridial medium

Meat extract 10.0 gPeptone 10.0 gYeast extract 3.0 gSoluble starch 1.0 gGlucose 5.0 gL-Cystein hydrochloride monohydrate 0.5 gSodium chloride 5.0 gSodium acetate trihydrate 3.0 gAgar 15.0 g

Water 1000 mLFor fluid medium, add 0.5 g of agar. Sterilize by heatingin an autoclave at 1219C for 15 to 20 min. pH aftersterilization: 6.7 � 6.9.

*1 Bovine brain extract powder Dried extract of bovinefresh brain. A yellow-brown powder having a characteristicodor.

Loss on drying: not more than 5z.*2 Bovine heart extract powder Dried extract of bovinefresh heart. A yellow-brown powder having a characteristicodor.

Loss on drying: not more than 5z.

8. Rinsing Liquids(i) Phosphate buffer (pH 7.2)See Microbial Limit Test.

(ii) Buffered sodium chloride-peptone solution (pH 7.0)See Microbial Limit Test.

(iii) Ringer's solution, 1/4 concentrationSodium chloride 2.25 gPotassium chloride 0.105 gCalcium chloride dihydrate 0.16 gWater 1000 mL

Sterilize by heating in an autoclave at 1219C for 15 to 20min. pH after sterilization: 7.0.

(iv) Thiosulfate-Ringer's solutionSodium thiosulfate pentahydrate 0.8 gRinger's solution, 1/4 concentration 1000 mL


(v) LP liquidCasein peptone 1.0 gSoybean lecithin 0.7 gPolysorbate 80 1.0 � 20.0 gWater 1000 mL


Preservatives-Effectiveness TestsThe purpose of the Preservatives-Effectiveness Tests is to

assess microbiologically the preservative efficacy, either dueto the action of product components themselves or any ad-ded preservative(s), for multi-dose containers. The efficacyof the preservatives is assessed by direct inoculation and mix-ing of the test strains in the product, and titration of survivalof the test strains with time.

Preservatives must not be used solely to comply with GMPfor drugs or to reduce viable aerobic counts. In addition,preservatives themselves are toxic substances. Therefore,preservatives must not be added to products in amountswhich might jeopardize the safety of human beings, and con-sideration must be given to minimizing the amounts ofpreservative used. These tests are commonly used to verifythat products maintain their preservative effectiveness at thedesign phase of formulation or in the case of periodicmonitoring. Although these tests are not performed for lot


release testing, the efficacy of the preservative present in theproduct packaged in the final containers should be verifiedthroughout the entire dating period.

1. Products and their CategoriesThe products have been divided into two categories for

these tests. Category I products are those made with aqueousbases or vehicles, and Category II products are those madewith nonaqueous bases or vehicles. Oil-in-water emulsionsare considered Category I products, and water-in-oil emul-sions Category II products. Category I is further divided intothree subtypes depending on the dosage forms.

Category IA: Injections and other sterile parenteralsCategory IB: Non-sterile parenteralsCategory IC: Oral products in liquid forms (including

syrup products to be dissolved or suspended before use)Category II: All the dosage forms listed under Category I

made with non-aqueous bases or vehicles.

2. Test Microorganisms and Culture MediaThe following strains or those considered to be equivalent

are used as the test microorganisms.Escherichia coli ATCC 8739, NBRC 3972Pseudomonas aeruginosa ATCC 9027, NBRC 13275Staphylococcus aureus ATCC 6538, NBRC 13276Candida albicans ATCC 10231, NBRC 1594, JCM 2085Aspergillus brasiliensis ATCC 16404, NBRC 9455These test microorganisms are representative of those that

might be found in the environment in which the product ismanufactured, used or stored, and they are also recognizedas opportunistic pathogens. In addition to these strainsdesignated as test microorganisms, it is further recommend-ed to use strains that might contaminate the product andgrow on or in it, depending on its characteristics. For the testmicroorganisms received from coordinated collections ofmicroorganisms, one passage is defined as the transfer ofmicroorganisms from an established culture to fresh me-dium, and microorganisms subjected to not more than fivepassages should be used for the tests. Single-strain challengesrather than mixed cultures should be used. The test strainscan be harvested by growth on solid agar or liquid media.

Cultures on agar plate media: Inoculate each of the fivetest strains on the surface of agar plates or agar slants. Forgrowth of bacteria, use Soybean-Casein Digest Agar Medi-um, and for yeasts and moulds, use Sabouraud GlucoseAgar, Glucose-Peptone Agar or Potato Dextrose Agar Medi-um. Incubate bacterial cultures at 309C to 359C for 18 to 24hours, the culture of C. albicans at 209C to 259C for 40 to48 hours and the culture of A. brasiliensis at 209C to 259Cfor one week or until good sporulation is obtained. Harvestthese cultured cells aseptically using a platinum loop, etc.Suspend the collected cells in sterile physiological saline or in0.1z Peptone Water and adjust the viable cell count toabout 108 microorganisms per mL. In the case of A.brasiliensis, suspend the cultured cells in sterile physiologicalsaline or 0.1z Peptone Water containing 0.05 w/vz ofpolysorbate 80 and adjust the spore count to about 108 permL. Use these suspensions as the inocula.

Liquid cultures: After culturing each of the four strainsexcept for A. brasiliensis in a suitable medium, remove themedium by centrifugation. Wash the cells in sterile physio-logical saline or 0.1z Peptone Water and resuspend them inthe same solution with the viable cell or spore count of theinoculum adjusted to about 108 per mL.

When strains other than the five listed above are cultured,select a culture medium suitable for growth of the strain con-cerned. The cell suspension may also be prepared by amethod suitable for that strain. Use the inoculum suspen-sions within 24 hours after they have been prepared from thecultivations on agar plate media or in liquid media. Store theinoculum suspensions in a refrigerator if it is not possible toinoculate them into the test specimens within 2 hours. Titratethe viable cell count of the inocula immediately before use,and then calculate the theoretical viable cell count per mL (g)of the product present just after inoculation.

3. Test Procedure3.1. Category I products

Inject each of the cell suspensions aseptically into five con-tainers containing the product and mix uniformly. When it isdifficult to inject the cell suspension into the container asep-tically or the volume of the product in each container is toosmall to be tested, transfer aseptically a sufficient volume ofthe product into each of alternative sterile containers, andmix the inoculum. When the product is not sterile, incubateadditional containers containing the uninoculated product ascontrols and calculate their viable cell counts (the viablecounts of bacteria and those of yeasts and moulds). A sterilesyringe, spatula or glass rod may be used to mix the cell sus-pension uniformly in the product. The volume of the suspen-sion mixed in the product must not exceed 1/100 of thevolume of the product. Generally, the cell suspension is in-oculated and mixed so that the concentration of viable cellsis 105 to 106 cells per mL or per gram of the product. Incu-bate these inoculated containers at 209C to 259C with pro-tection from light, and calculate the viable cell count of 1mL or 1 g of the product taken at 0, 14 and 28 days subse-quent to inoculation. Record any marked changes (e.g.,changes in color or the development of a bad odor) when ob-served in the mixed samples during this time. Such changesshould be considered when assessing the preservative effi-cacy of the product concerned. Express sequential changes inthe viable counts as percentages, with the count at the startof the test taken as 100. Titration of the viable cell counts isbased, in principle, on the Pour Plate Methods in ``Micro-bial Limit Tests''. In this case, confirm whether any anti-microbial substance is present in the test specimen. If aconfirmed antimicrobial substance needs to be eliminated,incorporate an effective inactivator of the substance in thebuffer solution or liquid medium to be used for dilution ofthe test specimen, as well as in the agar plate count medium.However, it is necessary to confirm that the inactivator hasno effect on the growth of the microorganisms. When theoccurrence of the preservative or the product itself affectstitration of the viable cell count and there is no suitable inac-tivator available, calculate the viable cell counts by the Mem-brane Filtration Method in ``Microbial Limit Tests''.3.2. Category II products

The procedures are the same as those described for Cate-gory I products, but special procedures and considerationsare required for both uniform dispersion of the test microor-ganism in the product and titration of viable cell counts inthe samples.

For semisolid ointment bases, heat the sample to 459C to509C until it becomes oily, add the cell suspension anddisperse the inoculum uniformly with a sterile glass rod orspatula. Surfactants may also be added to achieve uniform


dispersion, but it is necessary to confirm that the surfactantadded has no effect on survival or growth of the testmicroorganisms and that it does not potentiate the preserva-tive efficacy of the product. For titration of the viable cellcount, a surfactant or emulsifier may be added to dispersethe product uniformly in the buffer solution or liquid me-dium. Sorbitan monooleate, polysorbate 80 or lecithin maybe added to improve miscibility between the liquid mediumand semisolid ointments or oils in which test microorganismswere inoculated. These agents serve to inactivate or neutral-ize many of the most commonly used preservatives.

Table 1 Interpretation criteria by product category

Productcategory Microorganisms

Interpretation criteria

After 14 days After 28 days

Category IA

Bacteria 0.1z of inocu-lum count orless

Same or lessthan level after14 days

Yeasts/moulds Same or lessthan inoculumcount

Same or lessthan inoculumcount

Category IB

Bacteria 1z of inoculumcount or less




Category IC

Bacteria 10z of inocu-lum count orless




Category II

Bacteria Same or lessthan inoculumcount




4. InterpretationInterpret the preservative efficacy of the product accord-

ing to Table 1. When the results described in Table 1 are ob-tained, the product examined is considered to be effectivelypreserved. There is a strong possibility of massive microbialcontamination having occurred when microorganisms otherthan the inoculated ones are found in the sterile product tobe examined, and caution is required in the test proceduresand/or the control of the manufacturing process of theproduct. When the contamination level in a nonsterileproduct to be examined exceeds the microbial enumerationlimit specified in ``Microbial Attributes of Nonsterile Phar-maceutical Products'' in General Information, caution isalso required in the test procedures and/or the control of themanufacturing process of the product.

5. Culture MediaCulture media and buffer solution used for Preservatives-

Effectiveness Tests are described below. Other media may beused if they have similar nutritive ingredients and selectiveand growth-promoting properties for the microorganisms tobe tested.

(i) Soybean Casein Digest Agar MediumCasein peptone 15.0 gSoybean peptone 5.0 gSodium chloride 5.0 gAgar 15.0 gWater 1000 mL

Mix all of the components and sterilize at 1219C for 15 �20 minutes in an autoclave. pH after sterilization: 7.1 � 7.3.

(ii) Sabouraud Glucose Agar MediumPeptone (animal tissue and casein) 10.0 gGlucose 40.0 gAgar 15.0 gWater 1000 mL


(iii) Glucose Peptone (GP) Agar MediumGlucose 20.0 gYeast extract 2.0 gMagnesium sulfate heptahydrate 0.5 gPeptone 5.0 gMonobasic potassium phosphate 1.0 gAgar 15.0 gWater 1000 mL


(iv) Potato Dextrose Agar MediumPotato extract 4.0 gGlucose 20.0 gAgar 15.0 gWater 1000 mL


(v) 0.1z Peptone WaterPeptone 1.0 gSodium chloride 8.0 gWater 1000 mL


Rapid Counting of Microbes usingFluorescent Staining

This chapter provides rapid methods using fluorescencestaining for the quantitative estimation of viable microor-ganisms. Incubation on an agar medium has been widelyused for quantitative estimation of viable microorganisms,but a number of environmental microorganisms of interestare not easy to grow in culture under usual conditions, thusnew microbial detection methods based on fluorescence orluminescence have been developed. In the fluorescence stain-ing method, microorganisms are stained with fluorescentdye, and can easily be detected and counted with varioussorts of apparatus, such as a fluorescence microscope orflow cytometer. Methods are available to detect total


microorganisms, including both dead and viable cells, or todetect only cells with a specified bioactivity by choosing thedye reagent appropriately. Nucleic acid staining reagents,which bind with DNA or RNA, detect all cells containingnucleic acids, whether they are live or dead. This technique isthe most fundamental for the fluorescence staining method.On the other hand, fluorescent vital staining methods targetthe respiratory activity of the microorganism and the activityof esterase, which is present universally in microorganisms.In the microcolony method, microcolonies in the early stageof colony formation are counted. The CFDA-DAPI doublestaining method and the microcolony method are describedbelow. These methods can give higher counts than the othertechniques, because these rapid and accurate techniques pro-vide quantitative estimation of viable microorganisms basedon a very specific definition of viability, which may bedifferent from that implicit in other methods. The proce-dures of these methods described here may be changed as ex-perience with the methods is accumulated. Therefore, otherreagents, instruments and apparatus than those describedhere may also be used if there is a valid reason for so doing.

1. CFDA-DAPI double staining methodFluorescein diacetate (FDA) reagents are generally used

for the detection of microorganisms possessing esteraseactivity. These reagents are hydrolyzed by intracellularesterase, and the hydrolyzed dye exhibits green fluorescenceunder blue excitation light (about 490 nm). Modified FDAssuch as carboxyfluorescein diacetate (CFDA) are used be-cause of the low stainability of gramnegative bacteria withFDA. The principle of the CFDA-DAPI double stainingmethod, which also employs a nucleic acid staining reagent,4?,6-diamidino-2-phenylindole (DAPI), is as follows. Thenonpolar CFDA penetrates into the cells and is hydrolyzedto fluorescent carboxyfluorescein by intracellular esterase.The carboxyfluorescein is accumulated in the living cells dueto its polarity, and therefore green fluorescence due to car-boxyfluorescein occurs when cells possessing esterase activityare illuminated with blue excitation light. No fluorescent car-boxyfluorescein is produced with dead cells, since they areunable to hydrolyze CFDA. On the other hand, DAPI bindspreferentially to the adenine and thymine of DNA afterpenetration into both viable and dead microorganisms, andconsequently all of the organisms containing DNA exhibitblue fluorescence under ultraviolet excitation light. There-fore, this double staining method enables to count spe-cifically only live microorganisms possessing esterase activityunder blue excitation light, and also to determine the totalmicrobial count (viable and dead microorganisms) under ul-traviolet excitation light.1.1. Apparatus1.1.1. Fluorescence microscope or fluorescence observa-tion apparatus

Various types of apparatus for counting fluorescen-cestained microorganisms are available. Appropriate filtersare provided, depending on the fluorescent dye reagentsused. A fluorescence microscope, laser microscope, flowcytometer, and various other types of apparatus may be usedfor fluorescence observation.1.2. Instruments

(i) Filtering equipment (funnels, suction flasks, suctionpumps)

(ii) Membrane filters (poresize: 0.2 mm); A suitable filter

that can trap particles on the surface can be used such aspolycarbonate filter, alumina filter, etc.

(iii) Glass slide(iv) Cover glass(v) Ocular micrometer for counting (with 10× 10 grids)

1.3. ProcedureAn example of the procedure using fluorescence micro-

scope is described below.1.3.1. Preparation of samples

Prepare samples by ensuring that microbes are dispersedevenly in the liquid (water or buffer solution).1.3.2. Filtration

Set a membrane filter (poresize: 0.2 mm) on the funnel ofthe filtering equipment. Filter an appropriate amount ofsample to trap microbes in the sample on the filter.1.3.3. Staining

Pour sufficient amount of buffer solution for CFDAstaining, mixed to provide final concentration of 150 mg/mLof CFDA and 1 mg/mL of DAPI, into the funnel of thefiltering equipment and allow staining in room temperaturefor 3 minutes, then filter the liquid by suction. Pour insufficient amount of aseptic water, suction filter and removeexcess fluorescent reagent left on the filter. Thoroughly drythe filter.1.3.4. Slide preparation

Put one drop of immersion oil for fluorescence micro-scope on the glass slide. Place the air dried filter over it, withthe filtering side on the top. Then put one drop of immersionoil for fluorescence microscope on the surface of the filter,place a cover glass. Put another drop of immersion oil forfluorescence microscope on the cover glass when using anoilimmersion objective lens.1.3.5. Counting

Observe and count under fluorescence microscope, with1000 magnification. In case of CFDA-DAPI double stainingmethod, count the microorganisms (with esterase activity)exhibiting green fluorescence under the blue excitation lightfirst to avoid color fading by the ultraviolet light, then countthe microorganisms (with DNA) exhibiting blue fluorescenceunder the ultraviolet excitation light in the same microscopicfield. Count the organisms exhibiting fluorescence on morethan 20 randomly selected fields among 100 grids observedthrough an ocular micrometer of the microscope, and calcu-late the total number of organisms using the following for-mula. The area of the microscopic field should be previouslydetermined with the ocular and objective micrometers. Theamount of the sample to be filtered must be adjusted so thatthe cell number per field is between 10 and 100. It might benecessary to reprepare the sample in certain instances. (Insuch case that the average count number is not more than 2organisms per field, or where more than 5 fields are foundwhich have no organism per field, it is assumed that themicroorganism count is below the detection limit.)

Number of microbes (cells/mL)＝s(average number of microbes per visual field)× (area of filtration)t/s(amount of samplefiltered)× (area of one microscopic field)t

1.4. Reagents and test solutions(i) Aseptic water: Filter water through a membrane filter

with 0.2 mm pore size to remove particles, then sterilize it byheating in an autoclave at 1219C for 15 minutes. Water forinjection may be used.


(ii) CFDA solution, 10 mg/mL: Dissolve 50 mg ofCFDA in dimethylsulfoxide to prepare a 5 mL solution.Store at －209C in light shielded condition.

(iii) Buffer solution for CFDA staining: Dissolve 5 g ofsodium chloride with 0.5 mL of 0.1 mol/L disodium dihy-drogen ethylenediamine tetraacetate TS and diluted disodi-um hydrogen phosphate TS (1 in 3) to prepare 100 mL of so-lution. Add sodium dihydrogen phosphate dihydrate solu-tion (1 in 64) to adjust the pH level to 8.5. Filter the solutionthrough a membrane filter with a pore size of 0.2 mm.

(iv) DAPI solution, 10 mg/mL: Dissolve 10 mg of DAPIin 100 mL of aseptic water. Dilute this solution 10 times withaseptic water and filter through a membrane filter with apore size of 0.2 mm. Store at 49C in light shielded condition.

(v) Immersion oil for fluorescence microscope

2. Microcolony methodMicrocolonies, which are in early stages of colony forma-

tion, are fluorescently stained, then observed and countedunder fluorescence microscope or other suitable systems.This method enables to count the number of proliferativemicroorganisms, with short incubation time. In this method,the organisms are trapped on a membrane filter, the filter isincubated on a medium for a short time, and the microcolo-nies are counted. By this method, even colonies which areundetectable with the naked eye can be identified, so viableorganisms can be counted rapidly and with high precision.Various nucleic acid staining reagents can be used for stain-ing of microcolonies.2.1. Apparatus2.1.1. Fluorescence microscope or fluorescence observa-tion apparatus

Various types of apparatus for counting fluorescen-cestained microorganisms are available. Appropriate filtersare provided, depending on the fluorescence dye reagentsused. A fluorescence microscope, laser microscope and vari-ous other types of apparatus may be used for fluorescenceobservation.2.2. Instruments

(i) Filtering equipment (funnels, suction flasks, suctionpumps)

(ii) Membrane filters (pore size: 0.2 mm); A suitable filterthat can trap particles on the surface can be used such aspolycarbonate filter, alumina filter, etc.

(iii) Glass slide(iv) Cover glass(v) Filter paper (No. 2)(vi) Ocular micrometer for counting (with 10× 10 grids)

2.3. ProcedureAn example of the procedure using a fluorescence micro-

scope is described below.2.3.1. Preparation of samples

Prepare samples by ensuring that microbes are dispersedevenly in the liquid (water or buffer solution).2.3.2. Filtration

Set a membrane filter (pore size: 0.2 mm) on the funnel ofthe filtering equipment. Filter an appropriate amount ofsample to trap microbes in the sample on the filter.2.3.3. Incubation

Remove the filter from the filtering equipment and place itwith filtering side facing up on a culture medium avoidingformation of airbubbles between the filter and the medium.Incubate at a suitable temperature for appropriate hours in a

dark place. It should be noted that the appropriate incuba-tion conditions (such as medium, incubation temperatureand/or incubation time) are different, depending on thesample.2.3.4. Fixation

Soak filter paper with an appropriate amount of neutralbuffered formaldehyde test solution, then place the filterthat has been removed from the culture medium on top withfiltering side up, and allow to remain at room temperaturefor more than 30 minutes to fix the microcolonies.2.3.5. Staining

Soak filter paper with an appropriate amount of stainingsolution (such as 1 mg/mL of DAPI, 2z polyox-yethylenesorbitan monolaurate), then place the filter on topwith filtering side up, and then leave at room temperature,light shielded for 10 minutes to stain microcolonies. Washthe filter by placing it with the filtering side facing up on topof a filter paper soaked with aseptic water for 1 minute.Thoroughly air dry the filter.2.3.6. Slide preparation

Put one drop of immersion oil for fluorescence micro-scope on the slide glass. Place an air dried filter over it, withthe filtering side on the top. Then, put one drop of immer-sion oil for fluorescence microscope on top, place a coverglass.2.3.7. Counting

Count the organisms exhibiting fluorescence on more than20 randomly selected fields among the 100 grids observedthrough an ocular micrometer of the microscope with 400 or200 magnification, and calculate the total number of organ-isms using the following formula. The area of themicroscopic field should be previously determined with theocular and objective micrometers. In such case that theaverage count number is not more than 2 microcolonies perfield, or where more than 5 fields are found which have nomicrocolony per field, it is assumed that the microorganismcount is below the detection limit.

Number of microcolonies (cells/mL)＝s(average number of microcolonies per visual field)× (area of filtration)t/s(amount of samplefiltered)× (area of one microscopic field)t

2.4. Reagents and test solutions(i) Aseptic water: Filter water through a membrane filter

with 0.2 mm pore size to remove particles, and sterilize it byheating in an autoclave at 1219C for 15 minutes. Water forinjection may be used.

(ii) Staining solution: Dissolve 10 mg of DAPI in 100 mLof aseptic water. Dilute the solution 10 times with asepticwater and filter through a membrane filter with pore size of0.2 mm. Store at 49C in light shielded condition. Dissolvepolyoxyethylene sorbitan monolaurate to the final concen-tration of 2z, when using.

(iii) Neutral buffered formaldehyde solution (4w/vzformaldehyde solution; neutrally buffered).

(iv) Immersion oil for fluorescence microscope


Rapid Identification ofMicroorganisms Based on

Molecular Biological MethodThis chapter describes the methods for the identification

or estimation of microorganisms (bacteria and fungi), foundin in-process control tests or lot release tests of pharmaceuti-cal products, at the species or genus level based on theirDNA sequence homology. The identification of isolatesfound in the sterility test or aseptic processing can be helpfulfor investigating the causes of contamination. Furthermore,information on microorganisms found in raw materials usedfor pharmaceutical products, processing areas of phar-maceutical products, and so on is useful in designing meas-ures to control the microbiological quality of drugs. For theidentification of microorganisms, phenotypic analysis iswidely used, based on morphological, physiological, andbiochemical features and analysis of components. Commer-cial kits based on differences in phenotype patterns havebeen used for the identification of microorganisms, but arenot always applicable to microorganisms found in raw mate-rials used for pharmaceutical products and in processingareas of pharmaceutical products. In general, the identifica-tion of microorganisms based on phenotypic analysis needsspecial knowledge and judgment is often subjective. It isconsidered that the evolutionary history of microorganisms(bacteria and fungi) is memorized in their ribosomal RNAs(rRNAs), so that systematic classification and identificationof microorganisms in recent years have been based on theanalysis of these sequences. This chapter presents a rapidmethod to identify or estimate microorganisms based on par-tial sequences of divergent regions of the 16S rRNA gene forbacteria and of the internal transcribed spacer 1 (ITS1)region located between 18S rRNA and 5.8S rRNA for fungi,followed by comparison of the sequences with those in thedatabase. Methods described in this chapter do not take theplace of usual other methods for the identification, and canbe modified based on the examiner's experience, and on theavailable equipment or materials. Other gene regions besidesthose mentioned in this chapter can be used if appropriate.

1. Apparatuses(i) DNA sequencerVarious types of sequencers used a gel board or capillary

can be used.(ii) DNA amplifierTo amplify target DNA and label amplified (PCR) prod-

ucts with sequencing reagents.

2. ProceduresThe following procedures are described as an example.

2.1. Preparation of template DNAIt is important to use a pure cultivated bacterium or fun-

gus for identification. In the case of colony samples, colo-nies are picked up with a sterilized toothpick (in the case offungi, a small fragment of colony sample is picked up), andsuspended in 0.3 mL of DNA releasing solution in a 1.5 mLcentrifuge tube. In the case of culture fluid, a 0.5 mL por-tion of fluid is put in a 1.5 mL centrifuge tube and centri-fuged at 10,000 rpm for 10 min. After removal of the super-natant, the pellet is suspended in 0.3 mL of DNA releasing

solution, and then heated at 1009C for 10 min. In general,PCR can be run for bacteria and yeasts heated in DNAreleasing solution. For fungi, DNA extraction from culturefluid is better because some of colony samples can disturbPCR reaction.2.2. PCR

Add 2 mL of template DNA in PCR reaction solution. Use10F/800R primers (or 800F/1500R primers in the case toanalyze also a latter part of 16S rRNA) for bacteria andITS1F/ITS1R primers for fungi, and then perform 30 am-plification cycles at 949C for 30 sec, 559C for 60 sec, and729C for 60 sec. DNA fragments are amplified about 800 bpin the case of bacteria and about 150 � 470 bp depending onthe strain in the case of fungi. Include a negative control(water instead of the test solution) in the PCR.2.3. Confirmation of PCR products

Mix 5 mL of PCR product with 1 mL of loading buffer so-lution, place it in a 1.5 w/vz agarose gel well, and carry outelectrophoresis with TAE buffer solution (1-fold concentra-tion). Carry out the electrophoresis together with appropri-ate DNA size markers. After the electrophoresis, observePCR products on a trans-illuminator (312 nm) and confirmthe presence of a single band of the targeted size. If multiplebands are observed, cut the targeted band out of the gel, andextract DNA by using appropriate commercial DNA ex-traction kit.2.4. Purification of PCR products

Remove unincorporated PCR primers and deoxynucleo-side triphosphates (dNTP) from PCR products by using ap-propriate purification methods.2.5. Quantification of purified DNA

When purified DNA is measured by spectrophotometer,calculate 1 OD260 nm as 50 mg/mL.2.6. Labeling of PCR products with sequencing reagents

Use an appropriate fluorescence-labeled sequencing rea-gent suitable for the available DNA sequencer or its programand label the PCR products according to the instructionsprovided with the reagent.2.7. Purification of sequencing reagent-labeled PCR prod-ucts

Transfer the product in 75 mL of diluted ethanol (7 in 10)into a 1.5 mL centrifuge tube, keep in an ice bath for 20 min,and centrifuge at 15,000 rpm for 20 min. After removal ofsupernatant, add 250 mL of diluted ethanol (7 in 10) to theprecipitate and centrifuge at 15,000 rpm for 5 min. Removethe supernatant and dry the precipitate.2.8. DNA homology analysis

Place sequencing reagent-labeled PCR products in theDNA sequencer and read the nucleotide sequences of thePCR products. Compare the partial nucleotide sequencewith those in the BLAST database.

3. JudgmentIf sequencing data show over 90z identity with a se-

quence in the database, in general, judgment may be made asfollows.

( i ) In the case of bacteria, compare the nucleotides inthe product obtained with the 10F primer (the 800Fprimer when 800F/1500R primers are used) with theBLAST database. Higher ranked species are judgedas identified species or closely related species.

(ii) In the case of fungi, compare sequencing data for theproduct obtained with the ITS1F primer with the


BLAST database. Higher ranked species are judgedas identified species or closely related species.

4. Reagents, Test Solutions(i) 0.5 mol/L Disodium dihydrogen ethylenediaminetetraacetate TS: Dissolve 18.6 g of disodium dihydrogenethylenediamine tetraacetate dihydrate in water to make 100mL.(ii) 1 mol/L Tris buffer solution, pH 8.0: Dissolve 24.2 gof 2-amino-2-hydroxymethyl-1,3-propanediol in a suitableamount of water, adjust the pH to 8.0 with 0.2 mol/L hy-drochloric acid TS, and add water to make 200 mL.(iii) TE buffer solution: Mix 1.0 mL of 1 mol/L tris buffersolution, pH 8.0 and 0.2 mL of 0.5 mol/L disodium dihy-drogen ethylenediamine tetraacetate TS, and add water tomake 100 mL.(iv) DNA releasing solution: Divide TE buffer solutioncontaining 1 volz of polyoxyethylene (10) octylphenyl etherinto small amounts and store frozen until use.(v) PCR reaction solution

10-fold buffer solution* 5 mLdNTP mixture** 4 mL10 mmol/L Sense primer 1 mL10 mmol/L Anti-sense primer 1 mLHeat-resistant DNA polymerase (1 U/mL) 1 mLWater 36 mL

* Being composed of 100 mmol/L 2-amino-2-hydrox-ymethyl-1,3-propanediol hydrochloride, pH 8.4, 500 mmol/L potassium chloride, 20 mmol/L magnesium chloride and0.1 g/L gelatin.** A solution containing 2.5 mmol/L each of dGTP (sodi-um 2?-deoxyguanosine 5?-triphosphate), dATP (sodium 2?-deoxyadenosine 5?-triphosphate), dCTP (sodium 2?-deox-ycytidine 5?-triphosphate) and dTTP (sodium 2?-deoxythymi-dine 5?-triphosphate). Adequate products containing thesecomponents as described above may be used.(vi) Sequencing reagent: There are many kinds of sequenc-ing methods, such as the dye-primer method for labeling ofprimer, the dye-terminator method for labeling of dNTP ter-minator and so on. Use an appropriate sequencing reagentkit for the apparatus and program to be used.(vii) 50-Fold concentrated TAE buffer solution: Dissolve242 g of 2-amino-2-hydroxymethyl-1,3-propanediol in 57.1mL of acetic acid (100) and 100 mL of 0.5 mol/L disodiumdihydrogen ethylenediamine tetraacetate TS, and add waterto make 1000 mL.(viii) 1-Fold concentrated TAE buffer solution: Diluted 50-fold concentrated TAE buffer solution (1 in 50) prepared be-fore use is referred to as 1-fold concentrated TAE buffer so-lution.(ix) Agarose gel: Mix 1.5 g of agarose, 2.0 mL of 50-foldconcentrated TAE buffer solution, 10 mL of a solution ofethidium bromide (3,8-diamino-5-ethyl-6-phenylphenan-thridinium bromide) (1 in 100) and 100 mL of water. Afterdissolving the materials by heating, cool the solution toabout 609C, and prepare gels.(x) Loading buffer solution (6-fold concentrated): Dissolve0.25 g of bromophenol blue, 0.25 g of xylene cyanol FF and1.63 g of disodium dihydrogen ethylenediamine tetraacetatedihydrate in 50 mL of water, and add 30 mL of glycerol andwater to make 100 mL.(xi) PCR primers

For Primer

Bacteria 10F800R

5?-GTTTGATCCTGGCTCA-3?5?-TACCAGGGTATCTAATCC-3?

800F 5?-GGATTAGATACCCTGGTA-3?1500R 5?-TACCTTGTTACGACTT-3?

Fungi ITS1FITS1R

5?-GTAACAAGGT(T/C)TCCGT-3?5?-CGTTCTTCATCGATG-3?

(xii) Polyoxyethylene(10)octylphenyl ether: A pale yellow,viscous liquid.

Sterility Assurance for TerminallySterilized Pharmaceutical ProductsAs indicated in the ``Terminal Sterilization and Steriliza-

tion Indicators'', the pharmaceuticals to which terminalsterilization can be applied, generally must be sterilized sothat a sterility assurance level of 10－6 or less is obtained. Thesterility assurance level of 10－6 or less can be proven byusing a sterilization process validation based on physical andmicrobiological methods, but cannot be proven by sterilitytests of the sterilized products. This chapter deals with thenecessary requirements for the appropriate management ofthe important control points of the sterilization process forthe parametric release of products, without performingsterility tests on products which have been subjected toterminal sterilization (in the case of radiation sterilization,called dosimetric release). Parametric release is a methodthat can be applied in cases where the sterilization system isclearly defined, important control points are clearly speci-fied, and the sterilization system process can be validated bymicrobiological methods using appropriate biological indica-tors.

1. DefinitionsThe definitions of the terminology used in this chapter are

provided below.1.1. Terminal sterilization

A process whereby a product is sterilized in its final con-tainer or packaging, and which permits the measurement andevaluation of quantifiable microbial lethality.1.2. Validation

A documented procedure for obtaining, recording andinterpreting the results needed to show that a process willconsistently yield a product complying with predeterminedspecifications.1.3. Periodic re-validation

Validation that is regularly performed to reconfirm that aprocess is consistently yielding a product complying withpredetermined specifications. It should confirm that varia-bles and the acceptable ranges are permissible to yield aproduct consistently of the required quality.1.4. Facility/equipment qualification

This is to provide evidence that the manufacturing facili-ties/equipment, measuring equipment, and manufacturingenvironment control facilities, etc. have been properly select-ed, correctly installed, and are operated in conformity withthe specifications at the time of installation and during oper-ation.


1.5. Operation qualificationThis is to provide evidence to confirm physically, chemi-

cally and microbiologically that equipment, operated in ac-cordance with its operational instructions, operates as speci-fied and affords a product meeting the specifications.1.6. Support system for sterilization process

This refers to the facility/equipment that is associatedwith the sterilization devices, such as the preconditioningand aeration for ethylene oxide sterilization, the steam supp-ly equipment for moist heat sterilization, and the loadingdevices for radiation sterilization.1.7. Quality system

The procedures, resources and organizational structure ofa manufacturer (responsibilities, authorities and relation-ships between these) required to implement quality manage-ment.1.8. Change control system

A system designed to evaluate all of the changes that mayaffect the quality of the pharmaceutical product, in order toensure that the process is continuously controlled.1.9. F0 value

Assume a value of 109C for the Z value defined as thenumber of degrees of temperature required for a 10-foldchange in the D value. The F0 value indicates the time(minutes) required to give the equivalent lethality at Tb of thesterilization heat obtained by integrating the lethality rate (L)over an entire heating cycle.

L＝ log－1T0－ Tb

Z＝ 10

T0－Tb

Z

T0＝ Temperature inside the chamber or inside theproduct to be sterilized

Tb＝ Reference temperature (1219C)

F0＝ft1

t0Ldt

t1－ t0＝ Processing time (minutes)

1.10. Control deviceA general term for the devices and measurement equip-

ment, including the equipment for controlling, measuringand recording the physical parameters that can be measured(temperature, humidity, pressure, time, radiation dose, etc.).1.11. Parametric release

A release procedure based on an evaluation of the produc-tion records and critical parameters of the sterilizationprocess (temperature, humidity, pressure, time, radiationdose, etc.) based on the results of validation, in lieu ofrelease based on testing results of the final product.

2. Sterilization Validation2.1. Subject of the Implementation

A manufacturer of sterile pharmaceuticals (hereafter,``manufacturer'') must establish a quality system, implementproduct sterilization validation for the categories below as ageneral rule, and continuously control the sterilizationprocess based on the results of the sterilization validation.

a) Sterilization processb) Sterilization process support system

2.2. Documenting Sterilization Validation Procedure2.2.1. The manufacturer must prepare a ``SterilizationValidation Procedure'' defining the items listed belowregarding the procedures for managing the sterilization

process.a) Details related to the range of duties of the persons

responsible for the validation, as well as the extent of theirauthority

b) Details related to the implementation period for thesterilization validation

c) Details related to the creation, modification, and ap-proval of the sterilization validation plan documents

d) Details related to the reporting, evaluation, and ap-proval of the sterilization validation implementation results

e) Details related to the storage of documentation con-cerning the sterilization validation

f) Other required matters2.2.2. The sterilization validation procedure must list thenames of the enactors, the date of enactment, and whenthere are revisions, must also list the revisers, date of revi-sions, revised sections and reasons for the revisions.2.2.3. The manufacturer must properly store and maintainthe sterilization validation procedure after clarifying theprocedures related to alterations and deletions of the con-tents of the sterilization validation procedure.2.3. Persons Responsible for the Validation

The manufacturer must assign persons to be responsiblefor the sterilization validation. The responsible parties mustperform each of the duties listed below according to thesterilization validation procedure.2.3.1. For products that are to be produced according tothe sterilization validation procedure, a written sterilizationvalidation implementation plan must be prepared. The im-plementation plan will specify the following points based ona consideration of the implementation details of the steriliza-tion validation.

a) Subject pharmaceutical name (product name)b) Purpose of the applicable sterilization validationc) Expected resultsd) Verification methods (including inspection results and

evaluation methods)e) Period of verification implementationf) Names of persons performing the sterilization valida-

tion (persons-in-charge)g) Names of the persons who created the plan, creation

date, and in the event of revisions, the names of the revisers,date of the revisions, revised sections, and reasons for revi-sion.

h) Technical requirements for the applicable sterilizationvalidation

i) Other required matters for the implementation of theapplicable sterilization validation2.3.2. The following sterilization validation is implementedaccording to the plan defining the items above.

a) When the manufacturing license and additional(modification) licenses for product production are obtained,implementation items for the sterilization validation to beexecuted

1 Product qualification2 Facility/equipment qualification

1) Installation qualification2) Operation qualification

3 Performance qualification1) Physical performance qualification2) Microbiological performance qualification

b) Sterilization validation to be executed until it is timeto renew the manufacturing license


1 Re-validation when there are changes2 Periodic re-validation (The items implemented, etc.

must be determined based on a consideration ofrelevant factors such as the sterilization method.)

2.3.3. Evaluate the results of the sterilization validationand verify that sterility is assured.2.3.4. Make a written report of the results of the steriliza-tion validation to the manufacturer's authorized person.2.3.5. Perform the day-to-day management of the steriliza-tion process.

3. Microorganism Control ProgramWhen parametric release is adopted, it is important to

control the bioburden in the raw materials of the product,the containers and stoppers, and in the product beforesterilization. The bioburden is measured with a previouslyspecified method and frequency, and when required, surveysof the characteristics of the isolated microorganisms aremade to investigate their resistance to the applicable steriliza-tion method. Refer to the ``Microbiological Evaluation ofProcess Areas for Sterile Pharmaceutical Products'' regard-ing the method for evaluating the environmental microor-ganisms in the processing areas of pharmaceutical products.

4. Sterilization IndicatorsBiological indicators (BI), chemical indicators (CI), and

dosimeters are among the means used to monitor a steriliza-tion process and as indices of sterility (refer to TerminalSterilization and Sterilization Indicators). When using sterili-zation indicators it is important to consider environmentaland human safety, and to take all necessary precautions. TheBI used for sterilization validation and daily process controlmust be defined in the specification, and recorded in writing.When BI are used for daily process control it must be veri-fied that the loading pattern on the form, product, or simu-lated product has a resistance equal to or greater than thatused for the microbiological performance qualification.

5. Establishment of a Change Control SystemChanges which have a large effect on the sterile quality,

such as changes in sterilization equipment, loading pattern,and sterilization conditions, correspond to changes of theparametric release conditions for the relevant pharmaceuti-cal product. A change control system must be defined in thesterilization validation procedure; and when there arechanges in the causes of variation that have been previouslyspecified, there must be an investigation of the causes ofvariation and of acceptable conditions to verify that thepharmaceutical product is guaranteed always to conform tothe quality standards. Furthermore, before modifications aremade to a sterilization process that has been validated, it ismandatory to obtain approval for the implementation of themodifications in question from the appropriate authorizedperson.

6. Release ProcedureA release procedure must be created to clarify the condi-

tions required for shipment based on parametric release ofterminally sterilized products. The following points must beevaluated and recorded when a product is released.

Depending on the sterilization method, some of theseitems may be omitted or modified.

a) Batch recordb) Microorganism evaluation data of production en-

vironment

c) Bioburden data for the raw materials and product be-fore sterilization

d) Data related to the sterilization indicatorse) Data on the maintenance management of the steriliza-

tion process and sterilization process support systemsf) Data on the management of sterilization parametersg) Data on the calibrations of measurement equipmenth) Re-validation datai) Other

7. Critical Control PointsThe important control points for each sterilization method

are presented.7.1. Moist heat sterilization

Moist heat sterilization is a method for killing microorgan-isms in which saturated water vapor is generated or intro-duced into a sterilization chamber at the appropriate temper-ature and pressure, and the chamber is then heated for a cer-tain period of time. It is roughly classified into saturatedvapor sterilization, in which the target microorganisms aredirectly exposed to the saturated vapor, and unsaturatedvapor sterilization, in which the fluid inside a container,such as an ampule, is subjected to moist heat energy orhighfrequency energy from the outside.7.1.1. Important control points

A process control procedure must be created, specifyingthe process parameters that affect the sterile quality of thepharmaceutical product, and the permissible range of varia-tion for each parameter. The important control points forthe moist heat sterilization are indicated below.

a) Heating history (usually indicated by F0 value)b) Temperaturec) Pressured) Timee) Product loading format/loading densityf) Other necessary matters

7.1.2. UtilitiesThe utilities and control devices required for moist heat

sterilization determine the quality and precision.a) Quality of the vapor usedb) Quality of the air introduced into the sterilization

chamber to restore pressure, etc.c) Quality of the water used for coolingd) Precision of the temperature control devicese) Precision of the pressure control devicesf) Precision of the time control devicesg) Other

7.2. Ethylene oxide gas sterilizationEthylene oxide gas allows sterilization at low tempera-

tures, so there is typically little injury to the substance beingsterilized; however, since the gas is toxic it must be handledwith extreme caution. The sterilization process consists ofpreconditioning, a sterilization cycle, and aeration. Thepreconditioning is performed before the sterilization cycle toprocess the product so that temperature and relative hu-midity in the room or container are within the range in thespecifications. The sterilization cycle indicates the stage atwhich the actual sterilization is performed, and consists ofremoval of the air, conditioning (when used), injection ofthe sterilization gas, maintenance of the sterilization condi-tions, removal of the sterilization gas, and replacement ofthe air. The aeration is the process of eliminating the residualethylene oxide gas from the product, either inside the sterili-


zation chamber or in a separate location.7.2.1. Important control points

The important control points for the ethylene oxide gassterilization are indicated below.7.2.1.1. Preconditioning (when performed)

a) Time, temperature, humidityb) Product loading pattern/loading densityc) Sterilization loading temperature and/or humidityd) Time from the end of preconditioning until the start

of the sterilizatione) Other necessary matters

7.2.1.2. Conditioninga) If pressure reduction is performed, the pressure

achieved and required timeb) Reduced pressure maintenance periodc) Time, temperature, pressure, humidityd) Sterilization loading temperature and humiditye) Other necessary matters

7.2.1.3. Sterilization cyclea) Pressure increase, injection time, and final pressure

for the injection of the sterilization gasb) Concentration of the ethylene oxide gas (it is desirable

to analyze directly the gas concentration inside the steriliza-tion chamber, but the following alternatives are acceptable ifdirect analysis is difficult)

i) Mass of gas usedii) Volume of gas usediii) Conversion calculation using the initial low pressure

level and the gas injection pressurec) Temperature within the sterilization chamberd) Temperature of the loaded products to be sterilizede) Effect time (exposure time)f) Product loading pattern/loading densityg) BI placement points and cultivation resultsh) Other necessary matters

7.2.1.4. Aerationa) Time, temperatureb) Loaded sterilized substance temperaturec) Pressure variation in the sterilization chamber and/or

the aeration roomd) Rate of change of the air or other gases in the aeration

roome) Other necessary matters

7.2.2. UtilitiesThe utilities and control devices required for ethylene

oxide sterilization determine the quality and precision.a) Quality of the ethylene oxide gasb) Quality of the injected vapor or waterc) Quality of the replacement air after the completion of

sterilizationd) Quality of the BIe) Precision of the temperature control devicesf) Precision of the pressure control devicesg) Precision of the humidity control devicesh) Precision of the time control devicesi) Other

7.3. Irradiation SterilizationIrradiation sterilization refers to methods of killing

microorganisms through exposure to ionizing radiation. Thetypes of ionizing radiation used are gamma-rays (g-rays)emitted from a radioisotope such as 60Co or 137Cs, or elec-tron beams and bremsstrahling (X-ray) generated from anelectron accelerator. In the case of g-rays, the cells are killed

by secondarily generated electrons, while in the case of theelectron beam, the cells are killed by the electrons generateddirectly from the electron accelerator. For this reason, theprocessing time for electron beam sterilization is generallyshorter than that for g-ray sterilization; but, since thepenetration of the g-rays is better than that of the electronbeam, there must be appropriate consideration of the densityand thickness of the substance being sterilized when choos-ing between these methods. For an irradiation sterilizationprocess, the control procedures primarily make use ofdosimeters and measure the absorbed dose in the substancebeing sterilized. This is called dosimetric release.7.3.1. Important control points

The important control points for the irradiation steriliza-tion are indicated below.7.3.1.1. g-ray radiation

a) Irradiation time (timer setting or conveyor speed)b) Absorbed dosec) Product loading patternd) Other necessary matters

7.3.1.2. Electron beam and X-ray radiationa) Electron beam characteristics (average electron beam

current, electron energy, scan width)b) Conveyor speedc) Absorbed dosed) Product loading patterne) Other necessary matters

7.3.2. UtilitiesA traceable calibration, performed according to national

standards, must be performed for the radiation devices anddose measurement systems. This calibration must be per-formed as specified in a written plan in order to verify thatthe equipment is kept within the required range of accuracy.7.3.2.1. Required calibration items for gamma-radiationequipment

a) Cycle time or conveyor speedb) Weighing devicec) Dose measurement systemd) Other

7.3.2.2. Required calibration items for electron-beam andX-ray radiation equipment

a) Electron beam characteristicsb) Conveyor speedc) Weighing deviced) Dose measurement systeme) Other

References1) Validation Standards, PAB Notification No.158,

Ministry of Health and Welfare 19952) Sterilization Validation Standards, PMSB/IGD Notifi-

cation No.1, Ministry of Health and Welfare 19973) Quality Assurance Standards for Medical Devices,

PAB Notification No.1128, Ministry of Health andWelfare 1994

4) ISO 9000 series, International Standards for QualityAssurance

5) ISO 11134 Industrial moist heat sterilization6) ISO 11135 Ethylene oxide sterilization7) ISO 11137 Radiation sterilization8) ISO 11138 Biological indicators9) ISO 11140 Chemical indicators

10) ISO 11737-1 Microbiological Methods Part 1: Estima-


tion of population of microorganisms on products11) USP 〈1222〉 Terminally Sterilized Pharmaceutical

Products - Parametric Release

Terminal Sterilization andSterilization Indicators

Sterilization is a process whereby the killing or removal ofall forms of viable microorganisms in substances is accom-plished. It is achieved by terminal sterilization or a filtrationmethod. For substances to which terminal sterilization canbe applied, an appropriate sterilization method should beselected in accordance with the properties of the product, in-cluding the packaging, after full consideration of the advan-tages and disadvantages of each sterilization method, fromamong the heat method, irradiation method and gas method.After installation of the sterilizer (including design and de-velopment of the sterilization process), validation is requiredto confirm that the sterilization process is properly per-forming its designed function, under conditions of loadingand unloading of the product, on the basis of sufficientscientific evidence. After the process has been validated andthe sterilization of the product commenced, the process mustbe controlled correctly, and qualification tests of the equip-ment and procedures must be performed regularly.

The bioburden per product, prior to terminal sterilization,must be evaluated periodically or on the basis of batches.Refer to the ISO standard (ISO 11737-1) relevant to biobur-den estimation. For a substance to which terminal steriliza-tion can be applied, generally use sterilization conditionssuch that a sterility assurance level of less than 10－6 can beobtained. The propriety of the sterilization should be judgedby employing an appropriate sterilization process control,with the use of a suitable sterilization indicator, and if neces-sary, based on the result of the sterility test. The filtrationprocedure is used for the sterilization of a liquid product, towhich terminal sterilization can not be applied. Concerningthe disinfection and/or sterilization necessary for processingequipment and areas of pharmaceutical products, and per-forming microbiological tests specified in the monographs,see Disinfection and Sterilization Methods.

1. DefinitionsThe definitions of the terms used in this text are as

follows.(i) Terminal sterilization: A process whereby a product is

sterilized in its final container or packaging, and whichpermits the measurement and evaluation of quantifiablemicrobial lethality.

(ii) Product: A generic term used to describe raw mate-rials, intermediate products, and finished products, to besterilized.

(iii) Bioburden: Numbers and types of viable microor-ganisms in a product to be sterilized.

(iv) Sterility assurance level (SAL): Probability of a via-ble microorganism being present in a product unit after ex-posure to the proper sterilization process, expressed as 10－n.

(v) Integrity test: A non-destructive test which is used topredict the functional performance of a filter instead of themicroorganism challenge test.

(vi) D value: The value which shows the exposure time

(decimal reduction time) or absorbed dose (decimal reduc-tion dose) required to cause a 1-logarithm or 90z reductionin the population of test microorganisms under stated ex-posure conditions.

(vii) Sterilization indicator: Indicators used to monitorthe sterilization process, or as an index of sterility, includingbiological indicators (BI), chemical indicators (CI),dosimeters and the like.

2. Sterilization2.1. Heat Method

In the heat method, microorganisms are killed by heating.2.1.1. Moist heat method

Microorganisms are killed in saturated steam under pres-sure. In this method, factors which may affect the steriliza-tion include temperature, steam pressure and exposure time.Therefore, in routine sterilization process control, it is re-quired to monitor continuously the temperature, steam pres-sure and exposure time, and they should be included in thespecifications of the sterilizer.2.1.2. Dry-heat method

Microorganisms are killed in dry heated air. This methodis usually conducted in a batch-type dry heat sterilizer or atunnel-type dry heat sterilizer. In this method, factors whichmay affect the sterilization include temperature and ex-posure time. Therefore, in routine sterilization process con-trol, it is required to monitor continuously the temperatureand exposure time, and they should be included in the speci-fications of the sterilizer.2.2. Irradiation method

Microorganisms are directly killed by ionizing radiation,or by the heat generated by microwave radiation.2.2.1. Radiation method

Ionizing radiations which may be used are gamma (g) raysemitted from a radioisotope such as cobalt 60, an electronbeam and bremsstrahlung (X rays) generated from an elec-tron accelerator. Although any procedure can be applied tothermally unstable products with no radioactivity residue, itis necessary to consider the possibility of material degrada-tion. Although a 25 kGy dose is traditionally used as asterilization dose, there are some ways to calculate the doseas follows: the bioburden of the substance to be sterilized ismeasured and the sterilization dose is calculated based on themean bioburden and the standard resistance distribution(Method 1 in ISO 11137), the dose is calculated from thefraction positive information from a sterility test in whichrepresentative product samples are exposed to a substeriliz-ing dose (Method 2 in ISO 11137), or the dose is calculatedbased on the bioburden and D value of the most resistantmicroorganisms (Log method) (see 5.3.). In the case of theradiation sterilization procedure, factors which may affectthe sterilization include dose (absorbed dose) and exposuretime. Therefore, in g ray sterilization process control, it is re-quired to determine the dose (the absorbed dose) at ap-propriate intervals and to monitor continuously the exposuretime in terms of the operating parameters (the conveyerspeed, the cycle time). The dose control mechanism shouldbe included in the specifications of the sterilizer. In the caseof electron beam or bremsstrahlung irradiation, it is requiredto monitor the acceleration voltage, the beam current andbeam scanning width besides the above-mentioned items.2.2.2. Microwave method

Microorganisms are killed by the heat generated by micro-


wave radiation, usually at the frequency of 2450± 50 MHz.This method is applied to liquids or water-rich products insealed containers. Since a glass or plastic container may bedestroyed or deformed due to the rise of the inner pressure,the containers must be certified to be able to withstand theheat and the inner pressure generated during microwavesterilization. Leakage of electromagnetic radiation must beat a sufficiently low level to cause no harm to humans andno interference with radio communications and the like. Inthis method, factors which may affect the sterilization in-clude temperature, processing time and microwave outputpower. Therefore, in routine sterilization process control, itis required to monitor continuously the temperature, timeand the microwave output power, and they should be in-cluded in the specifications of the sterilizer.2.3. Gas method

Ethylene oxide (EO) is widely used as a sterilization gas.Since EO gas has an explosive nature, a 10 � 30z mixturewith carbon dioxide is commonly used. Also, as EO gas is astrong alkylating agent, it can not be applied to the productswhich are likely to react with or absorb it. Furthermore, be-cause EO gas is toxic, the residual concentration of EO gasand other secondarily generated toxic gases in products steri-lized with EO gas must be reduced to less than the safe levelsthereof by means of aeration and the like before the productis shipped. In this method, factors which may affect thesterilization include temperature, gas concentration (pres-sure), humidity and exposure time. Therefore, in routinesterilization process control, it is required to monitor con-tinuously the temperature, gas concentration (pressure), hu-midity and exposure time, and they should be included in thespecifications of the sterilizer.

3. Filtration methodMicroorganisms are removed by using a sterilizing filter

made of an appropriate material. However, this method isnot intended for microorganisms smaller than bacteria.Generally, a sterilizing filter challenged with more than 107

microorganisms of a strain of Brevundimonas diminuta(ATCC 19146, NBRC 14213, JCM 2428), cultured under theappropriate conditions, per square centimeter of effectivefilter area should provide a sterile effluent. In this method,factors which may affect the sterilization include pressure,flow rate, filter unit characteristics and the like. In routinefiltration process control, it is required to perform integritytests of the sterilizing filter after each filtration process (alsoprior to the filtration process, if necessary).

4. Sterilization Indicators4.1. Biological indicator (BI)

A BI is prepared from specific microorganisms resistant tothe specified sterilization process and is used to develop and/or validate a sterilization process. The dry type BI is classi-fied into two kinds. In one, bacterial spores are added to acarrier such as filter paper, glass or plastic and then the car-riers are dried and packaged. In the other, bacterial sporesare added to representative units of the product to be steri-lized or to simulated products. Packaging materials of the BIshould show good heat penetration in dry heat sterilizationand good gas or steam penetration in ethylene oxide andmoist heat sterilizations. It should be confirmed that anycarrier does not affect the D value of the spores. In the caseof a liquid product, the spores may be suspended in the samesolution as the product or in a solution showing an equiva-

lent effect in the sterilization of biological indicator.However, when the spores are suspended in liquid, it is nec-essary to ensure that the resistance characteristics of thespores are not affected due to germination.

Typical examples of biological indicator

SterilizationRepresentative

microorganisms* Strain name

Moist heatmethod

Geobacillusstearo-thermophilus

ATCC 7953, NBRC13737, JCM 9488ATCC 12980, NBRC12550, JCM 2501

Dry heatmethod

Bacillusatrophaeus ATCC 9372, NBRC

13721Gas method Bacillus

atrophaeus ATCC 9372, NBRC13721

* In addition to these microorganisms, other microorgan-isms with the greatest resistance to the sterilization proce-dure concerned, found in the bioburden, can be used asthe biological indicator.

4.1.1. D value of BIMethods for determination of the D value include the sur-

vival curve method and the fraction negative method(Stumbo, Murphy & Cochran procedure, Limited Spearman-Karber procedure and the like). In using marketed BIs, it isusually unnecessary to determine the D value before use ifthe D value indicated on the label has been determined by astandardized biological indicator evaluation resistometer(BIER) under strictly prescribed conditions in accordancewith ISO 11138-1. It is acceptable that the D value indicatedon the label shows a scattering of not more than ±30seconds.4.1.2. Setting up procedure of BI4.1.2.1. In the case of dry materials

A Dry type BI is placed at predetermined cold spots in theproduct to be sterilized or a suitable product showing anequivalent effect in the sterilization. The BIs are usually pri-mary packaged in the same way as the product, including asecondary packaging, if applicable.4.1.2.2. In the case of wet materials

Spores are suspended as the BI in the same solution as theproduct or in an appropriate similar solution, and should beplaced at cold spots in the sterilizer.4.1.3. Culture conditions of BI

Soybean casein digest medium is generally used. Generalculture conditions are at 55 � 609C for 7 days in the case ofG. stearothermophilus and at 30 � 359C for 7 days in thecase of B. atrophaeus.4.2. Chemical indicator (CI)

CI is an indicator which shows a color change of a sub-stance applied to a paper slip, etc. as a result of physicaland/or chemical change due to exposure to heat, gas or ra-diation. The CI can be classified into three types. The first isemployed to identify whether or not sterilization has alreadybeen implemented, the second is employed to control thesterilization process (for example, its color changes aftersterilization for a sufficient time), and the third is the Bowie

22272227JP XVI General Information / Crude Drugs

& Dick type used to evaluate the effectiveness of air removalduring the pre-vacuum phase of the pre-vacuum sterilizationcycle.4.3. Dosimeter

In the radiation (g-ray) method, the sterilization effect de-pends on the absorbed radiation dose, so the sterilizationprocess control is mainly performed by measuring the dose.A dosimeter is installed at a position corresponding to theminimum dose region of an exposed container or a positionwhere the dose is in a known relation to that in the aboveregion. Measurement should be done for each radiationbatch. If there are many containers in the same batch,dosimeters should be employed so that more than onedosimeter is always installed at the effective radiation sectionof the irradiation chamber. It should be noted thatdosimeters may be affected by environmental conditions(temperature, humidity, ultraviolet light, time until reading,etc.) before and during irradiation. Practical dosimeters forg-ray and bremsstrahlung sterilization include the dyed poly-methylmethacrylate dosimeter, clear polymethylmethacry-late dosimeter, ceric-cerous sulfate dosimeter, alanine-EPRdosimeter and the like. A dosimeter for gamma radiationcan not generally be used for sterilization process controlwith an electron beam of less than 3 MeV energy. Dosimetersfor electron beam sterilization include the cellulose acetatedosimeter, radiochromic film dosimeter and the like. A prac-tical dosimeter must be calibrated against an appropriate na-tional or international standard dosimetry system.

5. Determination of sterilization conditions using microor-ganism as an indicator

Taking account of the characteristics upon the sterilizationconcerned, bioburden, etc. of a product to be sterilized,chose a suitable method from the followings and determinethe conditions.5.1. Half-cycle method

In this method, a sterilization time of twice as long as thatrequired to inactive all of 106 counts of BI placed in theproduct is used, regardless of bioburden count in theproduct being sterilized or the resistance of the objectivemicroorganisms to the sterilization.5.2. Overkill method

In this method, a sterilization condition giving a sterilityassurance level of not more than 10－6 counts is used, regard-less of bioburden count in the product being sterilized or theresistance of the objective microorganisms to the steriliza-tion. Generally, a sterilization condition providing 12logarithmic reduction (12D) of a known count of BI of morethan 1.0 D value is used.5.3. Combination of BI and bioburden

Generally, a count of mean bioburden added three timesof its standard deviation obtained by an extensive bioburdenestimation is considered as the maximum bioburden count,and the sterilization time (or radiation dose) is calculatedwith the bioburden count based on an objective sterility as-surance level. When this procedure is used, it is required todetermine the resistance of the bioburden to the sterilizationas well as the bioburden count in the product being steri-lized. If a more resistant microorganism than the BI spore isfound in the bioburden estimation, it should be used as theBI.

Sterilization time (or radiation dose)＝ D× logN0

N

D: D value of the BIN: Sterility assurance levelN0: Maximum bioburden count in the product

5.4. Absolute bioburden methodThe sterilization conditions are determined by employing

the D value of the most resistant microorganism found in theproduct or environment by the resistant estimation and beingbased on the bioburden count in the product. Generally, acount of mean bioburden added three times of its standarddeviation obtained by an extensive bioburden estimation isemployed as the bioburden count. When this procedure isused, it is required to make frequent counting and resistancedetermination of microorganisms in daily bioburden estima-tion.

6. References( i ) ISO 11134 Industrial moist heat sterilization( ii ) ISO 11135 Ethylene oxide sterilization(iii) ISO 11137 Radiation sterilization(iv) ISO 11138 Biological indicators( v ) ISO 11140 Chemical indicators(vi) ISO 11737 Microbiological methods

Part 1: Estimation of population of mi-croorganisms on products

G5 Crude Drugs

Aristolochic AcidAristolochic acid, which occurs in plants of Aristolochia-

ceae, is suspected to cause renal damage. It is also reportedto be oncogenic (see References).

Aristolochic acid toxicity will not be a problem if crudedrugs of the origin and parts designated in the JP are used,but there may be differences in crude drug nomenclature be-tween different countries, and it is known that crude drugpreparations not meeting the specifications of the JP are cir-culating in some countries. Consequently, when crude drugsor their preparations are used, it is important that the mate-rials should not include any plant containing aristolochicacid.

Since Supplement I to JP14, the test for aristolochic acid Iwas added to the Purity under Asiasarum Root, which con-sists of the rhizome and root. Because the aerial part of theplant may contain aristolochic acid and may have been im-properly contaminated in Asiasarum Root.

It is considered that Akebia Stem, Sinomenium Stem andSaussurea Root do not contain aristolochic acid, unlessplants of origin other than that designated in the JP areused. However, contamination of aristolochic acid mightoccur, as mentioned above. In this case, the test described inthe Purity under Asiasarum Root is useful for checking thepresence of aristolochic acid.References:

Drug & Medical Device Safety Information (No.161)(July, 2000).

New England Journal of Medicine (June 8, 2000).Mutation Research 515, 63 � 72 (2002).

22282228 JP XVICrude Drugs / General Information

Purity Tests on Crude DrugsUsing Genetic Information

The first step in the quality assurance of natural productsis the use of raw materials from the right part of the rightorigin (the right source). Therefore, it is clearly stated in Ar-ticle 4 of the General Rules For Crude Drugs that the sourceof a crude drug is the approval or rejection criteria. Thereare various methods for differentiating the sources of crudedrugs, such as morphological methods, organoleptic tests,and chemical methods, and appropriate methods for eachare described in the individual monographs. Morphologicalmethods, organoleptic tests, and chemical methods arediscrimination methods for species that are based on thephenotypic characteristics of the crude drugs. On the otherhand, together with recent progress in molecular biologytechniques and the accumulation of genetic information onplants, differentiating methods of crude drugs based ongenotypes have been established. Unlike morphological andother methods that are based on phenotypic characteristics,the genotypic methods are not affected by environmentalfactors. Also, the methods have several advantages, such asspecialized expertise and skill for classification are not need-ed, and objective results are easily obtained.

The evolution of living organisms is accomplished bygenetic mutation, and differences among the nucleotidesequences of genes of closely related species reflect the strainrelationships between the species. Based on this theory, inrecent years methods that classify species phylogeneticallyusing the nucleotide sequence of rDNA that codes forribosomal RNA (rRNA) on the nuclear genome have beenadopted. In the same way, the sequence of this rDNA is alsomost often used in the classification of higher plants basedon the genotype. In particular, it is very easy to classifyclosely related species using the intergenic transcriber space(ITS) region of the rDNA region, since by comparison withthe coded gene region nucleotide substitution is more oftenundertaken. Furthermore, since the genes on the nucleargenome originate from the parents' genom, there is anadvantage that interspecies hybrids can be detected. Higherplants also have mitochondrial genes and chloroplasticgenes. Although the genes on these genomes are also oftenused for classification, interspecies hybrids cannot be con-firmed because the genes are normally uniparental in-heritance.

The two methods presented here have been developedbased on the reported identification methods of AtractylodesLancea Rhizome and Atractylodes Rhizome1,2) utilizing thegene sequence of the ITS of rDNA. Inter-laboratory valida-tion study for purity test of Atractylodes Rhizome targetedfor Atractylodes Lancea Rhizome have been completed. Theplant sources for Atractylodes Lancea Rhizome stipulated inthe individual monographs are Atractylodes lancea DeCandolle or A. chinensis Koidzumi (Compositae), whilethose for Atractylodes Rhizome are A. japonica Koidzumiex Kitamura or A. ovata De Candolle (Compositae). The ap-proval or rejection of the source of Atractylodes LanceaRhizome is, in principle, determined by the description ofthe crude drug, including microscopy, while that of Atrac-tylodes Rhizome is determined by the description of thecrude drug, including microscopy, together with color reac-

tion, which is an identification test. In the above scientificpaper, it was shown that these 4 plant species can be clearlyclassified by comparing the nucleotide sequences of the ITSmentioned above, and that the species can be easily classifiedwithout performing sequence analysis by performing PCRusing a species specific primer set or by using a restrictionenzyme which recognizes species specific sequence.

In validation studies, the simplicity of the test is givenmaximum consideration. We examined methods that observePCR amplification bands using species specific primer sets(Mutant Allele Specific Amplification: Method 1) and thatobserve DNA fragments produced by restriction enzymetreatment of the PCR products, which are prepared using aprimer set common to each plant source (PCR-RestrictionFragment Length Polymorphism: Method 2), and do notinvolve nucleotide sequence analyses. In these methodsbased on PCR, an extremely small amount of template DNAis amplified to billions to hundred-billions times. Therefore,when using them as identification tests for powdered crudedrugs, the target DNA fragment can be observed even if thevast majority of the crude drug for analysis is not appro-priate plant species and there is only a minute amount ofpowder from a crude drug derived from a suitable plant.(Consequently, in identification tests, either a cut or a wholecrude drug must be used, as long as one is careful to avoidcontamination by powder originating from other crudedrugs.) On the other hand, when used as a purity test, theform of the crude drug is irrelevant as long as the geneamplification is performed properly and the target gene isnot polymorphic, so if DNA fragments of an inappropriateplant are confirmed in the purity test, regardless of the formof the crude drug, it becomes clear there is contamination byan inappropriate crude drug.

The methods shown here are reference information and atthe present stage results obtained using the methods do notaffect the approval or rejection of the crude drug in eachmonograph. Furthermore, by performing the sequence ana-lysis outlined in the previous paper, it goes without sayingthat more accurate decision concerning the source speciescan be made.

1. DNA Amplification EquipmentDNA amplification equipment is used to amplify the DNA

which is extracted from a crude drug and then purified.Since there are slight differences in the methods of tempera-ture control, and so on depending on the equipment used,there may be differences in the intensity, etc. of the PCRamplification bands even if PCR is carried out under thestipulated conditions. Therefore, when judging results basedsolely on the presence or absence of PCR amplificationbands as in 3. Methods 1, confirm that only proper amplifi-cation bands are obtained when performing PCR using DNAobtained from samples confirmed beforehand to be thesource species. If proper amplification bands are not ob-tained, the PCR temperature conditions should be slightlyadjusted. This equipment can be used for the restriction en-zyme treatment in 4. Method 2.

2. General precautionsCrude drugs are different from fresh plants in that they

are dried products and a certain amount of time has passedsince they were harvested. Therefore, in many cases theDNA has undergone fragmentation. Furthermore, varioussubstances that can block or interfere with the PCR reaction


may be present in the plant. For these reasons, the extractionand purification of template DNA is the process that shouldreceive the greatest amount of attention. In the case ofAtractylodes crude drugs, the periderm should be removedusing a clean scalpel or other clean instrument before pul-verizing the sample because very often there are inhibitorysubstances present in the periderm.

3. Method 1 (Mutant Allele Specific AmplificationMethod)

Generally, this method is referred to as Mutant AlleleSpecific Amplification (MASA) or Amplification RefractoryMutation System (ARMS), and it provides nucleotidesequence information of sample-derived template DNA,based upon the presence or absence of DNA amplification inPCR using a species specific primer set.3.1. Procedure

The following is an example procedure.3.1.1. Preparation of template DNA

There are various methods with which to extract and puri-fy DNA from the samples. It is recommended that commer-cially available DNA extraction kits be used when consider-ing their advantages of not using any noxious reagents andnot requiring any complicated purification procedures. Inthis case, attention should be paid to the final amount (con-centration) of DNA obtained, and the initial amount of ini-tial sample and the volume of liquid to elute the DNA needto be controlled. When extraction and purification are per-formed using silica gel membrane type kits stipulated innotifications3) related to inspection methods of the foodsproduced by recombinant DNA techniques, it is appropriateto use 200 mg of sample, 1 mL of AP1 buffer solution, 2 mLof RNase A, and 325 mL of AP2 buffer solution. Also, themost important things are that the supernatant loaded on thefirst column is clear and that there is no need to load 1 mLunreasonably. Furthermore, 50 mL is an appropriate volumeused in the final elution of the DNA, and normally the initialeluate is used as the DNA sample stock solution.3.1.2. Confirmation of purity of DNA in DNA samplestock solution and assay of DNA

The purity of the DNA in the stock solution can be con-firmed by the OD260 nm/OD280 nm ratio using a spectropho-tometer. A ratio of 1.5 indicates that the DNA has been ade-quately purified. The amount of DNA is calculated using1 OD260 nm＝ 50 mg/mL. The measurement mentioned aboveis performed using appropriately diluted DNA sample stocksolution. Based on the results obtained, dilute with water tothe concentration needed for the subsequent PCR reactions,dispense the solution into micro tubes as the sample DNAsolution, and if necessary store frozen at not over －209C.The dispensed DNA sample is used immediately after thaw-ing and any remaining solution should be discarded and notrefrozen. If the concentration of the DNA sample stock so-lution does not reach the concentration stipulated in PCR, itis used as a DNA sample solution.3.1.3. PCR

When a commercially available PCR enzyme mentioned inthe above notification4) is used, it is appropriate that 25 mLof reaction mixture consisting of 2.5 mL of the PCR buffersolution containing magnesium, dNTP (0.2 mmol/L), 5?and3? primer (0.4 mmol/L), Taq DNA polymerase (1.25 units),and 5 mL of 10 ng/mL sample DNA solution (50 ng of DNA)is prepared on ice. Among them, the PCR buffer solution

and dNTP are provided as adjuncts to the enzyme. Whenconducting purity tests on Atractylodes Lancea Rhizome inAtractylodes Rhizome, the primer sets used are C and D (Cis positive with A. lancea, D is positive with A. chinensis) asdescribed in the paper mentioned above (J. Nat. Med. 60,149 � 156, 2006), however, when a combination of primer Aand B is used, it is possible to confirm the source species ofeach of the respective specimens. In order to confirm thatthe DNA has been extracted correctly, the reaction solutioncontaining the positive control primer (Pf and Pr) as shownbelow should be prepared. In addition, the negative controlsolutions which are not containing DNA sample or either ofthe primer sets should be prepared and simultaneously con-duct PCR.

Pf: 5?-CAT TGT CGA AGC CTG CAC AGC A-3?Pr: 5?-CGA TGC GTG AGC CGA GAT ATC C-3?

The PCR reaction is performed under the following condi-tions: starting the reaction at 959C for 10 minutes, 30 cyclesof 0.5 minutes at 959C and 0.75 minutes at 689C (699C onlywhen using the primer set C), terminate reaction at 729C for7 minutes, and store at 49C. The resulting reaction mixture isused for the following process as PCR amplification reactionsolution.3.1.4. Agarose gel electrophoresis and detection of PCRproducts

After completion of the PCR reaction, mix 5 mL of thePCR amplification reaction solution with an appropriatevolume of gel loading buffer solution, add the mixture to thewells of 2 w/vz agarose gel, and then perform electrophore-sis using 1-fold TAE buffer solution (refer to General Infor-mation, Rapid Identification of Microorganisms Based onMolecular Biological Method). Run in parallel an appropri-ate DNA molecular mass standard. Electrophoresis is termi-nated when the bromophenol blue dye in the gel loadingbuffer has advanced to a point corresponding to 1/2 to 2/3the length of the gel.

Stain the gel after electrophoresis when not using gelstained in advance with ethidium bromide. Place the gel thathas undergone electrophoresis and staining in a gel imageanalyzer, irradiate with ultraviolet light (312 nm), and de-tected its electrophoresis pattern. Compare this to the DNAmolecular mass standard and determine the absence orpresence of the target amplification band.3.2. Judgement

Confirm at first that a 305 bp band is found with the reac-tion solution to which the positive control primer set hasbeen added, and confirm there are no bands in a solutionwith no primer sets and a solution with no sample DNA so-lution. Next, if a 226 bp band is confirmed when the primerset C is added or if a 200 bp band is confirmed when theprimer set D is added, the sample is judged to be Atrac-tylodes Lancea Rhizome (in the case of cut crude drug, con-tamination of Atractylodes Lancea Rhizome is observed)and it is rejected. The sample is judged not to be Atrac-tylodes Lancea Rhizome (in the case of cut crude drug, thereis no contamination of Atractylodes Lancea Rhizome) andthe purity test is acceptable if a 305 bp band is confirmedwith the positive control primer set, bands are not observedin reaction solution without primer and reaction solutionwithout DNA sample solution, and a 226 bp band is not ob-served with the primer set C and a 200 bp band is not ob-served with the primer set D. If a band is not observed with


the positive control primer, it is to be concluded that theDNA extraction failed and the procedure should be startedover again from the DNA extraction step. If bands are con-firmed in reaction solutions without primer sets or withoutDNA sample solution, it should be assumed that there wasan error in the PCR procedure and therefore the procedureshould be repeated again from the step 3.1.3. PCR.

4. Method 2 (PCR-Restriction Fragment Length Polymor-phism)

Generally, this method is referred to as PCR-RestrictionFragment Length Polymorphism (RFLP), and it providesnucleotide sequence information of sample-derived templateDNA, based upon the DNA fragment pattern produced byrestriction enzyme treatment of the PCR products, which areamplified by using a primer set common to the DNA se-quence of the objective plant.

The test is performed with 25 samples randomly takenfrom a lot, and each sample is designated with a numberfrom 1 to 25. Differentiation of the sources is performed byindividual PCR-RFLP measurement of the samples, and de-cision of the acceptability of the purity is dependent on howmany nonconforming samples are present in the first 20 sam-ples, taken in numerical order, for which judgement is possi-ble as described below.4.1. Procedure

The following is an example procedure.4.1.1. Preparation of template DNA

There are various methods with which to extract and puri-fy DNA from the samples. It is recommended that commer-cially available DNA extraction kits be used, when consider-ing their advantages of not using noxious reagents and notrequiring complicated purification procedures. Recently,PCR reagents that inhibit the effect of PCR enzyme-inhibiting substances present in samples have become com-mercially available, and by using these reagents, it is possibleto prepare the template DNA from the sample simply by in-cubating the sample with the DNA extraction reagent. Here,a recommended DNA preparing procedure using such PCRreagents is described for the convenience of experimenters.

Cut 20 mg of the sample into small pieces with a cleanknife, add 400 mL of the DNA extraction reagent, and incu-bate at 559C overnight (16 � 18 hours). Then heat at 959Cfor 5 minutes to inactivate the enzyme in the reagent. Centri-fuge to precipitate the sample, and use 50 mL of the superna-tant liquid as the template DNA solution. The DNA solutionprepared in this method can not be used for concentrationmeasurement based on OD260 nm, because it contains manyforeign substances affecting OD260 value from the sample.

The composition of the DNA extraction reagent is asfollows:

2-Amino-2-hydroxymethyl-1.3- 20 mmol/Lpropanediol-hydrochloric acid (pH 8.0)Ethylenediamine tetraacetate 5 mmol/LSodium chloride 400 mmol/LSodium dodecyl sulfate 0.3zProteinase K 200 mg/mL

4.1.2. PCRIn the method using the PCR enzyme and PCR reagent as

described2), the reaction mixture is prepared on an ice bath ina total volume of 20 mL of a solution containing 10.0 mL of2-fold concentrated PCR reagent, 5?- and 3?-primers (0.5mmol/L), Taq DNA polymerase (0.5 units) and 0.5 mL of

template DNA solution.The PCR reaction is performed under the following condi-

tions: 959C for 10 minutes, 40 cycles of 959C for 0.5 minute,659C for 0.25 minute, and 729C for 0.25 minute and 729Cfor 7 minutes. Store the solution at 49C, and use this solu-tion as the PCR amplified solution. A negative control (con-taining water instead of the template DNA solution) must beincluded in the procedure.

The sequence of each primer is as follows:

5?-primer: 5?-GGC ACA ACA CGT GCC AAG GAAAA-3?

3?-primer: 5?-CGA TGC GTG AGC CGA GAT ATC C-3?

4.1.3. Restriction enzyme treatmentThe treatment is performed on individual reaction solu-

tions using two enzymes, Fau I and Msp I. In the case ofFau I, to an appropriate amount of the reaction solution,composed of a reaction buffer containing 1.0 unit of en-zyme, add 3.0 mL of PCR products while cooling in an icebath to make 15.0 mL. In the case of Msp I, to an appropri-ate amount of the reaction solution, composed of a reactionbuffer containing 20.0 units of enzyme, add 3.0 mL of PCRproducts while cooling in an ice bath to make 15.0 mL. Incu-bate these solutions at the temperature recommended by themanufacturer for 2 hours, and then inactivate the enzyme byheating at 729C for 10 minutes. The negative control of thePCR reaction is also treated in the same manner.4.1.4. Agarose gel electrophoresis and detection of DNAfragments

After the restriction enzyme treatment, mix the totalamount of the reaction solution and an appropriate amountof the gel loading buffer solution, place it in a 4 w/vzagarose gel well, and carry out electrophoresis with 1-foldconcentrated TAE buffer solution (see ``Rapid Identificationof Microorganisms Based on Molecular BiologicalMethods'' under General Information). Carry out the elec-trophoresis together with appropriate DNA molecular massstandard. Stop the electrophoresis when the bromophenolblue included in the loading buffer solution has moved about2 cm from the well. The 4 w/vz agarose gel is sticky,difficult to prepare and hard to handle, so that it is better touse a commercially available precast gel.

After the electrophoresis, stain the gel, if it is not alreadystained, with ethidium bromide, and observe the gel on anilluminating device under ultraviolet light (312 nm) to con-firm the electrophoretic pattern.4.2. Judgement4.2.1. Judgment of each sample

Confirm that no band is obtained with the negative con-trol of the PCR, other than the primer dimer (about 40 bp)band. A sample treated with Fau I, showing bands of about80 bp and 60 bp, or that treated with Msp I, showing bandsof about 90 bp and 50 bp, is judged as Atractylodes LanceaRhizome. A sample not showing any band other than a bandat about 140 bp and the primer dimer band is judged asAtractylodes Rhizome. If a sample does not show any bandother than the primer dimer band, it is considered that PCRproducts were not obtained, and judgement is impossible forthe sample.4.2.2. Judgment of the purity

Judgment of the purity is based on the result of the judg-ment of each sample. If there is no sample that is judged asAtractylodes Lancea Rhizome among 20 samples taken in

2231

Scientific names used in the JP and the corresponding taxonomic names

Crude Drug

Scientific names used in the JP＝Scientific names being used taxonomically (Combined notation, Stan-dard form for author or authors)

FamilyScientific names that are different from those written in JP but identicalto them taxonomically or being regarded as identical, and typical sub-classified groups belonging to their species. The names marked with ``*''are those being written together in JP.

Acaciaアラビアゴム

Acacia senegal Willdenow＝Acacia senegal (L.) Willd. Leguminosae

Other species of the same genus

Achyranthes Rootゴシツ

Achyranthes fauriei Leveille et Vaniot＝Achyranthes fauriei H. Lev. & Vaniot Amaranthaceae

Achyranthes bidentata Blume

Agarカンテン

Gelidium elegans Kuetzing

GelidiaceaeOther species of the same genus

Other red algae

Akebia Stemモクツウ

Akebia quinata Decaisne＝Akebia quinata (Thunb. ex Houtt.) Decne.

LardizabalaceaeAkebia trifoliata Koidzumi＝Akebia trifoliata (Thunb.) Koidz.

Alisma Rhizomeタクシャ

Alisma orientale Juzepczuk＝Alisma orientale (Sam.) Juz. Alismataceae

Alisma plantago-aquatica L. var. orientale Sam.


order of the numbering, excluding any sample for which jud-gement is impossible, the lot is acceptable for purity. Whenthere is one sample that is judged as Atractylodes LanceaRhizome among the 20 samples, perform the same test with25 newly taken samples from the lot, and if there is no sam-ple that is judged as Atractylodes Lancea Rhizome, the lot isacceptable for purity. When there is a sample that is judgedas Atractylodes Lancea Rhizome in the second test, or thereis more than one sample that is judged as Atractylodes Lan-cea Rhizome in the first test, the lot is not acceptable forpurity.

5. Reference1) Y. Guo, et al., J. Nat. Med. 60, 149 � 156 (2006).2) K. Kondo, et al., J. Jpn. Bot. 84, 356 � 359 (2009).3) Notification No. 110, Director of Food Health Depart-

ment, March 2001; Partial Amendment: NotificationNo. 0629002, 2.2.1.2, Director of Food Safety Depart-ment, June 2006.

4) Notification No. 0629002, 2.1.3.1.1, Director of FoodSafety Department, June 2006.

On the Scientific Names of CrudeDrugs Listed in the JP

The notation system of the scientific names for the origi-nal plants and animals of crude drugs listed in JP is notnecessary the same as the taxonomic system used in theliterature. The reason for this is that the JP is not an aca-demic text, but an ordinance. The relationship between thescientific names used in the JP and those generally usedtaxonomically is indicated in the following table, to avoidmisunderstanding by JP users owing to differences in thenotation system.

2232

Aloeアロエ

Aloe ferox Miller＝Aloe ferox Mill.

LiliaceaeHybrid between Aloe ferox Miller and Aloe africana MillerAloe africana Miller＝Aloe africana Mill.

Hybrid between Aloe ferox Miller and Aloe spicata Baker

Alpinia Officinarum Rhizomeリョウキョウ

Alpinia officinarum Hance Zingiberaceae

Amomum Seedシュクシャ

Amomum xanthioides Wallich＝Amomum xanthioides Wall. Zingiberaceae

Amomum villosum Lour. var. xanthioides (Wall.) T. L. Wu & Senjen

Anemarrhena Rhizomeチモ

Anemarrhena asphodeloides Bunge Liliaceae

Angelica Dahurica Rootビャクシ

Angelica dahurica Bentham et Hooker filius ex Franchet et Savatier＝Angelica dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav.

Umbelliferae

Apricot Kernelキョウニン

Prunus armeniaca Linn áe＝Prunus armeniaca L.

RosaceaePrunus armeniaca Linn áe var. ansu Maximowicz＝Prunus armeniaca L. var. ansu Maxim.

Prunus sibirica Linn áe＝Prunus sibirica L.

Aralia Rhizomeドクカツ

Aralia cordata Thunberg＝Aralia cordata Thunb.

Araliaceae

Arecaビンロウジ

Areca catechu Linn áe＝Areca catechu L.

Palmae

Artemisia Capillaris Flowerインチンコウ

Artemisia capillaris Thunberg＝Artemisia capillaris Thunb.

Compositae

Asiasarum Rootサイシン

Asiasarum sieboldii F. Maekawa＝Asiasarum sieboldii (Miq.) F. Maek.

Aristolochiaceae

Asarum sieboldii Miq.

Asarum sieboldii Miq. var. seoulense Nakai

Asiasarum heterotropoides F. Maekawa var. mandshuricum F. Maekawa＝Asiasarum heterotropoides (F. Schmidt) F. Maek. var. mandshuricum(Maxim.) F. Maek.

Asarum heterotropoides F. Schmidt var. mandshuricum (Maxim.) Kitag.

Asparagus Tuberテンモンドウ

Asparagus cochinchinensis Merrill＝Asparagus cochinchinensis (Lour.) Merr.

Liliaceae

Astragalus Rootオウギ

Astragalus membranaceus Bunge＝Astragalus membranaceus (Fisch.) Bunge

LeguminosaeAstragalus mongholicus Bunge

Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge)Hsiao


2233

Atractylodes Lancea Rhizomeソウジュツ

Atractylodes lancea De Candolle＝Atractylodes lancea (Thunb.) DC.

CompositaeAtractylodes chinensis Koidzumi＝Atractylodes chinensis (DC.) Koidz.

Hybrid between above species

Atractylodes Rhizomeビャクジュツ

Atractylodes japonica Koidzumi ex Kitamura＝Atractylodes japonica Koidz. ex Kitam.

CompositaeAtractylodes macrocephala Koidzumi＝Atractylodes macrocephala Koidz.

* Atractylodes ovata De Candolle＝Atractylodes ovata (Thunb.) DC.

Bear Bileユウタン

Ursus arctos Linn áe＝Ursus arctos L. Ursidae

Other animals of the related genus

Bearberry Leafウワウルシ

Arctostaphylos uva-ursi Sprengel＝Arctostaphylos uva-ursi (L.) Spreng.

Ericaceae

Belladonna Rootベラドンナコン

Atropa belladonna Linn áe＝Atropa belladonna L.

Solanaceae

Benincasa Seedトウガシ

Benincasa cerifera Savi

CucurbitaceaeBenincasa hispida (Thunb.) Cogn.

Benincasa cerifera Savi forma emarginata K. Kimura et Sugiyama＝Benincasa cerifera Savi f. emarginata K. Kimura & Sugiyama

Benzoinアンソッコウ

Styrax benzoin Dryander＝Styrax benzoin Dryand. Styracaceae

Other species of the same genus

Bitter Cardamonヤクチ

Alpinia oxyphylla Miquel＝Alpinia oxyphylla Miq.

Zingiberaceae

Bitter Orange Peelトウヒ

Citrus aurantium Linn áe＝Citrus aurantium L.

RutaceaeCitrus aurantium Linn áe var. daidai Makino＝Citrus aurantium L. var. daidai Makino

Citrus aurantium L. `Daidai'

Brown Riceコウベイ

Oryza sativa Linn áe＝Oryza sativa L.

Gramineae

Bupleurum Rootサイコ

Bupleurum falcatum Linn áe＝Bupleurum falcatum L.

UmbelliferaeBupleurum chinense DC.Bupleurum scorzonerifolium Willd.

Burdock Fruitゴボウシ

Arctium lappa Linn áe＝Arctium lappa L.

Compositae

Calumbaコロンボ

Jateorhiza columba Miers Menispermaceae

Capsicumトウガラシ

Capsicum annuum Linn áe＝Capsicum annuum L.

Solanaceae


2234

Cardamonショウズク

Elettaria cardamomum Maton Zingiberaceae

Cassia Seedケツメイシ

Cassia obtusifolia Linn áe＝Cassia obtusifolia L.

LeguminosaeCassia tora Linn áe＝Cassia tora L.

Catalpa Fruitキササゲ

Catalpa ovata G. Don

BignoniaceaeCatalpa bungei C. A. Meyer＝Catalpa bungei C. A. Mey.

Chrysanthemum Flowerキクカ

Chrysanthemum morifolium Ramatuelle＝Chrysanthemum morifolium Ramat.

CompositaeChrysanthemum indicum Linn áe＝Chrysanthemum indicum L.

Cimicifuga Rhizomeショウマ

Cimicifuga simplex Turczaninow＝Cimicifuga simplex (DC.) Turcz.

Ranunculaceae

Cimicifuga dahurica Maximowicz＝Cimicifuga dahurica (Turcz.) Maxim.

Cimisifuga heracleifolia Komarov＝Cimisifuga heracleifolia Kom.

Cimicifuga foetida Linn áe＝Cimicifuga foetida L.

Cinnamon Barkケイヒ

Cinnamomum cassia Blume Lauraceae

Cinnamon Oilケイヒ油

Cinnamomum cassia BlumeLauraceae

Cinnamomum zeylanicum Nees

Citrus Unshiu Peelチンピ

Citrus unshiu Marcowicz＝Citrus unshiu (Swingle) Marcow.

RutaceaeCitrus reticulata Blanco `Unshiu'

Citrus reticulata Blanco

Clematis Rootイレイセン

Clematis chinensis Osbeck

RanunculaceaeClematis mandshurica Ruprecht＝Clematis mandshurica Rupr.

Clematis hexapetala Pallas＝Clematis hexapetala Pall.

CloveチョウジClove oilチョウジ油

Syzygium aromaticum Merrill et Perry＝Syzygium aromaticum (L.) Merr. & M. L. Perry

Myrtaceae* Eugenia caryophyllata Thunberg＝Eugenia caryophyllata Thunb.Eugenia caryophyllus (Spreng.) Bullock & S. G. Harrison

Cnidium Monnieri Fruitジャショウシ

Cnidium monnieri Cusson＝Cnidium monnieri (L.) Cusson

Umbelliferae

Cnidium Rhizomeセンキュウ

Cnidium officinale Makino Umbelliferae

Coix Seedヨクイニン

Coix lacryma-jobi Linn áe var. mayuen Stapf＝Coix lacryma-jobi L. var. mayuen (Rom. Caill.) Stapf

Gramineae


2235

Condurangoコンズランゴ

Marsdenia cundurango Reichenbach filius＝Marsdenia cundurango Rchb. f.

Asclepiadaceae

Coptis Rhizomeオウレン

Coptis japonica Makino＝Coptis japonica (Thunb.) Makino

Ranunculaceae

Coptis japonica (Thunb.) Makino var. dissecta (Yatabe) NakaiCoptis japonica (Thunb.) Makino var. japonicaCoptis japonica (Thunb.) Makino var. major (Miq.) Satake

Coptis chinensis Franchet＝Coptis chinensis Franch.

Coptis deltoidea C. Y. Cheng et Hsiao

Coptis teeta Wallich＝Coptis teeta Wall.

Cornus Fruitサンシュユ

Cornus officinalis Siebold et Zuccarini＝Cornus officinalis Siebold & Zucc.

Cornaceae

Corydalis Tuberエンゴサク

Corydalis turtschaninovii Besser forma yanhusuo Y. H. Chou et C. C. Hsu＝Corydalis turtschaninovii Besser f. yanhusuo (W. T. Wang) Y. H.Chou & C. C. Hsu Papaveraceae

Corydalis yanhusuo W. T. Wang

Crataegus Fruitサンザシ

Crataegus cuneata Siebold et Zuccarini＝Crataegus cuneata Siebold & Zucc.

RosaceaeCrataegus pinnatifida Bunge var. major N. E. Brown＝Crataegus pinnatifida Bunge var. major N. E. Br.

Cyperus Rhizomeコウブシ

Cyperus rotundus Linn áe＝Cyperus rotundus L.

Cyperaceae

Digeneaマクリ

Digenea simplex C. Agardh＝Digenea simplex (Wulfen) C. Agardh

Rhodomelaceae

Dioscorea Rhizomeサンヤク

Dioscorea japonica Thunberg＝Dioscorea japonica Thunb.

DioscoreaceaeDioscorea batatas Decaisne＝Dioscorea batatas Decne.

Dioscorea opposita Thunb.

Dolichos Seedヘンズ

Dolichos lablab Linn áe＝Dolichos lablab L.

Leguminosae

Eleutherococcus SenticosusRhizomeシゴカ

Eleutherococcus senticosus Maximowicz＝Eleutherococcus senticosus (Rupr. & Maxim.) Maxim.

Araliaceae* Acanthopanax senticosus Harms＝Acanthopanax senticosus (Rupr. & Maxim.) Harms

Ephedra Herbマオウ

Ephedra sinica Stapf

EphedraceaeEphedra intermedia Schrenk et C. A. Meyer＝Ephedra intermedia Schrenk & C. A. Mey.

Ephedra equisetina Bunge


2236

Epimedium Herbインヨウカク

Epimedium pubescens Maximowicz＝Epimedium pubescens Maxim.

Berberidaceae

Epimedium brevicornu Maximowicz＝Epimedium brevicornu Maxim.

Epimedium wushanense T. S. Ying

Epimedium sagittatum Maximowicz＝Epimedium sagittatum (Siebold & Zucc.) Maxim.

Epimedium koreanum Nakai

Epimedium grandiflorum Morren var. thunbergianum Nakai＝Epimedium grandiflorum Morr. var. thunbergianum (Miq.) Nakai

Epimedium sempervirens Nakai

Eucommia Barkトチュウ

Eucommia ulmoides Oliver＝Eucommia ulmoides Oliv.

Eucommiaceae

Euodia Fruitゴシュユ

Euodia ruticarpa Hooker filius et Thomson＝Euodia ruticarpa (A. Juss.) Hook. f. & Thomson

Rutaceae

* Evodia rutaecarpa Bentham＝Evodia rutaecarpa (A. Juss.) Benth.Tetradium ruticarpum (A. Juss.) Hartley

Euodia officinalis Dode

* Evodia officinalis DodeEvodia rutaecarpa (A. Juss.) Benth. var. officinalis (Dode) Huang

Euodia bodinieri Dode

* Evodia bodinieri DodeEvodia rutaecarpa (A. Juss.) Benth. var. bodinieri (Dode) Huang

Fennelウイキョウ

Foeniculum vulgare Miller＝Foeniculum vulgare Mill.

Umbelliferae

Fennel Oilウイキョウ油

Foeniculum vulgare Miller＝Foeniculum vulgare Mill.

Umbelliferae

Illicium verum Hooker filius＝Illicium verum Hook. f.

Illiciaceae

Forsythia Fruitレンギョウ

Forsythia suspensa Vahl＝Forsythia suspensa (Thunb.) Vahl

OleaceaeForsythia viridissima Lindley＝Forsythia viridissima Lindl.

Fritillaria Bulbバイモ

Fritillaria verticillata Willdenow var. thunbergii Baker＝Fritillaria verticillata Willd. var. thunbergii (Miq.) Baker Liliaceae

Fritillaria thunbergii Miq.

Gambirアセンヤク

Uncaria gambir Roxburgh＝Uncaria gambir (Hunter) Roxb.

Rubiaceae

Gardenia Fruitサンシシ

Gardenia jasminoides EllisRubiaceae

Gardenia jasminoides Ellis f. longicarpa Z. W. Xie & Okada

Gastrodia Tuberテンマ

Gastrodia elata Blume Orchidaceae

Gentianゲンチアナ

Gentiana lutea Linn áe＝Gentiana lutea L.

Gentianaceae


2237

Geranium Herbゲンノショウコ

Geranium thunbergii Siebold et Zuccarini＝Geranium thunbergii Siebold & Zucc.

Geraniaceae

Gingerショウキョウ

Zingiber officinale Roscoe Zingiberaceae

Ginsengニンジン

Panax ginseng C. A. Meyer＝Panax ginseng C. A. Mey. Araliaceae

* Panax schinseng Nees

Glehnia Root and Rhizomeハマボウフウ

Glehnia littoralis Fr. Schmidt ex Miquel＝Glehnia littoralis F. Schmidt ex Miq.

Umbelliferae

Glycyrrhizaカンゾウ

Glycyrrhiza uralensis Fischer＝Glycyrrhiza uralensis Fisch.

LeguminosaeGlycyrrhiza glabra Linn áe＝Glycyrrhiza glabra L.

Hemp Fruitマシニン

Cannabis sativa Linn áe＝Cannabis sativa L.

Moracea

Honeyハチミツ

Apis mellifera Linn áe＝Apis mellifera L. Apidae

Apis cerana Fabricius

Houttuynia Herbジュウヤク

Houttuynia cordata Thunberg＝Houttuynia cordata Thunb.

Saururaceae

Immature Orangeキジツ

Citrus aurantium Linn áe var. daidai Makino＝Citrus aurantium L. var. daidai Makino

Rutaceae

Citrus aurantium L. `Daidai'

Citrus natsudaidai Hayata

Citrus aurantium Linn áe＝Citrus aurantium L.

Citrus aurantium L. subsp. hassaku (Tanaka) Hiroe＝Citrus hassaku hort. ex Tanaka

Imperata Rhizomeボウコン

Imperata cylindrica Beauvois＝Imperata cylindrica (L.) P. Beauv. Gramineae

Imperata cylindrica (L.) P. Beauv. var. major (Nees) C. E. Hubb.

Ipecacトコン

Cephaelis ipecacuanha A. Richard＝Cephaelis ipecacuanha (Brot.) A. Rich.

RubiaceaeCephaelis acuminata Karsten＝Cephaelis acuminata H. Karst.

Japanese Angelica Rootトウキ

Angelica acutiloba Kitagawa＝Angelica acutiloba (Siebold & Zucc.) Kitag.

UmbelliferaeAngelica acutiloba Kitagawa var. sugiyamae Hikino＝Angelica acutiloba (Siebold & Zucc.) Kitag. var. sugiyamae Hikino


2238

Japanese Gentianリュウタン

Gentiana scabra Bunge

Gentianaceae

Gentiana scabra Bunge var. buergeri (Miq.) Maxim.

Gentiana manshurica Kitagawa＝Gentiana manshurica Kitag.

Gentiana triflora Pallas＝Gentiana triflora Pall.

Gentiana triflora Pall. var. japonica Hara

Japanese Valerianカノコソウ

Valeriana fauriei Briquet＝Valeriana fauriei Briq. Valerianaceae

Valeriana fauriei Briq. f. yezoensis Hara

Jujube Seedサンソウニン

Zizyphus jujuba Miller var. spinosa Hu ex H. F. Chou＝Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou

Rhamnaceae

Jujubeタイソウ

Zizyphus jujuba Miller var. inermis Rehder＝Zizyphus jujuba Mill. var. inermis (Bunge) Rehder

Rhamnaceae

Koiコウイ

Zea mays Linn áe＝Zea mays L.

Gramineae

Manihot esculenta Crantz Euphorbiacaea

Solanum tuberosum Linn áe＝Solanum tuberosum L.

Solanaceae

Ipomoea batatas Poiret＝Ipomoea batatas (L.) Poir Convolvulaceae

Ipomoea batatas (L.) Lam.

Oryza sativa Linn áe＝Oryza sativa L.

Gramineae

Leonurus Herbヤクモソウ

Leonurus japonicus Houttuyn＝Leonurus japonicus Houtt.

LabiataeLeonurus sibiricus Linn áe＝Leonurus sibiricus L.

Lilium Bulbビャクゴウ

Lilium lancifolium Thunberg＝Lilium lancifolium Thunb.

Liliaceae

Lilium brownii F. E. Brown var. colchesteri Wilosn＝Lilium brownii F. E. Br. var. colchesteri (Van Houtte) E. H. Wilson exElwes

Lilium brownii F. E. Brown var. viridulum Baker

Lilium brownii F. E. Brown＝Lilium brownii F. E. Br.

Lilium pumilum De Candolle＝Lilium pumilum DC.

Lindera Rootウヤク

Lindera strychnifolia Fernandez-Villar＝Lindera strychnifolia (Siebold & Zucc.) Fern.-Vill. Lauraceae

Lindera aggregata (Sims) Kosterm.

Lithospermum Rootシコン

Lithospermum erythrorhizon Siebold et Zuccarini＝Lithospermum erythrorhizon Siebold & Zucc.

Boraginaceae


2239

Longan Arilリュウガンニク

Euphoria longana Lamarck＝Euphoria longana Lam. Sapindaceae

Dimocarpus longan Lour.

Lonicera Leaf and Stemニンドウ

Lonicera japonica Thunberg＝Lonicera japonica Thunb.

Caprifoliaceae

Loquat Leafビワヨウ

Eriobotrya japonica Lindley＝Eriobotrya japonica (Thunb.) Lindl.

Rosaceae

Lycium Barkジコッピ

Lycium chinense Miller＝Lycium chinense Mill.

SolanaceaeLycium barbarum Linn áe＝Lycium barbarum L.

Lycium Fruitクコシ

Lycium chinense Miller＝Lycium chinense Mill.

SolanaceaeLycium barbarum Linn áe＝Lycium barbarum L.

Magnolia Barkコウボク

Magnolia obovata Thunberg＝Magnolia obovata Thunb.

Magnoliaceae

* Magnolia hypoleuca Siebold et Zuccarini＝Magnolia hypoleuca Siebold & Zucc.

Magnolia officinalis Rehder et Wilson＝Magnolia officinalis Rehder & E. H. Wilson

Magnolia officinalis Rehder et Wilson var. biloba Rehder et Wilson＝Magnolia officinalis Rehder & E. H. Wilson var. biloba Rehder & E.H. Wilson

Magnolia Flowerシンイ

Magnolia salicifolia Maximowicz＝Magnolia salicifolia (Siebold & Zucc.) Maxim.

Magnoliaceae

Magnolia kobus De Candolle＝Magnolia kobus DC.

Magnolia biondii Pampanini＝Magnolia biondii Pamp.

Magnolia sprengeri Pampanini＝Magnolia sprengeri Pamp.

Magnolia heptapeta Dandy＝Magnolia heptapeta (Buchoz) Dandy

* Magnolia denudata Desrousseaux＝Magnolia denudata Desr.

Mallotus Barkアカメガシワ

Mallotus japonicus Mueller Argoviensis＝Mallotus japonicus (Thunb.) M äull. Arg.

Euphorbiaceae

Mentha HerbハッカMentha Oilハッカ油

Mentha arvensis Linn áe var. piperascens Malinvaud＝Mentha arvensis L. var. piperascens Malinv.

LabiataeMentha haplocalyx Briq.

Hybrid originated from Mentha arvensis L. var. piperascens Malinv. asthe mother species

Moutan Barkボタンピ

Paeonia suffruticosa AndrewsPaeoniaceae

* Paeonia moutan Sims


2240

Mulberry Barkソウハクヒ

Morus alba Linn áe＝Morus alba L.

Moraceae

Nelumbo Seedレンニク

Nelumbo nucifera Gaertner＝Nelumbo nucifera Gaertn.

Nymphaeaceae

Notopterygiumキョウカツ

Notopterygium incisum Ting ex H. T. ChangUmbelliferae

Notopterygium forbesii Boissieu

Nuphar Rhizomeセンコツ

Nuphar japonicum De Candolle＝Nuphar japonicum DC.

Nymphaeaceae

Nutmegニクズク

Myristica fragrans Houttuyn＝Myristica fragrans Houtt.

Myristicaceae

Nux Vomicaホミカ

Strychnos nux-vomica Linn áe＝Strychnos nux-vomica L.

Loganiaceae

Ophiopogon Tuberバクモンドウ

Ophiopogon japonicus Ker-Gawler＝Ophiopogon japonicus (L. f.) Ker Gawl.

Liliaceae

Oriental Bezoarゴオウ

Bos taurus Linn áe var. domesticus Gmelin＝Bos taurus L. var. domesticus Gmelin

Bovidae

Oyster Shellボレイ

Ostrea gigas Thunberg＝Ostrea gigas Thunb.

Ostreidae

Panax Japonicus Rhizomeチクセツニンジン

Panax japonicus C. A. Meyer＝Panax japonicus C. A. Mey.

Araliaceae

Peach Kernelトウニン

Prunus persica Batsch＝Prunus persica (L.) Batsch

RosaceaePrunus persica Batsch var. davidiana Maximowicz＝Prunus persica (L.) Batsch var. davidiana (Carri àere) Maxim.

Prunus davidiana (Carri àere) Franch.

Peony Rootシャクヤク

Paeonia lactiflora Pallas＝Paeonia lactiflora Pall.

Paeoniaceae

Perilla Herbソヨウ

Perilla frutescens Britton var. acuta Kudo＝Perilla frutescens (L.) Britton var. acuta (Thunb.) Kudo

LabiataePerilla frutescens Britton var. crispa Decaisne＝Perilla frutescens (L.) Britton var. crispa (Thunb.) Decne.

Peucedanum Rootゼンコ

Peucedanum praeruptorum Dunn

UmbelliferaeAngelica decursiva Franchet et Savatier＝Angelica decursiva (Miq.) Franch. & Sav.

* Peucedanum decursivum Maximowicz＝Peucedanum decursivum (Miq.) Maxim.

Pharbitis Seedケンゴシ

Pharbitis nil Choisy＝Pharbitis nil (L.) Choisy

Convolvulaceae

Phellodendron Barkオウバク

Phellodendron amurense Ruprecht＝Phellodendron amurense Rupr.

RutaceaePhellodendron amurense Rupr. var. sachalinense F. SchmidtPhellodendron amurense Rupr. var. japonicum (Maxim.) OhwiPhellodendron amurense Rupr. var. lavallei (Dode) Sprangue

Phellodendron chinense Schneider＝Phellodendron chinense C. K. Schneid.


2241

Picrasma Woodニガキ

Picrasma quassioides Bennet＝Picrasma quassioides (D. Don) Benn.

Simaroubaceae

Pinellia Tuberハンゲ

Pinellia ternata Breitenbach＝Pinellia ternata (Thunb.) Breitenb.

Araceae

Plantago Herbシャゼンソウ

Plantago asiatica Linn áe＝Plantago asiatica L.

Plantaginaceae

Plantago Seedシャゼンシ

Plantago asiatica Linn áe＝Plantago asiatica L.

Plantaginaceae

Platycodon Rootキキョウ

Platycodon grandiflorum A. De Candolle＝Platycodon grandiflorum (Jacq.) A. DC.

Campanulaceae

Pogostemon Herbカッコウ

Pogostemon cablin Bentham＝Pogostemon cablin (Blanco) Benth.

Labiatae

Polygala Rootオンジ

Polygala tenuifolia Willdenow＝Polygala tenuifolia Willd.

Polygalaceae

Polygonatum Rhizomeオウセイ

Polygonatum falcatum A. Gray

Liliaceae

Polygonatum sibiricum Redout áe

Polygonatum kingianum Collett et Hemsley＝Polygonatum kingianum Collett & Hemsl.

Polygonatum cyrtonema Hua

Polygonum Rootカシュウ

Polygonum multiflorum Thunberg＝Polygonum multiflorum Thunb.

Polygonaceae

Polyporus Sclerotiumチョレイ

Polyporus umbellatus Fries＝Polyporus umbellatus (Pers.) Fries

Polyporaceae

Poria Sclerotiumブクリョウ

Wolfiporia cocos Ryvarden et Gilbertson＝Wolfiporia cocos (Schw.) Ryv. & Gilbn.

Polyporaceae* Poria cocos Wolf＝Poria cocos (Schw.) Wolf

Processed Aconite Rootブシ

Aconitum carmichaeli Debeaux

RanunculaceaeAconitum japonicum Thunberg＝Aconitum japonicum Thunb.

Processed Gingerカンキョウ

Zingiber officinale Roscoe Zingiberaceae

Prunella Spikeカゴソウ

Prunella vulgaris Linn áe var. lilacina Nakai＝Prunella vulgaris L. var. lilacina Nakai

Labiatae

Pueraria Rootカッコン

Pueraria lobata Ohwi＝Pueraria lobata (Willd.) Ohwi

Leguminosae

Quercus Barkボクソク

Quercus acutissima Carruthers＝Quercus acutissima Carruth.

FagaceaeQuercus serrata Murray

Quercus mongolica Fischer ex Ledebour var. crispula Ohashi＝Quercus mongolica Fisch. ex Ledeb. var. crispula (Blume) Ohashi

Quercus variabilis Blume

Red Ginsengコウジン

Panax ginseng C. A. Meyer＝Panax ginseng C. A. Mey. Araliaceae

* Panax schinseng Nees


2242

Rehmannia Rootジオウ

Rehmannia glutinosa Liboschitz var. purpurea Makino＝Rehmannia glutinosa Libosch. var. purpurea Makino

ScrophulariaceaeRehmannia glutinosa Liboschitz＝Rehmannia glutinosa Libosch.

Rhubarbダイオウ

Rheum palmatum Linn áe＝Rheum palmatum L.

Polygonaceae

Rheum tanguticum Maximowicz＝Rheum tanguticum Maxim.

Rheum officinale Baillon＝Rheum officinale Baill.

Rheum coreanum Nakai

Hybrid between above species

Rose Fruitエイジツ

Rosa multiflora Thunberg＝Rosa multiflora Thunb.

Rosaceae

Rosinロジン

Several plants of Pinus genus Pinaceae

Royal Jellyローヤルゼリー

Apis mellifera Linn áe＝Apis mellifera L. Apidae

Apis cerana Fabricius

Safflowerコウカ

Carthamus tinctorius Linn áe＝Carthamus tinctorius L.

Compositae

Saffronサフラン

Crocus sativus Linn áe＝Crocus sativus L.

Iridaceae

Saposhnikovia Root and Rhi-zomeボウフウ

Saposhnikovia divaricata Schischkin＝Saposhnikovia divaricata (Turcz.) Schischk.

Umbelliferae

Sappan Woodソボク

Caesalpinia sappan Linn áe＝Caesalpinia sappan L.

Leguminosae

Saussurea Rootモッコウ

Saussurea lappa Clarke＝Saussurea lappa (Decne.) C. B. Clarke Compositae

Aucklandia lappa Decne.

Schisandra Fruitゴミシ

Schisandra chinensis Baillon＝Schisandra chinensis (Turcz.) Baill.

Schisandraceae

Schizonepeta Spikeケイガイ

Schizonepeta tenuifolia Briquet＝Schizonepeta tenuifolia Briq.

Labiatae

Scopolia Rhizomeロートコン

Scopolia japonica Maximowicz＝Scopolia japonica Maxim.

SolanaceaeScopolia carniolica Jacquin＝Scopolia carniolica Jacq.

Scopolia parviflora Nakai＝Scopolia parviflora (Dunn) Nakai

Scutellaria Rootオウゴン

Scutellaria baicalensis Georgi Labiatae


2243

Senegaセネガ

Polygala senega Linn áe＝Polygala senega L.

PolygalaceaePolygala senega Linn áe var. latifolia Torrey et Gray＝Polygala senega L. var. latifolia Torr. & A. Gray

Senna Leafセンナ

Cassia angustifolia VahlLeguminosae

Cassia acutifolia Delile

Sesameゴマ

Sesamum indicum Linn áe＝Sesamum indicum L.

Pedaliaceae

Sinomenium Stem and Rhi-zomeボウイ

Sinomenium acutum Rehder et Wilson＝Sinomenium acutum (Thunb.) Rehder & E. H. Wilson

Menispermaceae

Smilax Rhizomeサンキライ

Smilax glabra Roxburgh＝Smilax glabra Roxb.

Liliaceae

Sophora Rootクジン

Sophora flavescens Aiton Leguminosae

Sweet Hydrangea Leafアマチャ

Hydrangea macrophylla Seringe var. thunbergii Makino＝Hydrangea macrophylla (Thunb.) Ser. var. thunbergii (Siebold) Makino

Saxifragaceae

Swertia Herbセンブリ

Swertia japonica Makino＝Swertia japonica (Shult.) Makino

Gentianaceae

Toad Venomセンソ

Bufo bufo gargarizans CantorBufonidae

Bufo melanostictus Schneider

Tragacanthトラガント

Astragalus gummifer Labillardi áere＝Astragalus gummifer Labill.

Leguminosae

Tribulus Fruitシツリシ

Tribulus terrestris Linn áe＝Tribulus terrestris L.

Zygophyllaceae

Trichosanthes Rootカロコン

Trichosanthes kirilowii Maximowicz＝Trichosanthes kirilowii Maxim.

CucurbitaceaeTrichosanthes kirilowii Maximowicz var. japonica Kitamura＝Trichosanthes kirilowii Maxim. var. japonica (Miq.) Kitam.

Trichosanthes bracteata Voigt＝Trichosanthes bracteata (Lam.) Voigt

Turmericウコン

Curcuma longa Linn áe＝Curcuma longa L.

Zingiberaceae

Uncaria Hookチョウトウコウ

Uncaria rhynchophylla Miquel＝Uncaria rhynchophylla (Miq.) Miq.

RubiaceaeUncaria sinensis Haviland＝Uncaria sinensis (Oliv.) Havil.

Uncaria macrophylla Wallich＝Uncaria macrophylla Wall.

Zanthoxylum Fruitサンショウ

Zanthoxylum piperitum De Candolle＝Zanthoxylum piperitum (L.) DC. Rutaceae

Zanthoxylum piperitum (L.) DC. f. inerme Makino

Zedoaryガジュツ

Curcuma zedoaria Roscoe Zingiberaceae

When ``Other species of the same genus'' is included as its original plants the scientific name is not written in Monograph,however, it is written in this table.Reference

Terabayashi S. et al.: Pharmaceutical and Medical Device Regulatory Science, 41(5), 407 � 418 (2010).


22442244 JP XVIDrug Formulation / General Information

G6 Drug Formulation

Tablet Friability TestThis test is harmonized with the European Pharmacopoeia


The Tablet Friability Test is a method to determine thefriability of compressed uncoated tablets. The test procedurepresented in this chapter is generally applicable to most com-pressed tablets. Measurement of tablets friability supple-ments other physical strength measurement, such as tabletcrushing strength.

Use a drum, with an internal diameter between 283 and291 mm and a depth between 36 and 40 mm, of transparentsynthetic polymer with polished internal surfaces, and sub-ject to minimum static build-up (see figure for a typical ap-paratus). One side of the drum is removable. The tablets aretumbled at each turn of the drum by a curved projectionwith an inside radius between 75.5 and 85.5 mm that extendsfrom the middle of the drum to the outer wall. The outer di-ameter of the central ring is between 24.5 and 25.5 mm. Thedrum is attached to the horizontal axis of a device thatrotates at 25± 1 rpm. Thus, at each turn the tablets roll orslide and fall onto the drum wall or onto each other.

For tablets with a unit mass equal to or less than 650 mg,take a sample of whole tablets n corresponding as near aspossible to 6.5 g. For tablets with a unit mass of more than650 mg, take a sample of 10 whole tablets. The tabletsshould be carefully dedusted prior to testing. Accuratelyweigh the tablet sample, and place the tablets in the drum.Rotate the drum 100 times, and remove the tablets. Removeany loose dust from the tablets as before, and accuratelyweigh.

Generally, the test is run once. If obviously cracked,cleaved, or broken tablets are present in the tablet sampleafter tumbling, the sample fails the test. If the results aredifficult to interpret or if the mass loss is greater than thetargeted value, the test should be repeated twice and themean of the three tests determined. A maximum mean massloss from the three samples of not more than 1.0z is consi-dered acceptable for most products.

If tablet size or shape causes irregular tumbling, adjust the

drum base so that the base forms an angle of about 109withthe horizontal and the tablets no longer bind together whenlying next to each other, which prevents them from fallingfreely.

Effervescent tablets and chewable tablets may have differ-ent specifications as far as friability is concerned. In the caseof hygroscopic tablets, an appropriate humidity-controlledenvironment is required for testing.

Drums with dual scooping projections, or apparatus withmore than one drum, for the running of multiple samples atone time, are also permitted.

G7 Containers and Package

Plastic Containers forPharmaceutical Products

Various kinds of plastics are used as the materials formanufacturing containers for pharmaceutical products.Such plastics should not have the properties to deterioratethe efficacy, safety or stability of the pharmaceutical prod-ucts to be packed in the container. In selecting a suitableplastic container, it is desirable to have sufficient informa-tion on the manufacturing processes of the plastic containerincluding that on the substances added. Since each plastichas specific properties and a wide variety of pharmaceuticalproducts may be packed in plastic containers, the com-patibility of plastic containers with pharmaceutical productsshould be judged for each combination of container and thespecific pharmaceutical product to be contained therein.Such judgement should be performed through a verificationthat the container for the pharmaceutical preparation cancomply with the essential requirements for the container,i.e., the design specifications, based on the data from the ex-periments on the prototype products of the container and/orinformation from scientific documentation, etc. In addition,such compatibility must be ensured based upon an appropri-ate quality assurance system.

Furthermore, in introducing a plastic container, it isdesirable that proper disposal method after use is taken intoconsideration.

1. Essential Requirements in Designing Plastic Containersfor Pharmaceutical Products

The plastic material used for the container should be ofhigh quality. Therefore, recycled plastic materials, which areof unknown constitution, must not be used.

The leachables or migrants from the container should notdeteriorate the efficacy or stability of the pharmaceuticalproducts contained therein. In addition, the possible toxichazards due to the leachables or migrants should not exceeda given level. Furthermore, the amounts of leachable ormigratable chemical substances, such as monomers and ad-ditives, from the containers to the pharmaceutical productscontained therein must be sufficiently small from the view-point of safety.

The container should have a certain level of physicalproperties such as hardness, flexibility, shock resistance, ten-sile strength, tear strength, bending strength, heat resistance

22452245JP XVI General Information / Containers and Package

and the like, in accordance with its intended usage.The quality of the pharmaceutical products packed in the

container must not deteriorate during storage. For example,in the case of pharmaceutical products which are unstable tolight, the container should provide a sufficient level of lightshielding. In the case of pharmaceutical products which areeasily oxidized, the container material should not allow thepermeation of oxygen. In the case of aqueous pharmaceuti-cal products and pharmaceutical products that must be keptdry, the container material should not allow the permeationof water vapor. In addition, it is necessary to pay attentionto the permeability of solvents other than water through thecontainer. The concentration of the pharmaceuticals mustnot be decreased by more than a certain level due to the ad-sorption of the pharmaceuticals on the surface of the con-tainer, the migration of the pharmaceuticals into the insideof the material of the container, or the loss of pharmaceuti-cals through the container. Also, the pharmaceutical prod-ucts contained therein must not be degraded by an interac-tion with the material of the container.

The container should not be deformed, should not de-teriorate and should not be degraded by the pharmaceuticalproducts contained therein. Unacceptable loss of function ofthe container should not result from a possible high tempera-ture or low temperature or their repetitions encountered dur-ing storage or transportation.

The container should be of a required level of transparen-cy, when it is necessary to examine foreign insoluble matterand/or turbidity of the pharmaceutical products by visualobservation.

In the case of pharmaceutical products which must besterilized, it is required to satisfy the above-mentioned essen-tial requirements of the container after the sterilization, ifthere is a possibility that the quality of the container maychange after the sterilization. There should not be anyresidue or generation of new toxic substances of more thancertain risk level after the sterilization. In addition, the con-tainer should not have any inappropriate structure and/ormaterial that might result in any bacterial contamination ofthe pharmaceutical products contained therein duringstorage and transportation after sterilization.

2. Toxicity Evaluation of Container at Design PhaseFor design verification, the toxicity of the container

should be evaluated. For the toxicity evaluation, it is desira-ble to select appropriate test methods and acceptance criteriafor the evaluation, and to explain the rationale for the selec-tion clearly. The tests should be conducted using samples ofthe whole or a part of the prototype products of the con-tainer. If the container consists of plural parts of differentmaterials, each part should be tested separately. Such mate-rials as laminates, composites, and the like are regarded as asingle material. To test containers made of such materials, itis recommended to expose the inner surface of the container,which is in contact with the pharmaceutical products con-tained therein, to the extraction media used in the tests as faras possible.

The tests required for the toxicity evaluation of the con-tainer are different depending upon the tissue to which thepharmaceutical products contained therein are to be applied.

The following tests are required for containers for( i ) preparations in contact with blood:

Acute toxicity test, cytotoxicity test, sensitization

test and hemolysis test( ii ) preparations in contact with skin or mucous mem-

branes:Cytotoxicity test and sensitization test

(iii) liquid orally administered preparations:Cytotoxicity test

It is recommended to conduct the tests in accordance withthe latest versions of the standard test methods on medicaldevices and materials published in Japan and other coun-tries.

Those standard test methods are listed for information:2.1. Examplification of Standard Tests2.1.1. Selection of Tests

(i) Guidelines for Basic Biological Tests of MedicalDevices and Materials (PAB Notification, YAKU-KI NO.99,June 27, 1995), Principles and selection of tests

(ii) ISO 10993-1: Biological evaluation of medicaldevices�Evaluation and testing2.1.2. Acute Toxicity Test

(i) ASTM F750-82: Standard practice for evaluating ma-terial extracts by systemic injection in the mice

(ii) BS5736: Part 3 Method of test for systemic toxicity;assessment of acute toxicity of extracts from medical devices

(iii) USP 24 <88> Biological reactivity tests, in vivo2.1.3. Cytotoxicity Test

(i) Guidelines for Basic Biological Tests of MedicalDevices and Materials, I. Cytotoxicity Test 10. Cytotoxicitytest using extract of medical device or material

(ii) ISO 10993-5: Biological evaluation of medicaldevices�Tests for cytotoxicity : in vitro methods

(iii) USP 24 <87> Biological reactivity tests, in vitro2.1.4. Hemolysis Test

(i) Guidelines for Basic Biological Tests of MedicalDevices and Materials, VII. Hemolysis Test

(ii) ISO 10993-4: Biological evaluation of medicaldevices�Selection of tests for interaction with blood. AnnexD

(iii) ASTM F756-82: Standard practice for assessment ofhemolytic properties of materials2.1.5. Sensitization Test

(i) Guidelines for Basic Biological Tests of MedicalDevices and Materials, II. Sensitization Test

(ii) ISO 10993-10: Biological evaluation of medicaldevices�Tests for irritation and sensitization

3. Test Results to be Recorded per Production UnitAt the commercial production phase, it is required to

establish acceptance criteria on at least the test items listedbelow and to record the test results of each production unitof plastic containers for pharmaceutical products. In addi-tion, it is desirable to explain the rationale for setting theacceptance criteria clearly. However, these requirementsshould not be applied to orally administered preparationsexcept for liquid preparations.

(i) Combustion Tests: Residue of ignition, heavy metals.If necessary, the amounts of specified metals (lead, cad-mium, etc.)

(ii) Extraction Tests: pH, ultraviolet absorption spectra,potassium permanganate-reducing substances, foaming,non-volatile residue

(iii) Cytotoxicity Test(iv) Any other tests necessary for the specific container

for aqueous infusions.

22462246 JP XVIWater / General Information

G8 Water

Quality Control of Water forPharmaceutical Use

Water used for manufacturing pharmaceutical productsand for cleaning their containers and equipments used in themanufacture of the products is referred to as ``pharmaceuti-cal water''. For assuring the quality of pharmaceutical waterconsistently, it is important to verify through appropriateprocess validation of water processing system that water withthe quality suitable for its intended use is produced and sup-plied, and to keep the quality of produced water throughroutine works for controlling the water processing system.

1. Types of Pharmaceutical Water1.1. Water

The specification for ``Water'' is prescribed in theJapanese Pharmacopoeia (JP) monograph. It is required forWater to meet the Quality Standards for Drinking Waterprovided under the Article 4 of the Japanese Water SupplyLaw. In the case that Water is produced at individual facili-ties using well water or industrial water as source water, it isnecessary for produced water to meet the Quality Standardsfor Drinking Water and an additional requirement for am-monium of ``not more than 0.05 mg/L''. Furthermore,when Water is to be used after storing for a period of time, itis necessary to prevent microbial proliferation.

Water is used as source water for Purified Water andWater for Injection. It is also used for manufacturing inter-mediates of active pharmaceutical ingredients (APIs), andfor pre-washing of the equipments used in the manufactureof pharmaceutical products.1.2. Purified Water

The specifications for ``Purified Water'' and ``PurifiedWater in Containers'' are prescribed in the JP monographs.Purified Water is prepared by distillation, ion-exchange,reverse osmosis (RO), ultrafiltration (UF) capable of remov-ing substances with molecular masses of not less than ap-proximately 6000, or a combination of these processes fromWater, after applying some adequate pretreatments if neces-sary. For the production of Purified Water, appropriate con-trol of microorganisms is required. Particularly, in the casethat Purified Water is prepared by ion-exchange, RO or UF,it is necessary to apply the treatments adequate for prevent-ing microbial proliferation, or to sanitize the system periodi-cally.

When Purified Water is treated with chemical agents forsterilizing, preventing microbial proliferation, or maintain-ing the endotoxin level within an appropriate control range,a specification suitable for the intended use of treated watershould be established individually, and a process control forkeeping the quality of treated water in compliance with thespecification thus established should be performed.

``Purified Water in Containers'' is prepared from PurifiedWater by introducing it in a tight container.1.3. Sterile Purified Water

The specification for ``Sterile Purified Water in Contain-ers'' (its alternative name is Sterile Purified Water) is

prescribed in the JP monograph.Sterile Purified Water in Container is prepared from Puri-

fied Water by 1) introducing it into a hermetic container,sealing up the container, then sterilizing the product, or 2)making it sterile by using a suitable method, introducing thesterilized water into a sterile hermetic container by applyingaseptic manipulation, then sealing up the container.

Plastic containers for aqueous injections may be used inplace of hermetic containers.1.4. Water for Injection

The specifications for ``Water for Injection'' and ``SterileWater for Injection in Containers'' are prescribed in the JPmonographs. Water for Injection is prepared by distillationor reverse osmosis and/or ultrafiltration (RO/UF), eitherfrom Water after applying some adequate pretreatmentssuch as ion exchange, RO, etc., or from Purified Water.

In the case of water processing systems based on distilla-tion, it is necessary to take care for avoiding contaminationof produced water by the impurities accompanied with theentrain.

In the case of water processing system based on RO/UF, itis required to provide water with equivalent quality to thatprepared by distillation consistently, based on substantialprocess validation through long-term operation andelaborate routine control of the system. It is essential toensure consistent production of water suitable for Water forInjection by the entire water processing system includingpretreatment facilities, in any systems based on RO/UF. Forthe water supplied to the system, it is also required to keepthe quality suitable as source water through adequate valida-tion and routine control on the water.

For the water processing system based on RO/UF, routinecontrol should be performed by analyzing water specimens,monitoring some quality attributes using in-line apparatusand checking the volume of water passed through the sys-tem. In addition, it is recommended to carry out periodicalappearance observation and air-leak test on the membranesbeing currently used. It is also recommended to establishprotocols for keeping the performance of membrane mod-ules within appropriate control ranges and for estimating thetiming to exchange the modules, through diagnosis on thedegree of deterioration based on the results of tensilestrength test on the used membrane modules, and visual ob-servation on those modules whether any leakages of mem-branes have occurred or not, and to what extent they haveoccurred. Furthermore, it is desirable to establish the fre-quency of membrane exchange considering with its actualcondition of use.

In the case that Water for Injection is stored in the waterprocessing system temporarily, a stringent control for micro-organisms and endotoxins should be taken. An acceptablecriterion of lower than 0.25 EU/mL for endotoxins is speci-fied in the JP monograph of Water for Injection.

``Sterile Water for Injection in Container'' is preparedfrom Water for Injection by 1) introducing it into a hermeticcontainer, sealing up the container, then sterilizing theproduct, or 2) making it sterile by using a suitable method,introducing the sterilized water into a sterile hermetic con-tainer by applying aseptic manipulation, then sealing up thecontainer.

Plastic containers for aqueous injections may be used inplace of hermetic containers.

22472247JP XVI General Information / Water

2. Reverse Osmosis and/or Ultrafiltration (RO/UF)RO/UF are the methods for refining water by using mem-

brane modules based on either reverse osmosis or ultrafiltra-tion, or the modules combining them, and used as the alter-native methods for distillation in the production of PurifiedWater or Water for Injection.

When Water for Injection is produced by RO/UF, a waterprocessing system equipped with pretreatment facilities,facilities for producing Water for Injection and facilities forsupplying Water for Injection is usually used. The pretreat-ment facilities are used to remove solid particles, dissolvedsalts and colloids in source water, and placed before thefacilities for producing Water for Injection so as to reducethe load on the facilities for producing Water for Injection.They consist of apparatus properly selected from aggrega-tion apparatus, precipitation-separation apparatus, filtrationapparatus, chorine sterilization apparatus, oxidation-reduc-tion apparatus, residual chlorine-removing apparatus,precise filtration apparatus, reverse osmosis apparatus,ultrafiltration apparatus, ion exchange apparatus, etc.,depending on the quality of source water. The facilities forproducing Water for Injection consist of apparatus for sup-plying pretreated water, sterilization apparatus with ultravio-let rays, heat exchange apparatus, membrane modules, ap-paratus for cleaning and sterilizing the facilities, etc. Thefacilities for supplying Water for Injection consist of a reser-voir tank for storing Water for Injection in the facilities tem-porarily, pipe lines, heat exchange apparatus, a pump forcirculating Water for Injection in the facilities, pressure con-trol apparatus, etc.

In the case that Water for Injection is stored in the waterprocessing system temporarily, it should usually be circu-lated in a loop consisting of a reservoir tank and pipe lines ata temperature not lower than 809C for preventing microbialproliferation.

When Purified Water is produced by RO/UF, basic com-position of water processing system is almost the same asthat for Water for Injection described above.

When RO/UF is utilized for preparing pharmaceuticalwater, it is necessary to select the most appropriate combina-tion of membrane modules in consideration of the quality ofsource water and the quality of produced water required forits intended use. When the ultrafiltration membrane is usedto prepare Purified Water or Water for Injection, membranemodules capable of removing microorganisms and sub-stances with molecular masses not less than approximately6000 should be used.

3. Selection of Pharmaceutical WaterDepending on the intended use of pharmaceutical water,

the water suitable for assuring the quality of final productswithout causing any trouble during their manufacturingprocesses, should be selected from the above 4 types (1.1. ¿1.4.) of pharmaceutical water specified in the JP. Table 1 ex-emplifies a protocol for such selection (in the case of phar-maceutical water used for the manufacture of drug prod-ucts).

Sterile Purified Water in Containers or Water for Injec-tion (or Sterile Water for Injection in Containers) may beused in place of Purified Water or Purified Water in Con-tainers.3.1. Drug Products

For the manufacture of sterile drug products, for which

contamination with microorganisms or endotoxins is notpermissible, Water for Injection (or Sterile Water for Injec-tion in Containers) should be used. For the manufacture ofophthalmics and eye ointments, Purified Water (or PurifiedWater in Containers) can also be used.

For the manufacture of non-sterile drug products, waterwith a quality not lower than that of Purified Water (orPurified Water in Containers) should be used. However, outof the non-sterile drug products such as liquids, ointments,suspensions, emulsions, suppositories and aerosols, for thosewhich require care against microbiological contamination,Purified Water (or Purified Water in Containers) adequatelycontrolled from microbiological viewpoints should be usedin consideration of the possible impacts of preservativesformulated in the drug products. For the manufacture ofproducts containing crude drugs, it is recommended to selectadequate type of water considering with viable counts of thecrude drugs used for manufacturing the product andmicrobial limit required for the product.

Water used for pre-washing of containers or equipmentsurfaces that comes in direct contact with the drug productsshould have the quality not lower than that of Water. Waterused for final rinsing should have an equivalent quality tothat of water used for manufacturing drug products.3.2. Active Pharmaceutical Ingredient (API)

Water used for manufacturing active pharmaceutical in-gredient (API) should be selected in consideration of thecharacteristics of drug product for which the API is to beused, and its manufacturing process, so that the quality ofthe final drug product is assured.

Water used for manufacturing API or for cleaning con-tainers or equipment surfaces that comes in direct contactwith the raw materials or API intermediates, should have thequality not lower than that of Water adequately controlledfrom the chemical and microbiological viewpoints, even ifthe water is used at an earlier stage of synthetic or extractionprocess in the manufacture of API. Water used in the finalpurification process should have the quality equal to orhigher than that of Purified Water (or Purified Water inContainers). Water used for final rinsing of containers orequipment surfaces that comes in direct contact with theAPIs should have an equivalent quality to that of water usedfor manufacturing the APIs.

For manufacturing sterile API, Sterile Purified Water inContainers or Water for Injection (or Sterile Water forInjection in Containers) should be used. Similarly, formanufacturing APIs used for drug products where endo-toxin control is required and there are no subsequentprocesses capable of removing endotoxins, Water for Injec-tion (or Sterile Water for Injection in Containers), or Puri-fied Water (or Purified Water in Containers) for whichendotoxins are controlled at a low level, should be used.

4. Quality Control of Pharmaceutical Water4.1. Outline

Verification that water with the quality required for its in-tended use has been produced by the pharmaceutical waterprocessing system through substantial validation studies atan earlier stage of its operation, is the prerequisite for con-ducting quality control on pharmaceutical water in a routineand periodical manner. If this prerequisite is fulfilled, thefollowing methods are applicable for quality control of phar-maceutical water.

2248

Table 1 An Exemplified Protocol for Selecting Pharmaceutical Water(Water Used in the Manufacture of Drug Products or APIs)

Classification Class of Pharmaceutical Water Application Remarks

Drug Product Water for Injection or SterileWater for Injection in Contain-ers

Injections, Ophthalmics, EyeOintments

Purified Water or PurifiedWater in Containers

Ophthalmics, Eye Ointment For ophthalmics and eye oint-ments for which precautionsshould be taken against microbialcontamination, Purified Water orPurified Water in Containers keptits viable counts at low levelsthrough sterilization, UF filtra-tion, etc. should be used.

Aerosols, Liquids and Solutions,Extracts, Elixirs, Capsules, Gran-ules, Pills, Suspensions andEmulsions, Suppositories, Pow-ders, Spirits, Tablets, Syrups, In-fusions and Decoctions, Plasters,Tinctures, Troches, Ointments,Cataplasms, Aromatic Waters,Liniments, Lemonades, Fluidex-tracts, Lotions, and Pharmaceuti-cal preparations of percutaneousabsorption type

For liquids, solutions, ointments,suspensions, emulsions, supposi-tories, aerosols and so on forwhich precautions should betaken against microbial contami-nation, Purified Water or Puri-fied Water in Containers ade-quately controlled frommicrobiological viewpointsshould be used.

ActivePharmaceuticalIngredient (API)

Water for Injection or SterileWater for Injection in Contain-ers

Sterile APIs, and APIs renderedsterile in the formulation process

Purified Water or PurifiedWater in Containers

APIs, APIs rendered sterile in theformulation process, and API in-termediates

In the manufacture of APIs to berendered sterile in the formula-tion process and have no subse-quent processes capable ofremoving endotoxins, PurifiedWater or Purified Water in Con-tainers controlled endotoxins at alow level should be used.

Water API Intermediates


For routine control, it is very useful to control quality ofproduced water based on the monitoring of electrical con-ductivity (conductivity) and total organic carbon (TOC). Inaddition, items to be monitored periodically, such as somespecified impurities, viable counts, endotoxins, insolubleparticulate matters, etc., should be determined according tothe intended use of pharmaceutical water. The frequency ofmeasurement should be determined considering with thevariation in the quality of water to be monitored.

The following are points to consider in controlling thequality of produced water from microbiological and phys-icochemical (conductivity and TOC) viewpoints. It is neces-sary to monitor other items if necessary, and to confirm thatthey meet the specifications established individually.4.2. Sampling

Monitoring should be conducted at an adequate frequencyto ensure that the pharmaceutical water processing system iswell-controlled and that water with acceptable quality is con-

tinuously produced and supplied. Specimens should be col-lected at the representative locations in the facilities forproducing and supplying water, with particular care so thatcollected specimens reflect the operating condition of thepharmaceutical water processing system. An adequate pro-tocol for the control of microorganisms at the sampling siteshould be established considering with the situation aroundthe site.

Sampling frequency should be established based on thedata from validation studies on the system. For microbiolog-ical monitoring, it is adequate to use the water specimens forthe test within 2 hours after sampling. In the case that it isnot possible to test within 2 hours, the specimens should bekept at 2 � 89C and be used for the test within 12 hours.4.3. Alert and Action Levels

In producing pharmaceutical water using a water process-ing system, microbiological and physicochemical monitoringis usually carried out to assure that water with required qual-

2249

Table 2 Methods for Assessment of Viable Counts in Pharmaceutical Water

MethodPharmaceutical Water

Water Purified Water Water for Injection

Measurement Method Pour Plate Method or Mem-brane Filtration

Pour Plate Method or Mem-brane Filtration

Membrane Filtration

Minimum Sample Size 1.0 mL 1.0 mL 100 mL

Media Standard Agar Medium R2A Agar Medium, StandardAgar Medium

R2A Agar Medium, StandardAgar Medium

Incubation Period Standard Agar Medium: 48 �72 hours (or longer)

R2A Agar Medium: 4 � 7 days(or longer)Standard Agar Medium: 48 �72 hours (or longer)

R2A Agar Medium: 4 � 7 days(or longer)Standard Agar Medium: 48 �72 hours (or longer)

Incubation Temperature Standard Agar Medium: 30 �359C

R2A Agar Medium: 20 � 259Cor 30 � 359CStandard Agar Medium: 30 �359C

R2A Agar Medium: 20 � 259Cor 30 � 359CStandard Agar Medium: 30 �359C


ity is being continuously produced when the system is oper-ating as it designed. The operating condition of the systemcan be estimated by the comparison of monitoring data thusobtained against the alert level, action level, other levels forcontrolling the system, and acceptance criteria specified forthe water required for its intended use, and also by the trendanalysis of monitoring data through plotting them in a con-trol chart. In this manner, the alert level and action level areused for controlling the process of water production, andnot used for judging pass/fail of produced water.4.3.1. Definition of Alert Level

``Alert level'' indicates that, when exceeded it, the systemis threatening to deviate from its normal operating range.Alert levels are used for giving a warning, and exceedingthem does not necessarily require a corrective action. Alertlevel is generally established either at a mean＋2s on thebasis of past trend analysis, or at a level of 70z (50z forviable counts) of action level, whichever is smaller.4.3.2. Definition of Action Level

``Action level'' indicates that, when exceeded it, the sys-tem has deviated from its normal operating range. Exceedingit indicates that corrective action must be taken to bring thesystem back within its normal operating range.

Alert and action levels should be established within thespecified acceptance criteria of the water required for its in-tended use in consideration of available technologies and thequality required for the water. Consequently, exceeding analert or action level does not necessarily indicate that thequality of produced water has become inadequate for its in-tended use.4.4. Microbiological Monitoring

The main purpose of microbiological monitoring programfor pharmaceutical water processing system is to foresee anymicrobiological quality deterioration of the produced water,and to prevent any adverse effects on the quality of phar-maceutical products. Consequently, detecting all of themicroorganisms present in the water to be monitored maynot be necessary. However it is required to adopt a monitor-ing technique able to detect a wide range of microorganisms,including slow growing microorganisms.

The followings indicate incubation-based microbiologicalmonitoring techniques for pharmaceutical water processingsystems. To adopt a rapid microorganism detection tech-nique, it is necessary to confirm in advance that the microbi-al counts obtained by such techniques are equivalent to thoseobtained by the incubation-based monitoring techniques.4.4.1. Media and Incubation Conditions

There are many mesophilic bacteria of heterotrophic typethat are adapted to poor nutrient water environments.Heterotrophic bacteria may form bio-films in many phar-maceutical water processing systems, and to cause qualitydeterioration of the produced water. Therefore, it is usefulto monitor microbiological quality of water by using theR2A Agar Medium, which is excellent for growing bacteriaof oligotrophic type. On the other hand, in routinemicrobiological monitoring, an approach by using theStandard Agar Medium, prescribed in the Quality Standardsfor Drinking Water provided under the Article 4 of theJapanese Water Supply Law, is widely employed. In thisapproach, the trend in microbiological change of waterprocessing system is estimated from the number of viablemicroorganisms capable of proliferating at 30 � 359C in theStandard Agar Medium in a comparatively short period oftime.

Table 2 shows examples of measurement methods,minimum sample sizes, media, and incubation periods forestimating viable counts.

The media shown in Table 2 are as follows.

(i) Standard Agar MediumCasein peptone 5.0 gYeast extract 2.5 gGlucose 1.0 gAgar 15.0 gWater 1000 mL

Mix all the ingredients, and sterilize by heating in anautoclave at 1219C for 15 � 20 minutes. pH after sterili-zation: 6.9 � 7.1.

(ii) R2A Agar MediumPeptone (casein and animal tissue) 0.5 g


Casamino acid 0.5 gYeast extract 0.5 gSodium pyruvate 0.3 gGlucose 0.5 gMagnesium sulfate heptahydrate 0.05 gSoluble starch 0.5 gDipotassium hydrogen phosphate 0.3 gAgar 15.0 gWater 1000 mL

Mix all the ingredients, and sterilize by heating in anautoclave at 1219C for 15 � 20 minutes. pH after sterili-zation: 7.1 � 7.3.

The following reagents should be used for preparing theR2A Agar Medium.

(i) Casamino acid Prepared for microbial test, by theacid hydrolysis of casein.

Loss on drying <2.41>: Not more than 8z (0.5 g, 1059C,constant mass)

Residue on ignition <2.44>: Not more than 55z (0.5 g)Nitrogen content <1.08>: Not less than 7z (1059C, con-

stant mass, after drying)(ii) Sodium pyruvate CH3COCOONa White to pale

yellow crystalline powder. Freely soluble in water, andslightly soluble in ethanol (99.5) and in acetone.

Identification (1) Determine the infrared absorptionspectrum as directed in the potassium bromide disk methodunder Infrared Spectrophotometry <2.25>: it exhibits absorp-tion maxima at the wave numbers around 1710 cm－1, 1630cm－1, 1410 cm－1, 1360 cm－1, 1190 cm－1, 1020 cm－1, 980cm－1, 830 cm－1, 750 cm－1, 630 cm－1 and 430 cm－1.

(2) A solution (1 in 20) responds to the Qualitative Tests<1.09> for sodium salt (1).

Content: Not less than 97.0z. Assay�Weigh accuratelyabout 0.4 g of sodium pyruvate and dissolve it in 200 mL ofwater. Transfer 20 mL of this solution into an iodine bottle,and cool to 109C or lower. Add 40 mL of 0.05 mol/L iodineVS and 20 mL of sodium hydroxide solution (17 in 100),then allow to stand in a dark place for 2 hours, and add 15mL of diluted sulfuric acid (1 in 6). Titrate <2.50> with 0.1mol/L sodium thiosulfate VS (indicator: starch TS). Per-form a blank determination in the same manner, and makeany necessary correction.

Each mL of 0.05 mol/L iodine VS＝ 1.834 mg of C3H3NaO3

4.4.2. Media Growth Promotion TestIn the media growth promotion test with the R2A Agar

Medium, use the strains listed below or other strains consi-dered equivalent to these strains. Prior to the test, inoculatethese strains into Sterile Purified Water and incubate at 20 �259C for 3 days.

Methylobacterium extorquens: NBRC 15911Pseudomonas fluorescens: NBRC 15842, ATCC 17386,

etc.Dilute the fluid containing the strain starved in Purified

Water with Sterile Purified Water to prepare a fluid contain-ing about 50 � 200 CFU/mL of viable counts. When pipet-ting 1 mL of the diluted fluid onto the R2A Agar Mediumand incubating at 20 � 259C for 4 � 7 days, sufficientproliferation of the inoculated strain must be observed.

In the media growth promotion test with the StandardAgar Medium, use the strains listed below or other strains

considered equivalent to these strains. Prepare the fluid con-taining the strain according to the procedure prescribed inthe Microbial Limit Test <4.05>. When pipetting 1 mL of thefluid onto the Standard Agar Medium and incubating at 30 �359C for 48 hours, sufficient proliferation of the inoculatedstrain must be observed.

Staphylococcus aureus: ATCC 6538, NCIMB 9518, CIP4.83 or NBRC 13276

Pseudomonas aeruginosa: ATCC 9027, NCIMB 8626,CIP 82.118 or NBRC 13275

Colon bacillus (Escherichia coli ): ATCC 8739, NCIMB8545, CIP 53.126 or NBRC 39724.4.3. Action Levels for Microorganisms for Pharmaceuti-cal Water Processing System

The following action levels are considered appropriate andgenerally applicable to pharmaceutical water processing sys-tems.

Action Levels for viable counts in various types of phar-maceutical water

Water: 100 CFU/mL* (Acceptance criterion prescribed inthe Quality Standards for Drinking Water provided underthe Article 4 of the Water Supply Law)

Purified Water: 100 CFU/mL**Water for Injection: 10 CFU/100 mL**(*Viable counts obtained using the Standard Agar Medi-

um, **Viable counts obtained using the R2A Agar Medium)

Although the action level for Purified Water shown aboveis set at the same level as that for Water, in near future, anadequate action level would be set for Purified Water in con-sideration of the current level of technologies. Therefore, forthe time being, it is recommended for each facility to per-form a higher level of microbiological control of waterprocessing system based on the action level established indi-vidually.

When actual counts in validation studies or routine con-trol exceed the above action levels, it is necessary to isolateand identify the microorganisms present in the water, and tosanitize or disinfect the affected system.4.5. Physicochemical Monitoring

Physicochemical monitoring of a pharmaceutical waterprocessing system is usually performed using conductivityand TOC as the indicators for water quality. By monitoringconductivity, total amounts of inorganic salts present in thewater can be estimated, and by monitoring TOC, totalamount of organic compounds present in the water can beestimated. Normally, the Conductivity Measurements <2.51>and the Test for Total Organic Carbon <2.59> specified inthe General Tests, Processes and Apparatus of the JP shouldbe applied to these physicochemical monitoring. However,since tests for monitoring are performed in the situationsdifferent from those for judging pass/fail to the acceptancecriteria prescribed in the monographs, supplements neces-sary to cover the situations to which the JP general tests can-not be applied, are described below.

To adopt the monitoring using conductivity and TOC asthe indicators for inorganic and organic impurities at in-dividual facility, appropriate alert and action levels, andcountermeasures against unexpected apparatus failuresshould be established for each indicator.4.5.1. Monitoring of Conductivity as the Indicator forInorganic Impurities

Measurement of conductivity for monitoring is usually

2251

Table 3 Stage 1 Allowable Conductivity for DifferentTemperatures*

Temperature(9C)

AllowableConductivity(mS･cm－1)

Temperature(9C)

AllowableConductivity(mS･cm－1)

0 0.65 0.8 55 2.1

10 0.9 60 2.215 1.0 65 2.420 1.1 70 2.525 1.3 75 2.730 1.4 80 2.735 1.5 85 2.740 1.7 90 2.745 1.8 95 2.950 1.9 100 3.1

* Applicable only to non-temperature-compensated con-ductivity measurements.


conducted continuously using an in-line apparatus with aflow-through type or pipe-insertion type cell. Alternatively,offline batch testing may be performed using a dip type cellwith water specimens taken at point-of-use sites or other ap-propriate locations of the pharmaceutical water processingsystem. For the operation control of a pharmaceutical waterprocessing system, guides for judging whether it is adequateto continue the operation of the system or not, based on theresults from monitoring of conductivity, are shown below,both for the cases of monitoring at the standard temperature(209C) by applying Conductivity Measurements <2.51> of theJP and monitoring at temperatures other than 209C byapplying <645> WATER CONDUCTIVITY of the UnitedStates Pharmacopeia (USP) with some modifications.4.5.1.1. Monitoring of Conductivity by applying the Con-ductivity Measurements <2.51> of the JP

The Conductivity Measurements <2.51> of the JP princi-pally requires to measure the conductivity at the standardtemperature (209C). However, measurement at a tempera-ture within a range of 15 � 309C may also be acceptable,when the results are corrected using the equation prescribedin the Conductivity Measurements <2.51>. In this case, therecommended allowable conductivity (action level) for Puri-fied Water and Water for Injection is as follows.・Action Level 1.0 mS･cm－1 (209C)Since the above allowable conductivity is established for

in-line monitoring, an alternative action level may be usedfor the monitoring based on offline batch testing.4.5.1.2. Monitoring of Conductivity by applying the <645>WATER CONDUCTIVITY of the USP with some modifi-cations

Usually, it is somewhat difficult to control the tempera-ture exactly in in-line conductivity monitoring. Therefore,the following approach can be applied for the monitoring attemperatures other than the standard temperature (209C) ofthe JP. This approach is based on the Stages 1 and 2 of thethree-stage approach described in ``<645> WATER CON-DUCTIVITY'' of the USP.

Stage 1 (In-line Measurement)( i ) Determine the temperature and the conductivity of

the water specimens using a non-temperature-com-pensated conductivity reading.

( ii ) From the Table 3, find the temperature value equalto or just lower than the measured temperature.Adopt the corresponding conductivity value on thistable as the allowable conductivity at the measuredtemperature.

(iii) If the observed conductivity is not greater than theallowable conductivity adopted above, the watertested meets the requirement for monitoring conduc-tivity. If the observed conductivity exceeds theallowable conductivity, proceed with Stage 2.

Stage 2 (Off-line Measurement)( i ) Measure the conductivity of the water specimen, by

transferring it into a container and agitating it vigor-ously in order to attain equilibrium between thewater specimen and the atmosphere on absorbing/desorbing carbon dioxide.

( ii ) Transfer a sufficient amount of water to be testedinto a suitable container, and stir the water speci-men. Adjust the temperature to 25± 19C, andbegin agitating the water specimen vigorously, while

observing the conductivity periodically. When thechange in conductivity, due to the uptake of at-mospheric carbon dioxide, becomes not greater than0.1 mS･cm－1 per 5 minutes, adopt the observedvalue as the conductivity (259C) of the water speci-men.

(iii) If the conductivity of the water specimen at 259Cobtained above is not greater than 2.1 mS･cm－1, thewater tested meets the requirement for monitoringconductivity. If the observed value exceeds 2.1 mS･cm－1, it should be judged that the water tested doesnot meet the requirement for monitoring conduc-tivity.

4.5.2. Monitoring of TOC as the Indicator for Organic Im-purities

The acceptance criterion of TOC is specified as ``notgreater than 0.50 mg/L (500 ppb)'' in the monographs ofPurified Water and Water for Injection. However it isrecommended for each facility preparing pharmaceuticalwater to conduct operation control of pharmaceutical waterprocessing system through TOC monitoring on producedwater based on its own alert and action levels for TOC deter-mined individually. The followings are the recommendedaction levels for TOC.・Action Level: ≦300 ppb (in-line)

≦400 ppb (off-line)The Quality Standards for Drinking Water provided under

the Article 4 of the Japanese Water Supply Law require thatTOC should be ``not greater than 3 mg/L (3 ppm)''. Takingthe above recommended action levels into consideration, it isalso recommended for each facility to conduct quality con-trol of source water through TOC monitoring based on itsown alert and action levels for TOC determined individually.

The JP specifies the Test for Total Organic Carbon <2.59>,and normally, TOC measurement should be conducted usingan apparatus which meets the requirements described in theJP method. However, if a TOC apparatus conforms to theapparatus suitability test requirements described in ``<643>TOTAL ORGANIC CARBON'' of the USP, or thosedescribed in the ``Methods of Analysis 2.2.44. TOTAL


ORGANIC CARBON IN WATER FOR PHARMACEU-TICAL USE'' of the European Pharmacopoeia (EP), theapparatus can be used for the monitoring of pharmaceuticalwater processing system, if sufficiently pure water not con-taminated with ionic organic substances, or organic sub-stances having nitrogen, sulfur, phosphorus or halogenatoms in their chemical structures, is used as the sourcewater supplied to the system.

A TOC apparatus, characterized by calculating theamount of organic carbon from the difference in conduc-tivity before and after the decomposition of organic sub-stances without separating carbon dioxide from the samplesolution, may be influenced negatively or positively, whenapplied to the water specimens containing ionic organic sub-stances, or organic substances having nitrogen, sulfur, phos-phorus or halogen atoms in their chemical structures. There-fore, the apparatus used for TOC monitoring should beselected appropriately in consideration of the purity of thewater to be monitored and the contamination risk in the caseof apparatus failure.4.6. Storage of Water for Injection

In storing Water for Injection temporarily, adequatemeasures able to prevent microbial proliferation stringently,such as circulating it in a loop at a high temperature must betaken, and an appropriate storage time should also be estab-lished based on the validation studies, in consideration of therisks of contamination and quality deterioration.

5. Points to Consider for Assuring the Quality of Phar-maceutical Water in Containers

There are some specific points to consider for assuring thequality of pharmaceutical water in containers (PurifiedWater in Containers, Sterile Purified Water in Containersand Sterile Water for Injection in Containers), which areavailable as commercially products.5.1. Methods for Preparing Sterile Pharmaceutical Waterin Containers and Their Sterilization Validation

The following 2 different preparation methods aredescribed in the monographs of Sterile Purified Water inContainers and Sterile Water for Injection in Containers.

( i ) Introduce Purified Water (or Water for Injection)into a hermetic container, seal up the container, thensterilize the product.

(ii) Make Purified Water (or Water for Injection) sterileby using a suitable method, introduce the sterilizedwater into a sterile hermetic container by applyingaseptic manipulation, then seal up the container.

For assuring the sterility of pharmaceutical water prod-ucts, only the validation of final sterilization process is re-quired in the case of preparation method (i), whereas valida-tions of all the processes are indispensable in the case ofpreparation method (ii), since the latter is based on the ideato assure the sterility of pharmaceutical water products by``aseptically'' introducing Purified Water (or Water for In-jection) treated in advance with filtration sterilization, etc.into a sterile hermetic container, and sealing it up.5.2. Deterioration of Water Quality during the Storage inContainers5.2.1. Conductivity (as the indicator for inorganic impuri-ties)

The conductivity of pharmaceutical water in containersmay increase to some higher levels due to the absorption ofcarbon dioxide from the atmosphere at the time of its prepa-

ration and that passed through plastic layer of the containersduring storage, and also due to ionic substances releasedfrom the containers, even if the conductivity of PurifiedWater or Water for Injection used for its production ismaintained at the level not more than 1.0 mS･cm－1. Particu-larly in the cases of pharmaceutical water products packed insmall scale glass containers, it is necessary to pay attention tothe change of conductivity during storage.5.2.2. Potassium Permanganate-reducing Substances orTotal Organic Carbon (TOC) (as the indicator for organicimpurities)

JP specifies the classical test of potassium permanganate-reducing substances in the monographs of Purified Water inContainers, Sterile Purified Water in Containers and SterileWater for Injection in Containers for controlling organicimpurities in pharmaceutical water in containers. It forms aremarkable contrast to the specifications of Purified Waterand Water for Injection, in which JP requires to controlorganic impurities in pharmaceutical water in bulk based onthe test of TOC (acceptance criterion: not more than 0.5mg/L (500 ppb)). This is because that it is considereddifficult to establish the specification of pharmaceuticalwater in containers for organic impurities based on the testof TOC from the facts that there were many cases ofremarkable increases in TOC values after storage of water incontainers. Particularly in the cases of pharmaceutical waterproducts packed in small scale plastic containers, it is neces-sary to pay attention to the increase of materials releasedfrom containers during storage.

The test of potassium permanganate-reducing substancesis retained in the specifications of pharmaceutical water incontainers, not as the most suitable method for the test oforganic impurities present in the water in containers, but as acounter measure for performing the test of the water in con-tainers with the same test method despite of the material(glass, polyethylene or polypropylene, etc) and the size (0.5 �2000 mL) of the containers, and the duration of storage.Therefore, it is recommended to adopt the test of TOC asthe alternative for the test of potassium permanganate-reducing substances, and to perform quality control of phar-maceutical water in containers based on TOC measurementsunder the responsibility of each manufacturer, if possible.

In such cases, it is recommended to adopt the followingvalues as the levels preferable to attain.

For products containing not more than 10 mL of water:TOC not greater than 1500 ppb

For products containing more than 10 mL of water:TOC not greater than 1000 ppb

As for the pharmaceutical water packed in the plastic con-tainers made of polyethylene, polypropylene, etc., in addi-tion to the concern for the release of materials such asmonomer, oligomers, plasticizers, etc. from plastics, it isnecessary to pay attention to the storage environment of theproducts to avoid the contaminations with low molecularvolatile organics such as ethanol, or low molecular air pol-lutants such as nitrogen oxides, since these plastics have theproperties of permeating various gases.5.2.3. Microbial Limit (Total Aerobic Viable Counts)

For Purified Water in Containers, it is not required to as-sure the sterility, but it is necessary to produce it by usingsanitary or aseptic processes in order to meet the acceptancecriterion of ``102 CFU/mL'' for total aerobic viable counts

22532253JP XVI General Information / Others

throughout the period of their storage. It is also necessary totake special care against microbial contamination during itscirculation. In addition, it is recommended to use them assoon as possible after opening their seals.

The acceptance criterion of ``102 CFU/mL'' for total aer-obic viable counts of Purified Water in Containers is at thesame level as the action level for viable counts in the produc-tion of Purified Water (in bulk). However, different fromthe case of microbiological monitoring of Purified Water,Soybean-Casein Digest Agar Medium is used for the test oftotal aerobic viable counts of Purified Water in Containersto detect microorganisms contaminated from the surround-ings during its storage and circulation.5.3. Points to consider in the case that commercially avail-able products of pharmaceutical water in containers are usedfor the manufacture of pharmaceutical products

It is allowable to use commercially available products ofpharmaceutical water in containers (Purified Water in Con-tainers, Sterile Purified Water in Containers or Sterile Waterfor Injection in Containers) for the manufacture of phar-maceutical products and products for clinical trial, and forthe tests of pharmaceutical products. In such cases, it is nec-essary to consider the following points.

( i ) When such products are used for manufacturingpharmaceutical products, it is recommended to usethem soon after confirming their compliances to therequirements of JP monograph from the test resultsat the time of its receipt or those offered from thesupplier of the products.

( ii ) In the case that such products are used for manufac-turing pharmaceutical products, it is necessary tovalidate the process in which the water was used as apart of process validation of pharmaceutical prod-ucts. In the case that they are used for manufactur-ing products for clinical trial, it is necessary to con-firm that the water doesn't give any adverse effectson the quality of the products.

(iii) The products of sterile pharmaceutical water in con-tainers should be used only once after opening theirseals, and it must be avoided to use them again afterstorage.

(iv) It is recommended to prepare a standard operationpractice (SOP) adequate for its intended use, con-sidering that the contamination and quality deterio-ration of the water due to human and laboratory en-vironmental origins might go on rapidly immediatelyafter opening the product seal.

Water to be used in theTests of Drugs

The water to be used in the tests of drugs is defined as``the water suitable for performing the relevant test'' in theparagraph 20 under General Notices of the JP. Therefore, itis necessary to confirm that the water to be used in a test of adrug is suitable for the purpose of the relevant test before itsuse.

Unless otherwise specified in the individual test method,Purified Water, Purified Water in Containers or the waterproduced by an appropriate process, such as ion exchange or

ultrafiltration, may be used for these purposes. Water pro-duced for these purposes at other individual facilities mayalso be used.

The water for tests specified in General Tests in the JP isas follows:・Water for ammonium limit test: <1.02> Ammonium

Limit Test (Standard Ammonium Solution)・Water for bacterial endotoxins test: <4.01> Bacterial En-

dotoxins Test・Water for particulate matter test (for injections): <6.07>

Insoluble Particulate Matter Test for Injections・Water for particulate matter test (for ophthalmic solu-

tions): <6.08> Insoluble Particulate Matter Test for Oph-thalmic Solutions・Water for particulate matter test (for plastic containers):<7.02> Test Methods for Plastic Containers

The water for tests specified in General Information in theJP is as follows:・Water for aluminum test: Test for Trace Amounts of

Aluminum in Trans Parenteral Nutrition (TPN) Solu-tions・Water for ICP analysis: Inductively Coupled Plasma

Emission Spectral AnalysisThe term ``water'' described in the text concerning tests of

drugs means ``the water to be used in the tests of drugs'' asdefined in the paragraph 20 under General Notices.

G9 Others

International HarmonizationImplemented in the Japanese

Pharmacopoeia Sixteenth EditionItems for which harmonization has been agreed among the

European Pharmacopoeia, the United States Pharmacopeiaand the Japanese Pharmacopoeia are implemented in theJapanese Pharmacopoeia Sixteenth Edition (JP 16). Theyare shown in the tables below.

The column headed Harmonized items shows the har-monized items written in the Pharmacopoeial Harmoniza-tion Agreement Document, and the column headed JP 16shows the items as they appear in JP 16. In the Remarkscolumn, notes on any differences between JP 16 and theagreement are shown as occasion demands.

The date on which the agreement has been signed is shownon the top pf each table. In the case where the harmonizeditems have been revised and/or corrected, this is indicated inparenthesis.

2254

Aug. 2005 (Rev. 2)

Harmonized items JP 16 Remarks

Residue on Ignition/Sulphated AshTest

(Introduction)

2.44 Residue on Ignition Test

(Introduction) JP's particular description: Explana-tion on this test.Explanation of the description in mon-graph, etc.

Procedure 1. Procedure

Oct. 2007


Characterisation of Crystalline andPartially Crystalline Solids by X-rayPowder Diffraction (XRPD)

2.58 X-Ray Powder DiffractionMethod

(Introduction) (Introduction) JP's particular description: Explana-tion on this method.

Principle 1. Principle

Instrument 2. Instrument

Instrument set-up 2.1 Instrument setup

X-ray radiation 2.2 X-ray radiation

Radiation protection 2.3 Radiation protection

Specimen preparation and mounting 3. Specimen preparation and mount-ing

Specimen preparation 3.1 Specimen preparation

Specimen mounting ``Specimen mounting'' is not stipulat-ed.

Effect of specimen displacement

Effect of specimen thickness andtransparency

Control of the instrument perfor-mance

4. Control of the instrument perfor-mance

Qualitative phase analysis(Identification of phases)

5. Qualitative phase analysis(Identification of phases)

Quantitative phase analysis 6. Quantitative phase analysis

Polymorphic samples 6.1 Polymorphic samples

Methods using a standard 6.2 Methods using a standard

Estimate of the amorphous and crys-talline fractions

7. Estimate of the amorphous andcrystalline fractions

Single crystal structure 8. Single crystal structure

Figure 1 Diffraction of X-rays by acrystal according to Bragg's law

Fig. 1 Diffraction of X-rays by a crys-tal according to Bragg's law

Figure 2 X-ray powder diffractionpatterns collected for 5 differentsolid phases of a substance (the in-tensities are normalized)

Fig. 2 X-ray powder diffraction pat-terns collected for 5 different solidphases of a substance (the intensitiesare normalized)

2254 JP XVIOthers / General Information

2255

Figure 3 Geometric arrangement ofthe Bragg-Brentano parafocusing ge-ometry

Fig. 3 Geometric arrangement of theBragg-Brentano parafocusing geo-metry

June 2009 (Rev. 1 � Corr. 1)


Bulk Density and Tapped Density ofPowders

3.01 Determination of Bulk andTapped Densities

(Introduction) JP's particular description: Explana-tion on this method.

Bulk density 1. Bulk density

Method 1: Measurement in a grad-uated cylinder

1.1. Method 1: Measurement in aGraduated Cylinder

Procedure 1.1.1. Procedure

Method 2: Measurement in avolumeter

1.2. Method 2: Measurement in aVolumeter

Apparatus 1.2.1. Apparatus


Method 3: Measurement in a vessel 1.3. Method 3: Measurement in aVessel



Tapped density 2. Tapped density

Method 1 2.1. Method 1







Measures of powder compressibility 3. Measures of powder compressibili-ty

Figure 1 Volumeter Fig. 1 Volumeter

Figure 2 Measuring vessel (left) andcap (right)

Fig. 2 Measuring vessel (left) and cup(right)

Figure 3 Fig. 3 Tapping apparatus

Nov. 2003


Specific Surface Area 3.02 Specific Surface Area by GasAdsorption

(Introduction) (Introduction) JP's particular description: Explana-tion on this test.

1. Measurememts

Multi-point measurement 1.1. Multi-point measurement

Single-point measurement 1.2. Single-point measurement


2256

Sample preparation 2. Sample preparationOutgassingAdsorbateQuantity of sample

3. Methods

Method 1: The dynamic flow method 3.1. Method 1: The dynamic flowmethod

Method 2: The volumetric method 3.2. Method 2: The volumetricmethod

Reference materilals 4. Reference materilals

Figure 1 Schematic diagram of thedynamic flow method apparatus

Fig. 1 Schematic diagram of the dy-namic flow method apparatus

Figure 2 Schematic diagram of thevolumetric method apparatus

Fig. 2 Schematic diagram of the volu-metric method apparatus

May 2007


Gas Pycnometric Density of Solids 3.03 Powder Particle Density Determi-nation

(Introduction) (Introduction) JP's particular description: Objects tobe measured by this method.

Apparatus 1. Apparatus

2. Calibration of apparatus The range of temperature during themeasurement is described in the Proce-dure.

Method 3. Procedure

Expression of the results

Figure 1 Schematic diagram of a gaspycnometer

Fig. 1 Schematic diagram of a gas py-cnometer

June 2004 (Method 1)/May 2007 (Rev. 1) (Method 2)


3.04 Particle Size Determination

(Introduction) JP's particular description: Explana-tion on this test.

Optical microscopy 1. Method 1. Optical microscopy JP's particular description: Explana-tion on this test.

Apparatus 1.1. Apparatus

Adjustment 1.1.1. Adjustment

Illumination 1.1.1.1. Illumination

Visual characterization 1.1.1.2. Visual characterization JP's particular description: Explana-tion on the method of particle sizemeasurement.

Photographic characterization 1.1.1.3. Photographic characteriza-tion

Preparation of the mount 1.2. Preparation of the mount

1.3. Characterization

Crystallinity characterization 1.3.1. Crystallinity characterization


2257

Limit test of particle size bymicroscopy

1.3.2. Limit test of particle size bymicroscopy

Particle size characterization 1.3.3. Particle size characterization

Particle shape characterization 1.3.4. Particle shape characteriza-tion

General observations 1.3.5. General observations

Figure 1 Commonly used measure-ments of particle size

Fig. 1 Commonly used measurementsof particle size

Figure 2 Commonly used descriptionsof particle shape

Fig. 2 Commonly used descriptions ofparticle shape

Analytical sieving 2. Method 2. Analytical sievingmethod

JP's particular description: Explana-tion on this method.

Principles of analytical sieving Principles of analytical sieving

2.1. Procedure

Test sieves 2.1.1. Test sieves

Test specimen 2.1.2. Test specimen

Agitation methods 2.1.3. Agitation methods

Endpoint determination 2.1.4. Endpoint determination

Sieving methods 2.2. Sieving methods

1) Methanical agitationdry sieving method

2.2.1. Methanical agitation(Dry sieving method)

2) Air entrainment methodsair jet and sonic sifter sieving

2.2.2. Air entrainment methodsAir jet and sonic shifter sieving

Interpretation 2.3. Interpretation

Table 1 Size of standard sieve series inrange of interest

Table 1 Size of standard sieve series inrange of interest

Oct. 2009 (Rev. 1 � Corr. 1)


Bacterial Endotoxins Test 4.01 Bacterial Endotoxins Test

(Introduction) (Introduction)


2. Preparation of solutions

Preparation of standard endotoxinstock solution

2.1. Standard endotoxin stock solu-tion

Preparation of standard endotoxin so-lution

2.2. Standard endotoxin solution

Preparation of sample solutions 2.3. Sample solutions JP's particular description: Deletion ofthe preparation of sample solutions formedical devices.

Determination of maximum valid di-lution

3. Determination of maximum validdilution

Gel-clot technique 4. Gel-clot techniques

(1) Preparatory testing 4.1. Preparatory testing

(i) Test for confirmation of labeledlysate sensitivity

4.1.1. Test for confirmation oflabeled lysate reagent sensitivity

(ii) Test for interfering factors 4.1.2. Test for interfering factors

(2) Limit test 4.2. Limit test

(i) Procedure 4.2.1. Procedure


2258

(ii) Interpretation 4.2.2. Interpretation

(3) Assay 4.3. Quantitative test


(ii) Calculation and interpretation 4.3.2. Calculation and interpretation

Photometric techniques 5. Photometric quantitative tech-niques

(1) Turbidimetric techniques 5.1. Turbidimetric technique

(2) Chromogenic technique 5.2. Chromogenic technique

(3) Preparatory testing 5.3. Preparatory testing

(i) Assurance of criteria for thestandard curve

5.3.1. Test for assurance of criteriafor the standard curve

(ii) Test for interfering factors 5.3.2. Test for interfering factors

(4) Assay 5.4. Quantitative test


(ii) Calculation 5.4.2. Calculation of endotoxin con-centration

(iii) Interpretation 5.4.3. Interpretation

Reagents, test solutions Prescribed in <9.41> Reagents, Test So-lutions

Amoebocyte lysate

Lysate TS

Water for bacterial endotoxins test(BET)

Table 1 Table 1

Table 2 Table 2

Table 3 Table 3

Table 4 Table 4

June 2009 (Rev. 1 � Corr. 1) (I. Microbial Enumeration Tests)/Jun. 2008 (Rev. 1) (II. Test for specified Micro-organisms)


4.05 Microbiological Examination ofNon-Sterile Produds

Microbiological Examination of Non-Sterile Products:Microbial Enumeration Tests

I. Microbiological Examination ofNon-Sterile Products: MicrobialEnumeration Tests

1 Introduction (Introduction)

2 General procedures 1. General Procedures

3 Enumeration methods 2. Enumeration Methods

4 Growth promotion test, suitabilityof the counting method and negativecontrols

3. Growth Promotion Test, Suitabili-ty of the Counting Method andNegative Controls

4-1 General considerations

4-2 Preparation of test strains 3.1. Preparation of test strains

4-3 Negative control 3.2. Negative control

4-4 Growth promotion of the me-dia

3.3. Growth promotion of the media

4-5 Suitability of the countingmethod in the presence of product

3.4. Suitability of the countingmethod in the presence of product


2259

4-6 Results and interpretation 3.5. Results and interpretation

5 Testing of products 4. Testing of Products

5-1 Amount used for the test 4.1. Amount used for the test

5-2 Examination of the product 4.2. Examination of the product

5-3 Interpretation of the results 4.3. Interpretation of the results

Table 1 Preparation and use of testmicro-organisms

Table 1-1 Preparation and use of testmicro-organisms

Table 2 Common neutralising agentsfor interfering substances

Table 1-2 Common neutralising agents/Method for interfering substances

Table 3 Most-probable-number valuesof micro-organisms

Table 1-3 Most-probable-numbervalues of micro-organisms

Microbiological Examination of Non-Sterile Products:Tests for Specified Micro-organisms

II. Microbiological Examination ofNon-Sterile Products: Tests forSpecified Micro-organisms

1 Introduction (Introduction)

2 General procedures 1. General Procedures

3 Growth promoting and inhibitoryproperties of the media, suitability ofthe test and negative controls

2. Growth Promoting and InhibitoryProperties of the Media, Suitabilityof the Test and Negative Controls

3-1 Preparation of test strains 2.1. Preparation of test strains

3-2 Negative control 2.2. Negative control

3-3 Growth promotion and inhibi-tory properties of the media

2.3. Growth promotion and inhibito-ry properties of the media

3-4 Suitability of the test method 2.4. Suitability of the test method

4 Testing of products 3. Testing of Products

4-1 Bile-tolerant gram-negativebacteria

3.1. Bile-tolerant gram-negative bac-teria

4-2 Escherichia coli 3.2. Escherichia coli

4-3 Salmonella 3.3. Salmonella

4-4 Pseudomonas aeruginosa 3.4. Pseudomonas aeruginosa

4-5 Staphylococcus aureus 3.5. Staphylococcus aureus

4-6 Clostridia 3.6. Clostridia

4-7 Candida albicans 3.7. Candida albicans

5 Recommended solutions and cul-ture media

4. Recommended Solutions and Cul-ture Media

Table II-1 Growth promoting, inhibi-tory and indicative properties of me-dia

Table II-1 Growth promoting, inhibi-tory and indicative properties of me-dia

Table II-2 Interpretation of results Table II-2 Interpretation of results

June 2009 (Rev. 1 � Corr. 3)


Sterility 4.06 Sterility Test


Precautions against microbial con-tamination

1. Precautions against microbial con-tamination

Culture media and incubation temper-atures

2. Culture media and incubation tem-peratures


2260

Media for the test may be prepared asdescribed below, or equivalent com-mercial media may be used providedthat they comply with the growth pro-motion test.

Fluid thioglycollate medium (i) Fluid thioglycollate medium

Soya-bean casein digest medium (ii) Soya-bean casein digest medium

The media used comply with the fol-lowing tests, carried out before or inparallel with the test on the product tobe examined

3. Suitability of the culture medium

Sterility 3.1. Sterility

Growth promotion test of aerobes,anaerobes and fungi

3.2. Growth promotion test of aer-obes, anaerobes and fungi

Method suitability test 4. Method suitability test

Membrane filtration (i) Membrane filtration

Direct inoculation (ii) Direct inoculation

Test for sterility of the product to beexamined

5. Test for sterility of the product tobe examined

The test may be carried out using thetechnique of membrane filtration or bydirect inoculation of the culture mediawith the product to be examined

Membrane filtration 5.1. Membrane filtration Note 7

Aqueous solutions (i) Aqueous solutions

Soluble solids (ii) Soluble solids

Oils and oily solutions (iii) Oils and oily solutions

Ointments and creams (iv) Ointments and creams

Direct inoculation of the culture medi-um

5.2. Direct inoculation of the culturemedium

Oily liquids (i) Oily liquids

Ointments and creams (ii) Ointments and creams

Catgut and other surgical sutures forveterinary use

The items not included in JP.

Observation and interpretation ofresults

6. Observation and interpretation ofresults

Application of the test to parenteralpreparations, ophthalmic and othernon-injectable preparations required tocomply with the test for sterility

7. Application of the test to parenter-al preparations, ophthalmic andother non-injectable preparations re-quired to comply with the test forsterility

Minimum number of items to be test-ed

8. Minimum number of items to betested

Table 1. Strains of the test micro-or-ganisms suitable for use in thegrowth promotion test and themethod suitability test

Table 1. Strains of the test micro-or-ganisms suitable for use in thegrowth promotion test and themethod suitability test

Table 2. Minimum quantity to be usedfor each medium

Table 2. Minimum quantity to be usedfor each medium

Table 3. Minimum number of items tobe tested

Table 3. Minimum number of items tobe tested

Non-harmonized item: The largevolume preparation is specified as notless than 100 mL labeled.


2261

Feb. 2004


Uniformity of Dosage Units 6.02 Uniformity of Dosage Units

(Introduction) (Introduction) JP's particular description: Additionalexplanation on Liquids.Additional explanation for the partnot containing drug substance.

Content uniformity 1. Content uniformity

Solid dosage forms (i) Solid dosage forms

Liquid dosage forms (ii) Liquid dosage forms

Calculation of acceptance value 1.1. Calculation of acceptance value

Mass variation 2. Mass variation JP's particular description: Assumingthat the concentration of drug sub-stance is uniform in each lot.

Uncoated or film-coated tablets (i) Uncoated or film-coated tablets

Hard capsules (ii) Hard capsules

Soft capsules (iii) Soft capsules

Solid dosage forms other than tabletsand capsules

(iv) Solid dosage forms other thantablets and capsules

Liquid dosage forms (v) Liquid dosage forms The phrase ``in conditions of normaluse. If necessary, compute the equiva-lent volume after determining the den-sity.'' is deleted.

Calculation of acceptance value 2.1. Calculation of acceptance value

Criteria 3. Criteria

Solid and liquid dosage forms (i) Solid and liquid dosage formsTable 1 Application of contentuniformity (CU) and mass variation(MV) test for dosage forms

Table 1 Application of contentuniformity (CU) and mass variation(MV) test for dosage forms

JP's particular description: Addition of``(divided forms, lyophilized forms)''and ``(true solution)''.

Table 2 Table 2 The phrases ``at time of manufacture''and ``For purposes of this Phar-macopoeia'' are deleted

June 2004 (Rev.1)


Test for Extractable Volume of Paren-teral Preparations

6.05 Test for Extractable Volume ofParenteral Preparations

(Introduction) (Introduction) JP's particular description: Explana-tion on this Test

Single-dose containers 1. Single-dose containers

Multi-dose containers 2. Multi-dose containers

Cartridges and prefilled syringes 3. Cartridges and pre-filled syringes

Parenteral infusions 4. Parenteral infusions

June 2004 (Rev.1)


Particulate Matter in Injectables 6.07 Insoluble Particulate Matter Testfor Injections



2262

Method 1.

Light obscuration particle count test

1. Method 1. Light obscuration parti-cle count test

1.1. Apparatus JP's particular description: Descriptionof evaluation frequency.

1.1.1. Calibration JP's particular description

1.1.1.1. Manual method JP's particular description

1.1.1.2. Electronic method JP's particular description

1.1.1.3. Automated method JP's particular description

1.1.2. Sample volume accuracy JP's particular description

1.1.3. Sample flow rate JP's particular description

1.1.4. Sensor JP's particular description

1.1.4.1. Sensor resolution (Particlesize resolution of apparatus)

JP's particular description

1.1.4.2. Particle counting accuracy JP's particular description

1.1.4.3. Threshold accuracy JP's particular description

General precautions 1.2. General precautions

Method 1.3. Method

Evaluation 1.4. Evaluation JP's particular description: ``equal toor more than 100 mL''.

Method 2.

Microscopic particle count test

2. Method 2. Microscopic particlecount test

2.1. Apparatus

General precautions 2.2. General precautions

Method 2.3. Method

Evaluation 2.4. Evaluation JP's particular description: ``equal toor more than 100 mL''.

3. Reagents JP's particular description

1. Circular diameter graticule Fig. 1 Circular diameter graticule

Oct. 2007 (Rev.1)


Disintegration 6.09 Disintegration Test JP's particular description: This test isapplied to Granules, Dry Syrups andPills.


Basket-rack assembly (i) Basket-rack assembly JP's particular description: The ap-paratus may be varied somewhatprovided the specifications.

Disk (ii) Disk

(iii) Auxiliary tube JP's particular description


2.1. Immediate-release preparations JP's particular description:Setting a test for Granules, Dry Syrupsand Pills.Allowing use of water as the test fluid.Setting each intervals of the immersion.Making a definition of complete disin-tegration.


2263

2.2. Enteric coated preparations Setting a procedure for Granules.JP's particular description:Setting a test for enteric coated prepa-rations.

Figure 1 Disintegration apparatus Fig. 1 Disintegration apparatus

Fig. 2 Auxiliary tube JP's particular description.

Nov. 2008 (Rev. 2)


Dissolution 6.10 Dissolution Test JP's particular description: The testalso aims at preventing significantbioinequivalence.


Apparatus 1 (Basket apparatus) 1.1. Apparatus for Basket Method(Apparatus 1)

Apparatus 2 (Paddle apparatus) 1.2. Apparatus for Paddle Method(Apparatus 2)

JP's particular description: The sinkeris allowed to use in case only when spe-cified in the monograph.

Apparatus 3 (Reciprocating cylinder) not specified

Apparatus 4 (Flow-through cell) 1.3. Apparatus for Flow-ThroughCell Method (Apparatus 3)

Apparatus suitability 2. Apparatus suitability


Apparatus 1 or 2 3.1. Basket Method or PaddleMethod

Immediate-release dosage forms 3.1.1. Immediate-release dosageforms

Procedure (i) Procedure

Dissolution medium (ii) Dissolution medium

Time (iii) Time

Extended-release dosage forms 3.1.2. Extended-release dosage forms



Time (iii) Time

Delayed-release dosage forms 3.1.3. Delayed-release dosage forms

Procedure (i) Procedure In the Harmonized text: Alternativeusage of Method A or B.

Method A

Method B

(ii) Dissolution medium JP's particular description.

Time (iii) Time JP's particular description: Time is spe-cified each for the 1st and 2nd fluids.

Apparatus 3 not specified

Immediate-release dosage forms

Procedure

Dissolution medium

Time


2264

Extended-release dosage forms

Procedure

Dissolution medium

Time

Delayed-release dosage forms

Procedure

Time

Apparatus 4 3.2. Flow-Through Cell Method

Immediate-release dosage forms 3.2.1. Immediate-release dosageforms



Time (iii) Time

Extended-release dosage forms 3.2.2. Extended-release dosage forms



Time (iii) Time

Delayed-release dosage forms

Procedure

Time

Interpretation 4. Interpretation JP's particular description: Follow In-terpretation 1 when the value Q is spe-cified in the individual monograph,otherwise follow Interpretation 2.

Immediate-release dosage forms 4.1. Immediate-release dosage forms JP's particular description: Setting In-terpretation 2.

4.1.1. Interpretation 1


Extended-release dosage forms 4.2. Extended-release dosage forms JP's particular description: Setting In-terpretation 2.



Delayed-release dosage forms 4.3. Delayed-release dosage forms Non-harmonized items: Different dis-solution medium. Deletion of dishar-monized part on the value Q.

JP's particular description: Setting In-terpretation 2.

4.3.1. Interpretation 1 The value Q is specified in theindividual monograph.


Acceptance Table 1 Acceptance Table 1




Figure 1 Apparatus 1, Basket stirringelement

Fig. 1 Apparatus 1, Basket stirring ele-ment

Figure 2 Paddle stirring element Fig. 2 Apparatus 2, Paddle stirringelement

Figure 2a Alternative sinker Fig. 2a Alternative sinker


2265

Figure 3 Apparatus 3 not specified

Figure 4 Apparatus 4(top) large cell tablets and capsules(bottom) tablet holder for the largecell

Fig. 3 Apparatus 3(top) large cell for tablets and cap-sules(bottom) tablet holder for the largecell

Figure 5 Apparatus 4(top) small cell tablets and capsules(bottom) tablet holder for the smallcell

Fig. 4 Apparatus 3(top) small cell for tablets and cap-sules(bottom) tablet holder for the smallcell

Sep. 2002 (Rev. 1)


Ethanol Ethanol

Identification A not specified as Identification Setting Specific gravity as specification.

Identification B Identification

Relative density Specific gravity Setting Specific gravity at 159C.

Appearance Purity (1) Clarity and color of solu-tion

Acidity or alkalinity Purity (2) Acid or alkali

Volatile impurities Purity (3) Volatile impurities

Absorbance Purity (4) Other impurities

Residue onevaporation Purity (5) Residue on evaporation

Storage Containers and storage

Sep. 2002 (Rev. 1)


Ethanol, Anhydrous Anhydrous Ethanol

Definition limits of content

Identification A not specified as Identification Setting Specific gravity as specification.

Identification B Identification

Relative density Specific gravity Setting Specific gravity at 159C.

Appearance Purity (1) Clarity and color of solu-tion


Volatile impurities Purity (3) Volatile impurities

Absorbance Purity (4) Other impurities

Residue onevaporation Purity (5) Residue on evaporation

Storage Containers and storage

Nov. 2003 (Rev. 2)


Sodium Chloride Sodium Chloride

Definition limits of the content


2266

Identification A Identification (1)

Identification B Identification (2)

Acidity or alkalinity Purity (2) Acidity or alkalinity

Sulphates Purity (3) Sulfates

Phosphates Purity (4) Phosphates

Bromides Purity (5) Bromides

Iodides Purity (6) Iodides

Ferrocyanides Purity (7) Ferrocyanides

Iron Purity (9) Iron

Barium Purity (10) Barium

Magnesium and alkaline-earth metals Purity (11) Magnesium and alkaline-earth materials

Aluminium not specified

Nitrites not specified

Potassium not specified

Loss on drying Loss on drying

Assay Assay

Nov. 2008


Carmellose Carmellose

Definition Definition

Identification (1) Identification (1)


Purity (1) Chloride Purity (1) Chloride

Purity (2) Sulfate Purity (2) Sulfate


Residue on ignition Residue on ignition

July 2003 (Rev. 1)


Carboxymethylcellulose Calcium Carmellose Calcium

Definition origin



Identification C Identification (3)

Identification D Identification (4)

Alkalinity Purity (1) Alkalinity



Limit of chloride Purity (2) Chloride

Limit of sulfate Purity (3) Sulfate


2267

Oct. 2001


Croscarmellose Sodium Croscarmellose Sodium

Definition origin

Identification A

Identification B

Identification C

Identification (1)

Identification (2)

Identification (3)

pH pH

Settling volume Precipitation test

Degree of substitution Degree of substitution



Packaging and storage Containers and storate

Nov. 2003 (Rev. 1)


Citric Acid, Anhydrous Anhydrous Citric Acid


Appearance of solution Purity (1) Clarity and color of solu-tion


Oxalic acid Purity (3) Oxalic acid

Readily carbonisable substances Purity (5) Readily carbonizable sub-stances


Water Water

Sulphated ash Residue on ignition

Assay Assay

Nov. 2003 (Rev. 1)


Citric Acid Monohydrate Citric Acid Hydrate


Appearance of solution Purity (1) Clarity and color of solu-tion


Oxalic acid Purity (3) Oxalic acid



Water Water


Assay Assay


2268

Feb. 2003


Saccharin Saccharin


Limit of benzoate and salicylate Purity (3) Benzoate and salicylate




Assay Assay

Packaging and storage Containers and storage

Feb. 2004 (Rev. 1)


Saccharin Sodium Saccharin Sodium Hydrate


Identification B, C Identification (2)


Limit of benzoate and salicylate Purity (4) Benzoate and salicylate


Water Water

Assay Assay

Oct. 2001


Cellacefate Collacefate

Definition content of the acetyl and carboxyben-zoyl groups

Identification Identification

Viscosity Viscosity

Limit of free acid Purity (2) Free acids

Water Water


Phthalyl content Assay (1) Carboxybenzoyl group

Content of acetyl Assay (2) Acetyl group


May 2005 (Rev. 1)


Microcrystalline Cellulose Microcrystalline Cellulose

Definition origin



pH pH


2269

Water-soluble substances Purity (2) Water- soluble substances

Ether-soluble substances Purity(3) Diethyl ether-soluble sub-stances

Conductivity Conductivity



Bulk density Bulk density

May 2005 (Rev. 1)


Powdered Cellulose Powdered Cellulose

Definition origin

Labeling Labeling of mean degree of polymeri-zation



pH pH

Water-soluble substances Purity (2) Water-soluble substances

Ether-soluble substances Purity (3) Diethyl ether- soluble sub-stances



June 2008 (Rev. 1)


Talc Talc

Definition origin, content of magnesium


Acidity and alkalinity Purity (1) Acidity or alkalinity

Aluminium Purity (5) Aluminum

Calcium Purity (7) Calcium


Lead Purity (6) Lead

Magnesium Assay

Loss on ignition Loss on ignition

Oct. 2007 (Rev. 1)


Wheat Starch Wheat Starch

Definition origin




pH pH


2270


Total protein not specified

Oxidising substances Purity (2) Oxidizing substances

Sulphur dioxide Purity (3) Sulfur dioxide



Oct. 2006


Rice Starch Rice Starch

Definition Origin




pH pH






Oct. 2007 (Rev. 2)


Corn Starch Corn Starch

Definition origin




pH pH



Limit of iron Purity (1) Iron

Limit of oxidizing substances Purity (2) Oxidizing substances

Limit of sulfur dioxide Purity (3) Sulfur dioxide

Oct. 2007 (Rev. 1)


Potato Starch Potato Starch

Definition origin




pH pH


2271






May 2005 (Rev.1)


Sodium Starch Glycolate Sodium Starch Glycolate

Definition origin, limit of sodium



pH pH


Limit of iron Purity (2) Iron

Limit of sodium chloride Purity (4) Sodium chloride

Limit of sodium glycolate Purity (3) Sodium glycolate

Assay Assay

June 2008 (Rev. 3)


Anhydrous Lactose Anhydrous Lactose

Definition origin

Clarity and color of solution Purity (1) Clarity and color of solu-tion

Specific rotation Optical rotation

Acidity or alkalinity Purity (2) Acidity or alkalinity



Water Water

Protein and light-absorbingimpurities

Purity (4) Proteins and light absorbingsubstances

Content of alpha and beta anomers Isomer ratio

June 2008 (Rev. 2)


Lactose Monohydrate Lactose Hydrate

Definition origin

Clarity and color of solution Purity (1) Clarity and color of solu-tion


Specific rotation Optical rotation

Acidity or alkalinity Purity (2) Acid and alkali


Water Water


2272

Protein and light-absorbing impurities Purity (4) Protein and light absorbingsubstances

Feb. 2004


Ethyl Parahydroxybenzoate Ethyl Parahydroxybenzoate



Appearance of solution Purity (1) Clarity and color of solution

Acidity Purity (2) Acidity

Related substances Purity (4) Related substances System suitability is not specified.


Assay Assay

Feb. 2004


Butyl Parahydroxybenzoate Butyl Parahydroxybenzoate







Assay Assay

Feb. 2004


Propyl Parahydroxybenzoate Propyl Parahydroxybenzoate







Assay Assay

Feb. 2004


Methyl Parahydroxybenzoate Methyl Parahydroxybenzoate







2273


Assay Assay

Nov. 2003


Hydroxypropyl Methylcellulose Hypromellose

Definition limits of content of methoxy groupand hydroxypropoxy group

Labeling labeling of viscosity






Viscosity Viscosity

Method 1 Method I

Method 2 Method II

pH pH

Heavy metals Purity Heavy metals



Assay Assay

June 2006


Hypromellose Phthalate Hypromellose Phthalate

Definition origin, limit of carboxybenzoyl


Viscosity Viscosity

Water Water


Chloride Purity (1) Chloride

Limit of free phthalic acid Purity (3) Phthalic acid

Phthalyl content Assay

June 2008 (Rev. 2)


Benzyl Alcohol Benzyl Alcohol


Refractive index Refractive index


Benzaldehyde and other relatedsubstances

Purity (3) Benzaldehyde and otherrelated substances

Peroxide value Purity (4) Peroxide value

Residue on evaporation Purity (5) Residue on evaporation


2274

Assay Assay

Nov. 2005 (Rev. 1)


Methylcellulose Methylcellulose

Definition limits of content of methoxy group

Labeling labeling of viscosity






Viscosity Viscosity

Method 1 Method I

Method 2 Method II

pH pH

Heavy metals Purity Heavy metals



Assay Assay

Nov. 2005


Anhydrous Dibasic Calcium Phosphate Anhydrous Dibasic Calcium Phosphate

Definition limit of content



Acidinsoluble substances Purity (1) Acid-insoluble substance


Sulfate Purity (3) Sulfate

Carbonate Purity (4) Carbonate



Assay Assay

Nov. 2005


Dibasic Calcium Phosphate Dibasic Calcium Phosphate Hydrate

Definition limit of content



Acidinsoluble substances Purity (1) Acid-insoluble substances


Sulfate Purity (3) Sulfate


2275

Carbonate Purity (4) Carbonate



Assay Assay

Nov. 2008


General Information

Particle-size Analysis by Laser LightDiffraction

Laser Diffraction Measurement of Par-ticle Size

Introduction Introduction

Principle 1. Principle

Instrument 2. Instrument

Development of the method 3. Development of the method

Sampling 3.1. Sampling

Evaluation of the dispersion proce-dure

3.2. Evaluation of the dispersionprocedure

Optimisation of the liquid disper-sion

3.3. Optimization of the liquid dis-persion

Optimisation of the gas dispersion 3.4. Optimization of the gas disper-sion

Determination of the concentra-tion range

3.5. Determination of the concentra-tion range

Determination of the measuringtime

3.6. Determination of the measuringtime

Selection of an appropriate opticalmodel

3.7. Selection of an appropriate op-tical model

Validation 3.8. Validation

Measurement 4. Measurement

Precautions 4.1. Precautions

Measurement of the light scatter-ing of dispersed sample(s)

4.2. Measurement of the light scat-tering of dispersed sample(s)

Conversion of scattering patterninto particle-size distribution

4.3. Conversion of scattering patterninto particle-size distribution

Replicates 4.4. Replicates

Reporting of results 5. Reporting of results

Control of the instrument perfor-mance

6. Control of the instrument perfor-mance

Calibration 6.1. Calibration

Qualification of the system 6.2. Qualification of the system

Figure 1 Example of a set-up of laserlight diffraction instrument

Fig. 1 Example of a set-up of laserlight diffraction instrument

Note Note 1

Note 2 The first sentence of the harmonizedtext is described as Note 2.


2276

May 2007


Powder FinenessGeneral InformationPowder Fineness

June 2004


General InformationPowder Flow Powder Flow


Angel of repose 1. Angel of repose

Basic methods for angel of repose 1.1 Basic methods for angel of repose

Variations in angel of reposemethods

1.2 Variations in angel of reposemethods

Angel of repose general scale offlowability

1.3 Angel of repose general scale offlowability

Experimental considerations for an-gle of repose

1.4 Experimental considerations forangle of repose

Recommended procedure for angleof repose

1.5 Recommended procedure for an-gle of repose

Compressibility index and Hausner ra-tio

2. Compressibility index and Hausnerratio

Basic methods for compressibility in-dex and Hausner ratio

2.1 Basic methods for compressibilityindex and Hausner ratio

Experimental considerations for thecompressibility index and Hausnerratio

2.2 Experimental considerations forthe compressibility index and Haus-ner ratio

Recommended procedure for com-pressibility index and Hausner ra-tio

2.3 Recommended procedure for com-pressibility index and Hausner ratio

Flow through an orifice 3. Flow through an orifice

Basic methods for flow through anorifice

3.1 Basic methods for flow through anorifice

Variations in methods for flowthrough an orifice

3.2 Variations in methods for flowthrough an orifice

General scale of flowability for flowthrough an orifice

3.3 General scale of flowability forflow through an orifice

Experimental considerations for flowthrough an orifice

3.4 Experimental considerations forflow through an orifice

Recommended procedure for flowthrough an orifice

3.5 Recommended procedure for flowthrough an orifice

Shear cell methods 4. Shear cell methods

Basic methods for shear cell 4.1 Basic methods for shear cell

Recommendations for shear cell 4.2 Recommendations for shear cell

Table 1 Flow properties and cor-responding angle of repose

Table 1 Flow properties and cor-responding angle of repose

Table 2 Scale of flowability Table 2 Scale of flowability


2277

Sep. 2002


General Information

Amino Acid Analysis Amino Acid Analysis

Apparatus Apparatus

General precautions General precautions

Reference standard material Reference standard material

Calibration of instrumentation Calibration of instrumentation

Repeatability Repeatability

Sample preparation Sample preparation

Internal standards Internal standards

Protein hydrolysis Protein hydrolysis

Method 1 Method 1

Hydrolysis solution Hydrolysis solution

Procedure Procedure

Method 2 Method 2


Vapor phase hydrolysis Vapor phase hydrolysis

Method 3 Method 3



Method 4 Method 4

Oxidation solution Oxidation solution

Procedure Procedure

Method 5 Method 5


Liquid phase hydrolysis Liquid phase hydrolysis

Method 6 Method 6



Method 7 Method 7

Reducing solution Reducing solution

Procedure Procedure

Method 8 Method 8

Stock solutions Stock solutions


Procedure Procedure

Method 9 Method 9

Stock solutions Stock solutions

Carboxymethylation solution Carboxymethylation solution

Buffer solution Buffer solution

Procedure Procedure

Method 10 Method 10


Procedure Procedure


2278

Method 11 Method 11

Reducing solutions Reducing solutions

Procedure Procedure

Methodologies of amino acid analysisgeneral principles

Methodologies of amino acid analysisgeneral principles

Method 1-Postcolumn ninhydrindetection general principle

Method 1-Postcolumn ninhydrin de-tection general principle

Method 2-Postcolumn OPA fluoro-metric detection general principle

Method 2-Postcolumn OPA fluoro-metric detection general principle

Method 3-Precolumn PITC derivati-zation general principle

Method 3-Precolumn PITC derivati-zation general principle

Method 4-Precolumn AQC derivati-zation general principle

Method 4-Precolumn AQC derivati-zation general principle

Method 5-Precolumn OPA derivati-zation general principle

Method 5-Precolumn OPA derivati-zation general principle

Method 6-Precolumn DABS-Clderivatization general principle

Method 6-Precolumn DABS-Clderivatization general principle

Method 7-Precolumn FMOC-Clderivatization general principle

Method 7-Precolumn FMOC-Clderivatization general principle

Method 8-Precolumn NBD-Fderivatization general principle

Method 8-Precolumn NBD-Fderivatization general principle

Data calculation and analysis Data calculation and analysis

Calculations Calculations

Amino acid mole percent Amino acid mole percent

Unknown protein samples Unknown protein samples

Known protein samples Known protein samples

June 2010 (Corr. 2)


General Information

Capillary Electrophoresis Capillary Electrophoresis

Apparatus Apparatus

Capillary zone electrophoresis

Optimisation

Instrumental parameters

Voltage

Polarity

Temperature

Capillary

Electrolytic solution parameters

Buffer type and concentration

Buffer pH

Organic solvents

Additives for chiral separations

1. Capillary Zone Electrophoresis

Optimization


(1) Voltage

(2) Polarity

(3) Temperature

(4) Capillary


(1) Buffer type and concentration

(2) Buffer pH

(3) Organic solvents

(4) Additives for chiral separations

Capillary gel electrophoresis

Characteristics of gels

2. Capillary Gel Electrophoresis

Characteristics of Gels

Capillary isoelectric focusing

Loading step

loading in one step

3. Capillary Isoelectric Focusing

(1) Loading step

(i) loading in one step


2279

sequential loading

Focusing step

Mobilisation step

Optimisation

Voltage

Capillary

Solutions

(ii) sequential loading

(2) Focusing step

(3) Mobilization step

Optimization

(1) Voltage

(2) Capillary

(3) Solutions

Micellar electrokinetic chro-matography (MEKC)

Optimisation


Voltage

Temperature

Capillary


4. Micellar Electrokinetic Chro-matography (MEKC)

Optimization


(1) Voltage

(2) Temperature

(3) Capillary


Surfactant type and concentration

Buffer pH

(1) Surfactant type and concentra-tion

(2) Buffer pH

Organic solvents

Additives for chiral separations

Other additives

(3) Organic solvents

(4) Additives for chiral separations

(5) Other additives

Quantification

Calculations

Quantification

Calculations

System Suitability

Apparent number of theoreticalplates

Resolution

Symmetry factor

Signal-to-noise ratio

System Suitability

Apparent Number of TheoreticalPlates

Resolution

Symmetry Factor

Signal-to-noise Ratio

Sep. 2002


General Information

Isoelectric Focusing Isoelectric Focusing

General principle

Theoretical aspects Theoretical Aspects

Practical aspects Practical Aspects

Apparatus Apparatus

Isoelectric focusing in polyacrylamidegels: detailed procedure

Preparation of the gels

1) 7.5 per cent polyacrylamide gel

2) Preparation of the mould

Isoelectric Focusing in PolyacrylamideGels: Detailed Procedure

Preparation of the Gels

7.5z Polyacrylamide gel

Preparation of the mould

Method Method

Variations to the detailed procedure(subject to validation)

Variations to the Detailed Procedure(Subject to Validation)


2280

Validation of iso-electric focusingprocedures

Validation of Iso-Electric FocusingProcedures

Specified variation to the generalmethod

Specified Variations to the GeneralMethod

Point to consider Points to Consider

Reagents

Fixing solution for isoelectric focus-ing in polyacrylamide gel

Reagents and Solutions

Fixing solution for isoelectric focus-ing in polyacrylamide gel

Coomassie staining TS JP's particular description

Destaining solution JP's particular description

Figure-Mould Figure. Mould

Sep. 2002


General Information

Peptide Mapping Peptide Mapping

Purpose and scope Purpose and scope

The peptide map 1. The peptide map

Isolation and purification 2. Isolation and purification

Selective cleavage of peptide bonds 3. Selective cleavage of peptide bonds

Pretreatment of sample 3.1. Pretreatment of sample

Pretreatment of the cleavage agent 3.2. Pretreatment of the cleavageagent

Pretreatment of the protein 3.3. Pretreatment of the protein

Establishment of optimal digestionconditions

pH

Temperature

Time

Amount of cleavage agent

3.4. Establishment of optimal diges-tion conditions

(i) pH

(ii) Temperature

(iii) Time

(iv) Amount of cleavage agent

Chromatographic separation

Chromatographic column

solvent

4. Chromatographic separation

4.1. Chromatographic column

4.2. Solvent

Mobile phase

Gradient selection

Isocratic selection

Other parameters

Validation

4.3. Mobile phase

4.4. Gradient selection

4.5. Isocratic selection

4.6. Other parameters

4.7. Validation

Analysis and identification of peptides 5. Analysis and identification of pep-tides

Table 1. Examples of cleavage agents.

Table 2. Techniques used for the sepa-ration of peptides.

Table 1. Examples of cleavage agents

Table 2. Techniques used for the sepa-ration of peptides


2281

Oct. 1999


Sodium Dodecyl Sulphate Poly-acrylamide Gel Electrophoresis(SDS-PAGE)

General Information

SDS-Polyacrylamide Gel Electrophore-sis

Characteristics of polyacrylamide gels 1. Characteristics of PolyacrylamideGels

Denaturing polyacrylamide gel elec-trophoresis

Reducing conditions

Non-reducing conditions

2. Polyacrylamide Gel Electrophoresisunder Denaturing Conditions

2.1. Reducing conditions

2.2. Non-reducing conditions

Characteristics of discontinuousbuffer system gel electrophoresis

3. Characteristics of DiscontinuousBuffer System Gel Electrophoresis

Preparing vertical discontinuousbuffer SDS polyacrylamide gels

Assembling of the gel moulding cas-sette

Preparation of the gel

4. Preparing Vertical DiscontinuousBuffer SDS-Polyacrylamide Gels

4.1. Assembling of the gel mouldingcassette

4.2. Preparation of the gel

Mounting the gel in the electrophore-sis apparatus and electrophoreticseparation

4.3. Mounting the gel in the electro-phoresis apparatus and electro-phoretic separation

Detection of protein in gels

Coomassie staining

Silver staining

5. Detection of Proteins in Gels

5.1. Coomassie staining

5.2. Silver staining

Drying of stained SDS polyacrylamidegels

6. Drying of Stained SDS-Polyacrylamide Gels

Molecular-mass determination 7. Molecular-Mass Determination

Validation of the test 8. Suitability of the Test (Validation)

Quantification of impurities 9. Quantification of Impurities

Reagents, test solutions 10. Test Solutions

Blocking solution Blocking TS

Coomassie staining solution Coomassie staining TS

Destaining solution Destaining TS

Developer solution Developer TS

Fixing solution Fixing TS

Silver nitrate reagent Silver nitrate TS for silver staining

Trichloroacetic acid reagent Trichloroacetic acid TS for fixing

Table 1－ Preparation of resolvinggel

Table 2－ Preparation of stacking gel

Table 1. Preparation of resolving gel

Table 2. Preparation of stacking gel

Sep. 2002


General Information

Total Protein Assay Total Protein Assay

Method 1

Standard solution

Method 1 (UV method)

Standard Solution


2282

Test solution

Procedure

Light-scattering

Calculations

Test Solution

Procedure

Light-Scattering

Calculations

Method 2

Standard solutions

Test solution

Blank

Reagents and solutions

Copper sulfate reagent

SDS Solution

Sodium hydroxide solution

Alkaline copper reagent

Diluted Folin-Ciocalteu's phenol ra-gent

Procedure

Calculations

Interfering substances

Sodium deoxycholate reagent

Trichloroacetic acid reagent

Procedure

Method 2 (Lowry method)

Standard Solutions

Test Solution

Blank


Copper Sulfate Reagent

5z SDS TS

Sodium Hydroxide Solution

Alkaline Copper Reagent

Diluted Folin's TS

Procedure

Calculations

Interfering Substances

Sodium Deoxycholate Reagent

Trichloroacetic Acid Reagent

Procedure

Explanatory footnote ``Example: theMinimum Requirements for BiologicalProducts and individual monograph ofJP'' is added.

Method 3

Standard solutions

Method 3 (Bradford method)

Standard Solutions

Test solution

Blank

Test Solution

Blank

Coomassie reagent Coomassie Reagent

Procedure

Calculations

Procedure

Calculations

Method 4

Standard solutions

Test solution

Blank

Method 4 (Bicinchonic acid method)

Standard Solutions

Test Solution

Blank

Reagents

BCA Reagent

Copper sulfate reagent

Copper-BCA reagent

Procedure

Calculations


BCA Reagent

Copper Sulfate Reagent

Copper-BCA Reagent

Procedure

Calculations

Method 5

Standard solutions

Test solution

Blank

Biuret reagent

Procedure

Calculations

Interfering substances

Method 5 (Biuret method)

Standard Solutions

Test Solution

Blank

Biuret Reagent

Procedure

Calculations

Interfering Substances


2283

Comments Comments

Method 6

Standard solutions

Test solution

Blank

Reagents

Borate buffer

Stock OPA reagent

OPA reagent

Procedure

Calculations

Method 6 (Fluorometric method)

Standard Solutions

Test Solution

Blank


Borate Buffer

Stock OPA Reagent

OPA Reagent

Procedure

Calculations

Method 7

Procedure A

Procedure B

Calculations

Method 7 (Nitrogen method)

Procedure A

Procedure B

Calculations

Nov. 2005


Microbiological Examination of Non-Sterile Products:

General InformationMicrobial Attributes of Non-SterilePharmaceutical Products

JP's particular description:1. Definitions2. Scope3. Sampling plan and frequency of

testing4. Microbial control program

Acceptance criteria for pharmaceuticalpreparations and substances for phar-maceutical use

5. Microbial acceptance criteria fornon-sterile pharmaceutical products

JP's particular description:

Explanation of the microbial accep-tance criteria

JP's particular description:

6. Acceptance criteria for crude drugsand crude drug preparations

Table 2. Acceptance criteria formicrobiological quality of non-sterilesubstances for pharmaceutical use

Table 1. Acceptance criteria formicrobiological quality of non-sterilesubstances for pharmaceutical use

Table 1. Acceptance criteria formicrobiological quality of non-steriledosage forms

Table 2. Acceptance criteria formicrobiological quality of non-steriledosage forms

Table 3. Acceptance criteria for crudedrugs and crude drug preparations

JP's particular description

Feb. 2004


General InformationTablet Friability Tablet Friability Test


22872287

Atomic Weight Table (2010)

In 1961 it was decided that the atomic weights of theelements would be based on values relative to the massnumber of 12 (no fractions) for carbon (12C). Eversince, there has been a marked improvement in thequality and quantity of data on the nuclide masses andisotope ratios of the elements using physical methodssuch as mass spectrometry. The Commission onIsotope Abundances and Atomic Weights (CIAAW)of the International Union of Pure and AppliedChemistry (IUPAC) collected and examined newlymeasured data and publishes a new atomic weighttable every two years (in the odd years). Based on thistable, in April of each year the Atomic Weight Sub-committee of the Chemical Society of Japan also pub-lishes an atomic weight table. The numbers of the fol-lowing Atomic Weight Table (2010) is based on thenumbers published by the IUPAC in 20071). For amore detailed explanation, the user is referred to areport2) and a review3) published by the CIAAW.

The atomic weight values of each of the elementsshown in the atomic weight tables are, as stated in thepreface to the table, for elements that originate onEarth and are present in substances that exist natural-ly. Atomic weights are, with the exception of singlenuclide elements (elements consisting of one stablenuclide), not natural constants like the speed of light,but rather change depending on a variety of factors,such as the method of treatment or the origin of thesubstance containing that element. This is because theatomic weight is dependent on the relative frequency(isotope ratio) of the stable nuclides comprising eachof the respective elements. Due to advancements inmeasurement techniques, the isotopic frequencies ofeach of the elements are not necessarily constant, andfluctuate due to a variety of processes that occur onthe Earth. We have come to learn that this is reflectedin the atomic weights. The result of this is that differ-ences have arisen in the accuracy of the atomic weightsbetween elements. The figures in parentheses thatfollow the atomic weight values in the atomic weighttables represent the uncertainty with respect to the lastdigit in the atomic weight. For example, in the case ofhydrogen, 1.00794(7) means 1.00794± 0.00007.

The atomic weight of a single nuclide element is themost accurate and the precision is also high. This isbecause it is not necessary to consider the isotope ratiosince single nuclide elements do not possess a multiplenumber of stable isotopes. The atomic weights of suchelements are determined based on the mass4) of eachnuclide determined by physical techniques, taking intoconsideration the uncertainty with constant criteria.

Among the elements, the majority of samplesgathered on Earth exhibit a constant isotope composi-tion, however, some specific samples have isotopecompositions that are different to these. These kindsof elements are indicated by a ``g'', which means thevalue in the atomic weight table cannot be used as is,depending on the sample, as the atomic weight of theseelements. In relation to this, oxygen for example existsin a number of forms on Earth, such as in air, saltwater, fresh water, and in rocks, and because theisotope compositions fluctuate among these sub-stances, oxygen is not an element for which only onevalue can be used. Thus, an ``r'' is attached to anelement for which a precise atomic weight cannot begiven, no matter how much progress is made intechniques for measuring the isotope composition. Onthe other hand, it is also possible, depending on theelement, to use an isotope that has undergone artificialfractionation as a reagent. Typical elements that arerepresentative include hydrogen, lithium, boron, anduranium. This type of element is identified by an``m'', and particularly in cases where the atomicweight is a problem, it is necessary to be careful byreferring to the label of the reagent.

1) IUPAC Inorganic Chemistry Division, CIAAW: Stan-dard Atomic Weights Revised. Chem. Int., 29, 18 (2007).

2) IUPAC Inorganic Chemistry Division, CIAAW: AtomicWeights of the Elements 2007. Pure Appl. Chem., 81,2131 (2009).

3) J. R. De Laeter et al.: Atomic Weights of the Elements:Review 2000. Pure Appl. Chem., 75, 683 (2003).

4) G. Audi et al.: The AME 2003 Atomic Mass Evaluation(II). Tables, graphs and references. Nucl. Phys. A, 729,337 (2003).

22882288 JP XVIAppendix

Standard Atomic Weights 2010

(Scaled to Ar(12C)＝12, where 12C is a neutral atom inits nuclear and electronic ground state)

The atomic weights of many elements are not invariantbut depend on the origin and treatment of the materi-al. The standard values of Ar(E) and the uncertainties(in parentheses, following the last significant figure towhich they are attributed) apply to elements of naturalterrestrial origin. The footnotes to this table elaboratethe types of variation which may occur for individualelements and which may be larger than the listed un-certainties of values of Ar(E). Names of elements withatomic number 112 to 118 are provisional.

22892289JP XVI Appendix

22912291

INDEX

A

Absorptive Cream, 313Acacia, 1593

Powdered, 1593Acebutolol Hydrochloride, 313Aceglutamide Aluminum, 314Acemetacin, 315

Capsules, 316Tablets, 317

Acetaminophen, 318Acetazolamide, 319Acetic Acid, 319

Glacial, 320Acetohexamide, 320Acetylcholine Chloride for Injection,

322Acetylcysteine, 322Acetylsalicylic Acid, 400

Tablets, 400Achyranthes Root, 1594Aciclovir, 323

for Syrup, 325Injection, 325Syrup, 326

Aclarubicin Hydrochloride, 327Acrinol

and Zinc Oxide Oil, 329and Zinc Oxide Oil, Compound,

330and Zinc Oxide Ointment, 330Hydrate, 328

Actinomycin D, 331Adrenaline, 331

Injection, 332Solution, 332

AdsorbedDiphtheria Toxoid for Adult Use,

738Diphtheria-Purified Pertussis-Teta-

nus Combined Vaccine, 739Diphtheria-Tetanus Combined Tox-

oid, 739Habu-venom Toxoid, 908Hepatitis B Vaccine, 909Purified Pertussis Vaccine, 1230Tetanus Toxoid, 1479

Afloqualone, 333Agar, 1594

Powdered, 1595Ajmaline, 334

Tablets, 334Akebia Stem, 1595Alacepril, 335

Tablets, 336

L-Alanine, 337Albumin Tannate, 338Alcohol, 802

Dehydrated, 803for Disinfection, 804

Aldioxa, 339Alendronate

Sodium Hydrate, 340Sodium Injection, 341Sodium Tablets, 342

Alimemazine Tartrate, 343Alisma Rhizome, 1595

Powdered, 1596Allopurinol, 344

Tablets, 344Alminoprofen, 345

Tablets, 346Aloe, 1596

Powdered, 1597Alpinia Officinarum Rhizome, 1598Alprazolam, 347Alprenolol Hydrochloride, 348Alprostadil, 349

Alfadex, 352Injection, 350

Alum, 357Solution, 358Powder, Salicylated, 1371

AluminumAcetylsalicylate, 401Monostearate, 356Potassium Sulfate Hydrate, 356Silicate Hydrate with Silicon Diox-

ide, 1598Silicate, Natural, 354Silicate, Synthetic, 356Sucrose Sulfate Ester, 1429

Amantadine Hydrochloride, 358Ambenonium Chloride, 359Amidotrizoic Acid, 360Amikacin Sulfate, 361

for Injection, 362Injection, 362

Aminoacetic Acid, 895Aminobenzylpenicillin

Anhydrous, 383Hydrate, 384Sodium, 385

AminophyllineHydrate, 363Injection, 363

Amiodarone Hydrochloride, 364Tablets, 366

Amitriptyline Hydrochloride, 367Tablets, 367

Amlexanox, 368

Tablets, 370Amlodipine Besilate, 370

Tablets, 372Ammonia Water, 373Amobarbital, 373

Sodium for Injection, 374Amomum Seed, 1599

Powdered, 1599Amosulalol Hydrochloride, 375

Tablets, 376Amoxapine, 377Amoxicillin, 378

Capsules, 379Hydrate, 378

Amphotericin B, 380for Injection, 381Syrup, 382Tablets, 382

AmpicillinAnhydrous, 383Ethoxycarbonyloxyethyl Hydrochlo-

ride, 415Hydrate, 384Sodium, 385Sodium for Injection, 386

Ampicillinphthalidyl Hydrochloride,1450

Amyl Nitrite, 387Anemarrhena Rhizome, 1599Anesthamine, 809Anesthetic Ether, 805Angelica Dahurica Root, 1599Anhydrous

Aminobenzylpenicillin, 383Ampicillin, 383Caffeine, 489Citric Acid, 645Dibasic Calcium Phosphate, 504Ethanol, 803Lactose, 1018Light, Silicic Acid, 1385

Antipyrine, 388Apricot Kernel, 1600

Water, 1601Aprindine Hydrochloride, 388

Capsules, 389Aralia Rhizome, 1601Arbekacin Sulfate, 390

Injection, 392Areca, 1602Argatroban Hydrate, 392L-Arginine, 394

Hydrochloride, 394Hydrochloride Injection, 395

Aromatic Castor Oil, 528Arotinolol Hydrochloride, 396

22922292 JP XVIIndex

Arsenic Trioxide, 397Arsenical Paste, 396Arsenous Acid, 397Artemisia Capillaris Flower, 1602Ascorbic Acid, 397

Injection, 398Powder, 398

Asiasarum Root, 1602Asparagus Tuber, 1603L-Aspartic Acid, 399Aspirin, 400

Aluminum, 401Tablets, 400

Aspoxicillin Hydrate, 402Astragalus Root, 1604Atenolol, 403Atorvastatin Calcium

Hydrate, 404Tablets, 405

AtractylodesLancea Rhizome, 1604Lancea Rhizome, Powdered, 1605Rhizome, 1605Rhizome, Powdered, 1606

Atropine SulfateHydrate, 407Injection, 407

Azathioprine, 408Tablets, 409

Azelastine Hydrochloride, 410Granules, 411

Azithromycin Hydrate, 412Aztreonam, 413

for Injection, 414

B

Bacampicillin Hydrochloride, 415Bacitracin, 416Baclofen, 417

Tablets, 418Bakumondoto Extract, 1606Bamethan Sulfate, 419Barbital, 419Barium Sulfate, 420Bear Bile, 1608Bearberry Leaf, 1608Beclometasone Dipropionate, 421Beef Tallow, 422Beeswax

White, 422Yellow, 423

Bekanamycin Sulfate, 423Belladonna

Extract, 1610Root, 1609

Benidipine Hydrochloride, 424Tablets, 425

Benincasa Seed, 1611Benoxinate Hydrochloride, 1195Benserazide Hydrochloride, 427Bentonite, 427Benzalkonium Chloride, 428

Solution, 429Solution 50, Concentrated, 429

Benzbromarone, 430Benzethonium Chloride, 431

Solution, 431Benzocaine, 809Benzoic Acid, 432Benzoin, 1611Benzyl

Alcohol, 432Benzoate, 434

BenzylpenicillinBenzathine Hydrate, 434Potassium, 436Potassium for Injection, 437

Beraprost Sodium, 438Tablets, 439

BerberineChloride Hydrate, 440Tannate, 441

Betahistine Mesilate, 443Tablets, 443

Betamethasone, 445Dipropionate, 447Sodium Phosphate, 448Tablets, 446Valerate, 449Valerate and Gentamicin Sulfate

Cream, 450Valerate and Gentamicin Sulfate

Ointment, 452Betamipron, 453Betaxolol Hydrochloride, 454Bethanechol Chloride, 455Bezafibrate, 455

Sustained Release Tablets, 456Bifonazole, 457Biotin, 458Biperiden Hydrochloride, 459Bisacodyl, 459

Suppositories, 460Bismuth

Subgallate, 461Subnitrate, 462

Bisoprolol Fumarate, 462Tablets, 463

BitterCardamon, 1611Orange Peel, 1612Tincture, 1613

BleomycinHydrochloride, 465Sulfate, 467

Boric Acid, 468Bromazepam, 469Bromhexine Hydrochloride, 470Bromocriptine Mesilate, 471Bromovalerylurea, 471Brown Rice, 1613Bucillamine, 472

Tablets, 473Bucumolol Hydrochloride, 474Bufetolol Hydrochloride, 475

Buformin Hydrochloride, 476Enteric-coated Tablets, 476Tablets, 478

Bumetanide, 479Bunazosin Hydrochloride, 479Bupleurum Root, 1613Bupranolol Hydrochloride, 480Buprenorphine Hydrochloride, 481Burdock Fruit, 1614Burnt Alum, 357Busulfan, 482Butenafine Hydrochloride, 483

Cream, 484Solution, 484Spray, 485

Butropium Bromide, 485Butyl Parahydroxybenzoate, 486

C

Cacao Butter, 487Cadralazine, 487

Tablets, 488Caffeine

and Sodium Benzoate, 491Anhydrous, 489Hydrate, 490

Calciferol, 787Calcitonin (Salmon), 492Calcium

Chloride Hydrate, 497Chloride Injection, 497Folinate, 498Gluconate Hydrate, 499Hydroxide, 500Lactate Hydrate, 500Leucovorin, 498Oxide, 501Pantothenate, 501Paraaminosalicylate Granules, 503Paraaminosalicylate Hydrate, 502Polystyrene Sulfonate, 506Stearate, 508

Calumba, 1615Powdered, 1615

Camellia Oil, 508Camostat Mesilate, 509Camphor

Synthetic, 510d-Camphor, 510dl-Camphor, 510Candesartan Cilexetil, 511

Tablets, 512Capsicum, 1615

and Salicylic Acid Spirit, 1618Powdered, 1616Tincture, 1617

Capsules, 514Capsules

Acemetacin, 316Amoxicillin, 379Aprindine Hydrochloride, 389Cefaclor, 529

22932293JP XVI Index

Cefadroxil, 535Cefalexin, 537Cefdinir, 553Cefixime, 561Cinoxacin, 643Clindamycin Hydrochloride, 651Clofibrate, 658Clorazepate Dipotassium, 665Doxifluridine, 754Droxidopa, 760Flopropione, 842Flurazepam, 854Indometacin, 961Methotrexate, 1093Nizatidine, 1172Rifampicin, 1348Roxatidine Acetate Hydrochloride

Extended-release, 1361Sodium Iodide (123I), 1404Sodium Iodide (131I), 1404Sulpiride, 1442Tranexamic Acid, 1519Ubenimex, 1547Vitamin A, 1566Vitamin A Oil, 1566

Captopril, 514Carbamazepine, 515Carbazochrome Sodium Sulfonate Hy-

drate, 516Carbetapentane Citrate, 1223Carbetapentene Citrate, 1223Carbidopa Hydrate, 517L-Carbocisteine, 518Carbolic Acid, 1236

for Disinfection, 1236Liquefied, 1237

Carbon Dioxide, 518Carboxymethylcellulose, 519

Calcium, 519Sodium, 520

Cardamon, 1618Carmellose, 519

Calcium, 519Sodium, 520

Carmofur, 521Carnauba Wax, 522Carteolol Hydrochloride, 522Carumonam Sodium, 523Carvedilol, 525

Tablets, 526Cassia Seed, 1618Castor Oil, 527

Aromatic, 528Catalpa Fruit, 1619Cefaclor, 528

Capsules, 529Compound Granules, 532Fine Granules, 531

Cefadroxil, 534Capsules, 535for Syrup, 535

Cefalexin, 536Capsules, 537

for Syrup, 539Cefalotin Sodium, 540Cefatrizine Propylene Glycolate, 541

for Syrup, 542Cefazolin Sodium, 543

for Injection, 544Hydrate, 545

Cefbuperazone Sodium, 546Cefcapene Pivoxil Hydrochloride

Fine Granules, 550Hydrate, 548Tablets, 551

Cefdinir, 552Capsules, 553Fine Granules, 554

Cefditoren Pivoxil, 555Fine Granules, 556Tablets, 556

Cefepime Dihydrochloridefor Injection, 559Hydrate, 557

CefiximeCapsules, 561Hydrate, 560

Cefmenoxime Hydrochloride, 562Cefmetazole Sodium, 564

for Injection, 565Cefminox Sodium Hydrate, 565Cefodizime Sodium, 566Cefoperazone Sodium, 568Cefotaxime Sodium, 569Cefotetan, 570Cefotiam

Hexetil Hydrochloride, 572Hydrochloride, 575Hydrochloride for Injection, 576

Cefozopran Hydrochloride, 576for Injection, 577

Cefpiramide Sodium, 578Cefpirome Sulfate, 580Cefpodoxime Proxetil, 581Cefroxadine

for Syrup, 584Hydrate, 582

Cefsulodin Sodium, 585Ceftazidime


Cefteram Pivoxil, 589Fine Granules, 590Tablets, 591

Ceftibuten Hydrate, 592Ceftizoxime Sodium, 593Ceftriaxone Sodium Hydrate, 594Cefuroxime Axetil, 596Cellacefate, 598Cellulose

Acetate Phthalate, 598methyl ether, 1096Microcrystalline, 599Powdered, 602

Celmoleukin (Genetical Recombina-tion), 602

Cetanol, 605Cetirizine Hydrochloride, 606

Tablets, 607Cetraxate Hydrochloride, 608Chenodeoxycholic Acid, 609Chloral Hydrate, 610Chloramphenicol, 610

Palmitate, 611Sodium Succinate, 612

Chlordiazepoxide, 613Powder, 613Tablets, 614

ChlorhexidineGluconate Solution, 616Hydrochloride, 616

Chlorinated Lime, 617Chlormadinone Acetate, 617Chlorobutanol, 618Chlorphenesin Carbamate, 619

Tablets, 620Chlorpheniramine

and Calcium Powder, 621Maleate, 622Maleate Injection, 623Maleate Powder, 623Maleate Tablets, 624

d-Chlorpheniramine Maleate, 625Chlorpromazine Hydrochloride, 626

Injection, 627Tablets, 627

Chlorpropamide, 629Tablets, 629

Cholecalciferol, 630Cholera Vaccine, 630Cholesterol, 631Chorionic Gonadotrophin, 897

for Injection, 899Chotosan Extract, 1619Chrysanthemum Flower, 1622Cibenzoline Succinate, 632

Tablets, 632Ciclacillin, 634Ciclosporin, 634

A, 634Cilastatin Sodium, 636Cilazapril


Cilostazol, 639Tablets, 640

Cimetidine, 641Cimicifuga Rhizome, 1622Cinchocaine Hydrochloride, 709Cinnamon

Bark, 1623Bark, Powdered, 1623Oil, 1623

Cinoxacin, 642Capsules, 643

Cisplatin, 644Citric Acid

Anhydrous, 645Hydrate, 645


Citrus Unshiu Peel, 1624Clarithromycin, 646

Tablets, 647Clebopride Malate, 649Clemastine Fumarate, 650Clematis Root, 1625Clindamycin

Hydrochloride, 650Hydrochloride Capsules, 651Phosphate, 652Phosphate Injection, 653

Clinofibrate, 654Clobetasol Propionate, 655Clocapramine Hydrochloride Hydrate,

656Clofedanol Hydrochloride, 657Clofibrate, 657

Capsules, 658Clomifene Citrate, 659

Tablets, 660Clomipramine Hydrochloride, 661Clonazepam, 661Clonidine Hydrochloride, 662Cloperastine Hydrochloride, 663Clorazepate Dipotassium, 664

Capsules, 665Clotiazepam, 666Clotrimazole, 667Clove, 1625

Oil, 1626Powdered, 1625

Cloxacillin Sodium Hydrate, 668Cloxazolam, 669Cnidium

Monnieri Fruit, 1626Rhizome, 1626Rhizome, Powdered, 1627

Cocaine Hydrochloride, 670Coconut Oil, 670Cod Liver Oil, 674Codeine Phosphate

Hydrate, 671Powder, 1z, 671Powder, 10z, 672Tablets, 672

Coix Seed, 1627Powdered, 1627

Colchicine, 674Colistin

Sodium Methanesulfonate, 676Sulfate, 677

CompoundAcrinol and Zinc Oxide Oil, 330Diastase and Sodium Bicarbonate

Powder, 706Hycodenone Injection, 1197Iodine Glycerin, 967Methyl Salicylate Spirit, 1107Oxycodone and Atropine Injection,

1197Oxycodone Injection, 1197Phellodendron Powder for

Cataplasm, 1709

Rhubarb and Senna Powder, 1725Salicylic Acid Spirit, 1372Scopolia Extract and Diastase Pow-

der, 1744Thianthol and Salicylic Acid Solu-

tion, 1488Vitamin B Powder, 1567

ConcentratedGlycerin, 893Glycerol, 893

Condurango, 1628Fluidextract, 1628

Coptis Rhizome, 1628Powdered, 1629

CornOil, 678Starch, 678

Cornus Fruit, 1630Cortisone Acetate, 679Corydalis Tuber, 1631

Powdered, 1632Crataegus Fruit, 1632Creams

Absorptive, 313Betamethasone Valerate and Gen-

tamicin Sulfate, 450Butenafine Hydrochloride, 484Hydrophilic, 934Ketoconazole, 1006Terbinafine Hydrochloride, 1473

Cresol, 680Solution, 680Solution, Saponated, 681

Croconazole Hydrochloride, 681Croscarmellose Sodium, 682Crude Glycyrrhiza Extract, 1651Crystal Violet, 1105Cyanamide, 683Cyanocobalamin, 684

Injection, 685Cyclopentolate Hydrochloride, 686Cyclophosphamide Hydrate, 686Cycloserine, 687Cyperus Rhizome, 1633

Powdered, 1633Cyproheptadine Hydrochloride Hy-

drate, 688L-Cysteine, 688

Hydrochloride Hydrate, 689Cytarabine, 690

D

Daiokanzoto Extract, 1634Danazol, 691Dantrolene Sodium Hydrate, 691Daunorubicin Hydrochloride, 692Deferoxamine Mesilate, 693Dehydrated Alcohol, 803Dehydrocholate Sodium Injection,

696Dehydrocholic Acid, 694

Injection, 696

Purified, 695Demethylchlortetracycline Hydrochlo-

ride, 696Dental

Antiformin, 387Iodine Glycerin, 968Paraformaldehyde Paste, 1215Phenol with Camphor, 1237Sodium Hypochlorite Solution, 387Triozinc Paste, 1540

Dermatol, 461Deslanoside, 698

Injection, 698Dexamethasone, 699Dextran

40, 70040 Injection, 70170, 702Sulfate Sodium Sulfur 5, 703Sulfate Sodium Sulfur 18, 704

Dextrin, 704Dextromethorphan Hydrobromide Hy-

drate, 705Diagnostic Sodium Citrate Solution,

1399Diastase, 706

and Sodium Bicarbonate Powder,706

and Sodium Bicarbonate Powder,Compound, 706

Diazepam, 706Tablets, 707

DibasicCalcium Phosphate, Anhydrous,

504Calcium Phosphate Hydrate, 504Sodium Phosphate Hydrate, 1408

Dibekacin Sulfate, 708Ophthalmic Solution, 709

Dibucaine Hydrochloride, 709Dichlorphenamide, 711

Tablets, 712Diclofenac Sodium, 710Diclofenamide, 711

Tablets, 712Dicloxacillin Sodium Hydrate, 713Diethylcarbamazine Citrate, 713

Tablets, 714Difenidol Hydrochloride, 715Diflucortolone Valerate, 715Digenea, 1635Digitoxin, 717

Tablets, 718Digoxin, 719


Dihydrocodeine Phosphate, 723Powder, 1z, 724Powder, 10z, 724

Dihydroergotamine Mesilate, 725Dihydroergotoxine Mesilate, 726Dilazep Hydrochloride Hydrate, 728Diltiazem Hydrochloride, 729


DiluteHydrochloric Acid, 925Iodine Tincture, 967

Diluted Opium Powder, 1184Dimemorfan Phosphate, 730Dimenhydrinate, 731

Tablets, 732Dimercaprol, 732

Injection, 733Dimorpholamine, 733

Injection, 734Dinoprost, 735Dionin, 813Dioscorea Rhizome, 1635

Powdered, 1636Diphenhydramine, 735

and Bromovalerylurea Powder, 736Hydrochloride, 737Phenol and Zinc Oxide Liniment,

737Tannate, 738

Diphenylhydantoin, 1242Powder, 1242Sodium for Injection, 1244Tablets, 1243

DiphtheriaAntitoxin, Equine, Freeze-dried,

738-Purified Pertussis-Tetanus Com-

bined Vaccine, Absorbed, 739-Tetanus Combined Toxoid, 739-Tetanus Combined Toxoid, Ab-

sorbed, 739Toxoid, 738Toxoid for Adult Use, Absorbed,

738Dipyridamole, 739Disodium Edetate Hydrate, 1400Disopyramide, 740Distigmine Bromide, 741

Tablets, 741Disulfiram, 742Dobutamine Hydrochloride, 743Dolichos Seed, 1636Domperidone, 744Donepezil Hydrochloride, 745

Fine Granules, 746Tablets, 747

Dopamine Hydrochloride, 749Injection, 749

Doxapram Hydrochloride Hydrate,750

Doxazosin Mesilate, 751Tablets, 752

Doxifluridine, 753Capsules, 754

Doxorubicin Hydrochloride, 755for Injection, 756

Doxycycline Hydrochloride Hydrate,757

DriedAluminum Hydroxide Gel, 353Aluminum Hydroxide Gel Fine

Granules, 354Aluminum Potassium Sulfate, 357Sodium Carbonate, 1395Sodium Sulfite, 1417Thyroid, 1494Yeast, 1580

Droperidol, 758Droxidopa, 759

Capsules, 760Fine Granules, 761

Dydrogesterone, 762Tablets, 762

E

Ebastine, 763Orally Disintegrating Tablets, 764Tablets, 765

Ecabet SodiumGranules, 768Hydrate, 767

Ecarazine Hydrochloride, 1510Ecothiopate Iodide, 769Edrophonium Chloride, 770

Injection, 770EDTA Sodium Hydrate, 1400Elcatonin, 771Eleutherococcus Senticosus Rhizome,

1636Emorfazone, 774

Tablets, 774Enalapril Maleate, 775

Tablets, 777Enflurane, 778Enoxacin Hydrate, 779Enviomycin Sulfate, 780Eperisone Hydrochloride, 781Ephedra Herb, 1636Ephedrine Hydrochloride, 782

Injection, 783Powder, 783Powder, 10z, 783Tablets, 784

Epimedium Herb, 1638Epinephrine, 331

Injection, 332Solution, 332

Epirizole, 785Epirubicin Hydrochloride, 786Ergocalciferol, 787Ergometrine Maleate, 788


Ergotamine Tartrate, 790Erythromycin, 791

Enteric-Coated Tablets, 792Ethylsuccinate, 793Lactobionate, 793Stearate, 794

Estazolam, 795Estradiol Benzoate, 796

Injection, 796Injection (Aqueous Suspension),

797Estriol, 798

Injection (Aqueous Suspension),798

Tablets, 799Etacrynic Acid, 800

Tablets, 801Ethacridine Lactate, 328Ethambutol Hydrochloride, 801Ethanol, 802

Anhydrous, 803for Disinfection, 804

Ethenzamide, 804Ether, 805

Anesthetic, 805Ethinylestradiol, 806

Tablets, 806Ethionamide, 807Ethosuximide, 808Ethoxybenzamide, 804Ethyl

Aminobenzoate, 809Cysteine Hydrochloride, 809L-Cysteine Hydrochloride, 809Icosapentate, 810Parahydroxybenzoate, 811

Ethylenediamine, 812Ethylmorphine Hydrochloride Hy-

drate, 813Etidronate Disodium, 813

Tablets, 814Etilefrine Hydrochloride, 815

Tablets, 815Etizolam, 817


Etodolac, 820Etoposide, 821Eucalyptus Oil, 822Eucommia Bark, 1638Euodia Fruit, 1639Exsiccated Gypsum, 1655Extracts

Bakumondoto, 1606Belladonna, 1610Chotosan, 1619Crude Glycyrrhiza, 1651Daiokanzoto, 1634Glycyrrhiza, 1651Goshajinkigan, 1652Hachimijiogan, 1656Hangekobokuto, 1659Hochuekkito, 1661Juzentaihoto, 1671Kakkonto, 1674Kamishoyosan, 1676Keishibukuryogan, 1679Mukoi-Daikenchuto, 1691Nux Vomica, 1696Orengedokuto, 1698Rikkunshito, 1725Ryokeijutsukanto, 1729Saibokuto, 1732


Extracts (continued)Saikokeishito, 1734Saireito, 1737Scopolia, 1742Shakuyakukanzoto, 1752Shimbuto, 1753Shosaikoto, 1756Shoseiryuto, 1758

F

Famotidine, 822for Injection, 823Injection, 824Powder, 825Tablets, 826

Faropenem Sodiumfor Syrup, 828Hydrate, 827Tablets, 829

Felbinac, 831Fenbufen, 831Fennel, 1639

Oil, 1640Powdered, 1639

Fentanyl Citrate, 832Ferrous Sulfate Hydrate, 832Fexofenadine Hydrochloride, 833Fine Granules

Cefaclor, 531Cefcapene Pivoxil Hydrochloride,

550Cefdinir, 554Cefditoren Pivoxil, 556Cefteram Pivoxil, 590Donepezil Hydrochloride, 746Dried Aluminum Hydroxide Gel,

354Droxidopa, 761Etizolam, 817Haloperidol, 910Irsogladine Maleate, 977Pravastatin Sodium, 1278Precipitated Calcium Carbonate,

495Probucol, 1294Risperidone, 1351Sarpogrelate Hydrochloride, 1375Troxipide, 1541

Flavin Adenine Dinucleotide Sodium,834

Flavoxate Hydrochloride, 836Flecainide Acetate, 836

Tablets, 838Flomoxef Sodium, 839

for Injection, 840Flopropione, 841

Capsules, 842Fluconazole, 843Flucytosine, 844Fludiazepam, 845Fludrocortisone Acetate, 845

FluidextractsCondurango, 1628Platycodon, 1712Uva Ursi, 1609

Flunitrazepam, 846Fluocinolone Acetonide, 847Fluocinonide, 848Fluorescein Sodium, 849Fluorometholone, 850Fluorouracil, 851Fluoxymesterone, 852Fluphenazine Enanthate, 853Flurazepam, 853

Capsules, 854Hydrochloride, 855

Flurbiprofen, 855Flutamide, 856Flutoprazepam, 857

Tablets, 858Fluvoxamine Maleate, 859

Tablets, 860Foeniculated Ammonia Spirit, 1640Folic Acid, 861


Formalin, 864Water, 864

Formoterol Fumarate Hydrate, 865Forsythia Fruit, 1640Fosfomycin

Calcium Hydrate, 865Sodium, 867Sodium for Injection, 868

Fradiomycin Sulfate, 869Freeze-dried

BCG Vaccine (for PercutaneousUse), 421

Botulism Antitoxin, Equine, 469Diphtheria Antitoxin, Equine, 738Habu Antivenom, Equine, 908Inactivated Tissue Culture Rabies

Vaccine, 1333Japanese Encephalitis Vaccine, 995Live Attenuated Measles Vaccine,

1065Live Attenuated Mumps Vaccine,

1137Live Attenuated Rubella Vaccine,

1365Mamushi Antivenom, Equine, 1061Smallpox Vaccine, 1390Smallpox Vaccine Prepared in Cell

Culture, 1390Tetanus Antitoxin, Equine, 1479

Fritillaria Bulb, 1641Fructose, 870

Injection, 870Furosemide, 871


Fursultiamine Hydrochloride, 874

G

Gabexate Mesilate, 875b-Galactosidase

(Aspergillus), 876(Penicillium), 876

Gallium (67Ga) Citrate Injection, 878Gambir, 1641

Powdered, 1641Gardenia Fruit, 1642

Powdered, 1642Gas Gangrene Antitoxin, Equine, 878Gastrodia Tuber, 1643Gefarnate, 878Gelatin, 879

Purified, 880Gentamicin Sulfate, 881

Ophthalmic Solution, 882Gentian, 1644

and Sodium Bicarbonate Powder,1645

Powdered, 1644Geranium Herb, 1645

Powdered, 1645Ginger, 1645

Powdered, 1646Processed, 1719

Ginseng, 1646Powdered, 1647

Glacial Acetic Acid, 320Glehnia Root and Rhizome, 1649Glibenclamide, 883Gliclazide, 883Glimepiride, 884

Tablets, 886Glucose, 888

Injection, 888L-Glutamic Acid, 889L-Glutamine, 890Glutathione, 891Glycerin, 892

and Potash Solution, 894Concentrated, 893

Glycerol, 892Concentrated, 893Monostearate, 894

Glycine, 895Glycyrrhiza, 1649

Extract, 1651Extract, Crude, 1651Powdered, 1650

Gonadorelin Acetate, 895Goshajinkigan Extract, 1652Gramicidin, 903Granules

Azelastine Hydrochloride, 411Calcium Paraaminosalicylate, 503Cefaclor Compound, 532Ecabet Sodium, 768L-Isoleucine, L-Leucine and L-Valine,

984Pas-calcium, 503


Ursodeoxycholic Acid, 1554Griseofulvin, 904

Tablets, 905Guaiacol Glyceryl Ether, 906Guaifenesin, 906Guanabenz Acetate, 907Guanethidine Sulfate, 908Gypsum, 1655

H

Hachimijiogan Extract, 1656Haloperidol, 909


Halothane, 912Haloxazolam, 913Hangekobokuto Extract, 1659Hemp Fruit, 1661Heparin

Calcium, 914Sodium, 916Sodium Injection, 919

L-Histidine, 920Hydrochloride Hydrate, 920

Hochuekkito Extract, 1661Homatropine Hydrobromide, 921Homochlorcyclizine Hydrochloride,

922Honey, 1664Houttuynia Herb, 1665Human

Chorionic Gonadotrophin, 897Chorionic Gonadotrophin for Injec-

tion, 899Menopausal Gonadotrophin, 899Normal Immunoglobulin, 923

Hycoato Injection, 1197Hydralazine Hydrochloride, 923

for Injection, 923Powder, 924Tablets, 924

Hydrochloric Acid, 925Dilute, 925Lemonade, 926

Hydrochlorothiazide, 926Hydrocortisone, 927

Acetate, 928and Diphenhydramine Ointment,

929Butyrate, 929Sodium Phosphate, 930Sodium Succinate, 931Succinate, 932

Hydrocotarnine Hydrochloride Hy-drate, 933

Hydrogenated Oil, 934Hydrophilic

Cream, 934Petrolatum, 1231

Hydrous Lanolin, 1022Hydroxocobalamin Acetate, 935Hydroxypropylcellulose, 935

Hydroxypropylmethylcellulose, 940Hydroxyzine

Hydrochloride, 938Pamoate, 938

Hymecromone, 939Hypromellose, 940

Phthalate, 942

I

Ibudilast, 943Ibuprofen, 944Ichthammol, 944Idarubicin Hydrochloride, 945

for Injection, 946Idoxuridine, 947

Ophthalmic Solution, 947Ifenprodil Tartrate, 948Imidapril Hydrochloride, 949

Tablets, 950Imipenem

and Cilastatin Sodium for Injection,953

Hydrate, 952Imipramine Hydrochloride, 954

Tablets, 955Immature Orange, 1665Imperata Rhizome, 1665Indapamide, 956

Tablets, 957Indenolol Hydrochloride, 958Indigocarmine, 959

Injection, 959Indium (111In) Chloride Injection, 960Indometacin, 960

Capsules, 961Suppositories, 962

Influenza HA Vaccine, 963Injection

Acetylcholine Chloride for, 322Aciclovir, 325Adrenaline, 332Alendronate Sodium, 341Alprostadil, 350Amikacin Sulfate for, 362Amikacin Sulfate, 362Aminophylline, 363Amobarbital Sodium for, 374Amphotericin B for, 381Ampicillin Sodium for, 386Arbekacin Sulfate, 392L-Arginine Hydrochloride, 395Ascorbic Acid, 398Atropine Sulfate, 407Aztreonam for, 414Benzylpenicillin Potassium for, 437Calcium Chloride, 497Cefazolin Sodium for, 544Cefepime Dihydrochloride for, 559Cefmetazole Sodium for, 565Cefotiam Hydrochloride for, 576Cefozopran Hydrochloride for, 577Ceftazidime for, 588

Chlorpheniramine Maleate, 623Chlorpromazine Hydrochloride,

627Chorionic Gonadotrophin for, 899Clindamycin Phosphate, 653Compound Hycodenone, 1197Compound Oxycodone, 1197Compound Oxycodone and Atro-

pine, 1197Cyanocobalamin, 685Dehydrocholate Sodium, 696Dehydrocholic Acid, 696Deslanoside, 698Dextran 40, 701Digoxin, 720Dimercaprol, 733Dimorpholamine, 734Diphenylhydantoin Sodium for,

1244Dopamine Hydrochloride, 749Doxorubicin Hydrochloride for,

756Edrophonium Chloride, 770Ephedrine Hydrochloride, 783Epinephrine, 332Ergometrine Maleate, 789Estradiol Benzoate, 796Estradiol Benzoate, (Aqueous Sus-

pension), 797Estriol, (Aqueous Suspension), 798Famotidine for, 823Famotidine, 824Flomoxef Sodium for, 840Folic Acid, 862Fosfomycin Sodium for, 868Fructose, 870Furosemide, 872Gallium (67Ga) Citrate, 878Glucose, 888Heparin Sodium, 919Human Chorionic Gonadotrophin

for, 899Hycoato, 1197Hydralazine Hydrochloride for,

923Idarubicin Hydrochloride for, 946Imipenem and Cilastatin Sodium for,

953Indigocarmine, 959Indium (111In) Chloride, 960Iodinated (131I) Human Serum Albu-

min, 966Isepamicin Sulfate, 981Isoniazid, 986Isotonic Sodium Chloride, 1397Levallorphan Tartrate, 1029Lidocaine Hydrochloride, 1035Lidocaine, 1035Lincomycin Hydrochloride, 1038Magnesium Sulfate, 1059D-Mannite, 1064D-Mannitol, 1064Meglumine Iotalamate, 1073


Injection (continued)Meglumine Sodium Amidotrizoate,

1074Meglumine Sodium Iodamide, 1075Mepivacaine Hydrochloride, 1082Meropenem for, 1087Metenolone Enanthate, 1089Minocycline Hydrochloride for,

1124Mitomycin C for, 1127Morphine and Atropine, 1130Morphine Hydrochloride, 1131Neostigmine Methylsulfate, 1151Nicardipine Hydrochloride, 1153Nicotinic Acid, 1162Noradrenaline, 1174Noradrenaline Hydrochloride,

1174Norepinephrine Hydrochloride,

1174Norepinephrine, 1174Operidine, 1231Opium Alkaloids and Atropine,

1187Opium Alkaloids and Scopolamine,

1188Opium Alkaloids Hydrochlorides,

1186Oxytocin, 1205Ozagrel Sodium for, 1207Papaverine Hydrochloride, 1212Peplomycin Sulfate for, 1226Pethidine Hydrochloride, 1231Phenolsulfonphthalein, 1239Phenytoin Sodium for, 1244Piperacillin Sodium for, 1255Prednisolone Sodium Succinate for,

1290Procainamide Hydrochloride, 1296Procaine Hydrochloride, 1299Progesterone, 1304Protamine Sulfate, 1317Pyridoxine Hydrochloride, 1324Reserpine, 1338Riboflavin Phosphate, 1345Riboflavin Sodium Phosphate,

1345Roxatidine Acetate Hydrochloride

for, 1363Serum Gonadotrophin for, 903Sodium Bicarbonate, 1393Sodium Chloride, 0.9z, 1397Sodium Chloride, 10z, 1397Sodium Chromate (51Cr), 1398Sodium Citrate, for Transfusion,

1398Sodium Iodohippurate (131I), 1405Sodium Iotalamate, 1405Sodium Pertechnetate (99mTc), 1408Sodium Thiosulfate, 1417Sterile Water for, in Containers,

1573Streptomycin Sulfate for, 1428

Sulfobromophthalein Sodium,1439

Sulpyrine, 1444Suxamethonium Chloride, 1449Suxamethonium Chloride for, 1448Teceleukin for, (Genetical Recombi-

nation), 1465Testosterone Enanthate, 1477Testosterone Propionate, 1478Thallium (201Tl) Chloride, 1481Thiamine Chloride Hydrochloride,

1484Thiamylal Sodium for, 1487Thiopental Sodium for, 1490Tobramycin, 1506Tranexamic Acid, 1520Vancomycin Hydrochloride for,

1559Vasopressin, 1560Vinblastine Sulfate for, 1564Vitamin B1 Hydrochloride, 1484Vitamin B2 Phosphate Ester, 1345Vitamin B6, 1324Vitamin B12, 685Vitamin C, 398Water for, 1572Weak Opium Alkaloids and Scopola-

mine, 1189Xylitol, 1580

Insulin Human (Genetical Recombina-tion), 963

Iodamide, 965Iodinated (131I) Human Serum Albumin

Injection, 966Iodine, 966

Glycerin, Compound, 967Glycerin, Dental, 968Salicylic Acid and Phenol Spirit,

969Tincture, 966Tincture, Dilute, 967

Iodoform, 971Iopamidol, 971Iotalamic Acid, 972Iotroxic Acid, 973Ipecac, 1666

Powdered, 1667Syrup, 1667

Ipratropium Bromide Hydrate, 974Ipriflavone, 975

Tablets, 976Iproveratril Hydrochloride, 1561Irsogladine Maleate, 976


Isepamicin Sulfate, 980Injection, 981

Isoflurane, 982L-Isoleucine, 983

L-Leucine and L-Valine Granules,984

Isoniazid, 985Injection, 986

Tablets, 986l-Isoprenaline Hydrochloride, 987Isopropanol, 988Isopropyl Alcohol, 988Isopropylantipyrine, 989Isosorbide, 989

Dinitrate, 990Dinitrate Tablets, 991

IsotonicSalt Solution, 1397Sodium Chloride Injection, 1397Sodium Chloride Solution, 1397

Isoxsuprine Hydrochloride, 992Tablets, 993

Itraconazole, 994

J

JapaneseAngelica Root, 1668Angelica Root, Powdered, 1669Encephalitis Vaccine, 995Gentian, 1669Gentian, Powdered, 1669Valerian, 1670Valerian, Powdered, 1670

Josamycin, 995Propionate, 997Tablets, 996

Jujube, 1670Seed, 1671

Juzentaihoto Extract, 1671

K

Kainic Acidand Santonin Powder, 999Hydrate, 998

Kakkonto Extract, 1674Kallidinogenase, 1000Kamishoyosan Extract, 1676Kanamycin

Monosulfate, 1002Sulfate, 1003

Kaolin, 1004Keishibukuryogan Extract, 1679Ketamine Hydrochloride, 1005Ketoconazole, 1005

Cream, 1006Lotion, 1007Solution, 1008

Ketoprofen, 1008Fumarate, 1009

Kitasamycin, 1010Acetate, 1012Tartrate, 1013

Koi, 1681

L

Labetalol Hydrochloride, 1014Tablets, 1015

Lactic Acid, 1016


L-Lactic Acid, 1017Lactose, 1019

Anhydrous, 1018Hydrate, 1019

Lactulose, 1020Lanatoside C, 1021

Tablets, 1021Lanolin

Hydrous, 1022Purified, 1023

Lard, 1024Latamoxef Sodium, 1024Lauromacrogol, 1026Lemonades

Hydrochloric Acid, 926Lenampicillin Hydrochloride, 1026Leonurus Herb, 1682L-Leucine, 1028Leucomycin, 1010

Acetate, 1012Tartrate, 1013

Levallorphan Tartrate, 1029Injection, 1029

Levodopa, 1030Levofloxacin Hydrate, 1031Levomepromazine Maleate, 1032Levothyroxine Sodium


Lidocaine, 1034Hydrochloride Injection, 1035Injection, 1035

LightAnhydrous Silicic Acid, 1385Liquid Paraffin, 1214

Lilium Bulb, 1682Limaprost Alfadex, 1036Lincomycin Hydrochloride

Hydrate, 1037Injection, 1038

Lindera Root, 1683Liniments

Diphenhydramine, Phenol and ZincOxide, 737

Phenol and Zinc Oxide, 1237Liothyronine Sodium, 1039

Tablets, 1040Liquefied

Carbolic Acid, 1237Phenol, 1237Paraffin, 1213

LisinoprilHydrate, 1041Tablets, 1042

Lithium Carbonate, 1043Lithospermum Root, 1683Live Oral Poliomyelitis Vaccine, 1263Longan Aril, 1684Longgu, 1684

Powdered, 1684Lonicera Leaf and Stem, 1685Loquat Leaf, 1685Lorazepam, 1045

Losartan Potassium, 1045Lotions

Ketoconazole, 1007Sulfur and Camphor, 1440

Low Substituted Hydroxypropylcellu-lose, 937

Loxoprofen Sodium Hydrate, 1047Lycium

Bark, 1686Fruit, 1686

L-Lysine Acetate, 1048Lysine Hydrochloride, 1049L-Lysine Hydrochloride, 1049Lysozyme Hydrochloride, 1050

M

Macrogol400, 10501500, 10514000, 10526000, 105220000, 1053Ointment, 1053

MagnesiumCarbonate, 1054Oxide, 1055Silicate, 1056Stearate, 1057Sulfate Hydrate, 1058Sulfate Injection, 1059Sulfate Mixture, 1059

MagnoliaBark, 1687Bark, Powdered, 1687Flower, 1688

Mallotus Bark, 1688Maltose Hydrate, 1060Manidipine Hydrochloride, 1061

Tablets, 1062D-Mannite Injection, 1064D-Mannitol, 1063

Injection, 1064Maprotiline Hydrochloride, 1065Meclofenoxate Hydrochloride, 1065Mecobalamin, 1066Medazepam, 1067Medicinal

Carbon, 1068Soap, 1069

Mefenamic Acid, 1069Mefloquine Hydrochloride, 1070Mefruside, 1071

Tablets, 1072Meglumine, 1073

Iotalamate Injection, 1073Sodium Amidotrizoate Injection,

1074Sodium Iodamide Injection, 1075

Melphalan, 1076Menatetrenone, 1077Mentha

Herb, 1689

Oil, 1689Water, 1690

dl-Menthol, 1078l-Menthol, 1079Mepenzolate Bromide, 1079Mepirizole, 785Mepitiostane, 1080Mepivacaine Hydrochloride, 1081

Injection, 1082Mequitazine, 1083Merbromin, 1084

Solution, 1085Mercaptopurine Hydrate, 1083Mercurochrome, 1084

Solution, 1085Meropenem


Mestranol, 1087Metenolone

Acetate, 1088Enanthate, 1089Enanthate Injection, 1089

Metformin Hydrochloride, 1090Tablets, 1091

Methamphetamine Hydrochloride,1091

L-Methionine, 1092Methotrexate, 1093

Capsules, 1093Methoxsalen, 1095Methyl

Parahydroxybenzoate, 1103Salicylate, 1106Salicylate Spirit, Compound, 1107

Methylbenactyzium Bromide, 1096Methylcellulose, 1096Methyldopa


dl-Methylephedrine Hydrochloride,1100

Powder, 1101Powder, 10z, 1101

Methylergometrine Maleate, 1101Tablets, 1102

Methylprednisolone, 1104Succinate, 1104

Methylrosanilinium Chloride, 1105Methyltestosterone, 1107

Tablets, 1108Meticrane, 1109Metildigoxin, 1110Metoclopramide, 1111

Tablets, 1112Metoprolol Tartrate, 1113

Tablets, 1113Metronidazole, 1114

Tablets, 1115Metyrapone, 1116Mexiletine Hydrochloride, 1117Miconazole, 1118

Nitrate, 1118


Microcrystalline Cellulose, 599Micronomicin Sulfate, 1119Midecamycin, 1120

Acetate, 1121Migrenin, 1122Minocycline Hydrochloride, 1123

for Injection, 1124Tablets, 1125

Mitomycin C, 1126for Injection, 1127

Mizoribine, 1127Tablets, 1128

Monobasic Calcium Phosphate Hy-drate, 505

Monosodium Trichloroethyl Phos-phate, 1531

Syrup, 1532Morphine

and Atropine Injection, 1130Hydrochloride Hydrate, 1131Hydrochloride Injection, 1131Hydrochloride Tablets, 1132

Mosapride CitrateHydrate, 1133Powder, 1134Tablets, 1136

Moutan Bark, 1690Powdered, 1691

Mukoi-Daikenchuto Extract, 1691Mulberry Bark, 1693Mupirocin Calcium

Hydrate, 1137Ointment, 1139

N

Nabumetone, 1139Tablets, 1141

Nadolol, 1142Nafamostat Mesilate, 1143Nalidixic Acid, 1144Naloxone Hydrochloride, 1145Naphazoline

and Chlorpheniramine Solution,1146

Hydrochloride, 1145Nitrate, 1146

Naproxen, 1147Narcotine, 1179

Hydrochloride, 1180Natamycin, 1246Nateglinide, 1148

Tablets, 1149Natural Aluminum Silicate, 354Nelumbo Seed, 1693Neomycin Sulfate, 869Neostigmine Methylsulfate, 1151

Injection, 1151Nicardipine Hydrochloride, 1152

Injection, 1153Nicergoline, 1154

Powder, 1155Tablets, 1156

Niceritrol, 1157Nicomol, 1158

Tablets, 1159Nicorandil, 1159Nicotinamide, 1160Nicotinic Acid, 1161

Injection, 1162Nifedipine, 1162Nilvadipine, 1163

Tablets, 1164Nitrazepam, 1166Nitrendipine, 1166

Tablets, 1167Nitrogen, 1168Nitroglycerin Tablets, 1169Nitrous Oxide, 1170Nizatidine, 1171

Capsules, 1172Noradrenaline, 1173

Hydrochloride Injection, 1174Injection, 1174

Norepinephrine, 1173Hydrochloride Injection, 1174Injection, 1174

Norethisterone, 1175Norfloxacin, 1175Norgestrel, 1176

and Ethinylestradiol Tablets, 1177Nortriptyline Hydrochloride, 1178Noscapine, 1179

Hydrochloride Hydrate, 1180Notopterygium, 1694Nuphar Rhizome, 1694Nutmeg, 1695Nux Vomica, 1695

Extract, 1696Extract Powder, 1696Tincture, 1697

Nystatin, 1180

O

Ofloxacin, 1181Ointments

Acrinol and Zinc Oxide, 330Betamethasone Valerate and Gen-

tamicin Sulfate, 452Hydrocortisone and Diphenhydra-

mine, 929Macrogol, 1053Mupirocin Calcium, 1139Polyethylene Glycol, 1053Simple, 1388Sulfur, Salicylic Acid and Thianthol,

1441White, 1575Zinc Oxide, 1586

Olive Oil, 1182Omeprazole, 1182Operidine, 1230

Injection, 1231Ophiopogon Tuber, 1698

Ophthalmic SolutionDibekacin Sulfate, 709Gentamicin Sulfate, 882Idoxuridine, 947Silver Nitrate, 1387Zinc Sulfate, 1587

OpiumIpecac Powder, 1698Powder, Diluted, 1184Powdered, 1183Tincture, 1184

Opium Alkaloidsand Atropine Injection, 1187and Scopolamine Injection, 1188Hydrochlorides, 1185Hydrochlorides Injection, 1186

OrangeOil, 1190Peel Syrup, 1612Peel Tincture, 1612

Orciprenaline Sulfate, 1191Orengedokuto Extract, 1698Oriental Bezoar, 1700Oxapium Iodide, 1191Oxaprozin, 1192Oxazolam, 1193Oxetacaine, 1194Oxethazaine, 1194Oxprenolol Hydrochloride, 1194Oxybuprocaine Hydrochloride, 1195Oxycodone

and Atropine Injection, Compound,1197

Hydrochloride Hydrate, 1196Injection, Compound, 1197

Oxydol, 1199Oxygen, 1199Oxymetholone, 1200Oxytetracycline Hydrochloride, 1201Oxytocin, 1203

Injection, 1205Oyster Shell, 1701

Powdered, 1701Ozagrel Sodium, 1206

for Injection, 1207

P

Panax Japonicus Rhizome, 1702Powdered, 1702

Pancreatin, 1207Pancuronium Bromide, 1208Panipenem, 1208Pantethine, 1210Papaverine Hydrochloride, 1211

Injection, 1212Paracetamol, 318Paraffin, 1212

Light Liquid, 1214Liquid, 1213

Paraformaldehyde, 1214Paste, Dental, 1215

Parnaparin Sodium, 1216


Pas-calciumGranules, 503Hydrate, 502

PasteArsenical, 396Paraformaldehyde, Dental, 1215Triozinc, Dental, 1540

Peach Kernel, 1702Powdered, 1703

Peanut Oil, 1218Pemirolast Potassium, 1218

for Syrup, 1219Tablets, 1220

Penbutolol Sulfate, 1221Penicillin G Potassium, 436Pentazocine, 1221Pentobarbital Calcium, 1222Pentoxyverine Citrate, 1223Peony Root, 1704

Powdered, 1705Peplomycin Sulfate, 1224

for Injection, 1226Perilla Herb, 1706Perphenazine, 1226

Maleate, 1228Maleate Tablets, 1228Tablets, 1227

Pethidine Hydrochloride, 1230Injection, 1231

PetrolatumHydrophilic, 1231White, 1232Yellow, 1232

Petroleum Benzin, 1233Peucedanum Root, 1706Pharbitis Seed, 1707Phellodendron

Albumin Tannate and Bismuth Sub-nitrate Powder, 1709

Bark, 1707Bark, Powdered, 1708Powder for Cataplasm, Compound,

1709Phenazone, 388Phenethicillin Potassium, 1233Phenobarbital, 1234

Powder, 1235Powder, 10z, 1235

Phenol, 1236and Zinc Oxide Liniment, 1237for Disinfection, 1236Liquefied, 1237with Camphor, Dental, 1237

Phenolated Water, 1238for Disinfection, 1238

Phenolsulfonphthalein, 1238Injection, 1239

L-Phenylalanine, 1240Phenylbutazone, 1240Phenylephrine Hydrochloride, 1241Phenytoin, 1242

Powder, 1242Sodium for Injection, 1244

Tablets, 1243Phytomenadione, 1244Phytonadione, 1244Picrasma Wood, 1710

Powdered, 1710Pilocarpine Hydrochloride, 1245Pimaricin, 1246Pimozide, 1247Pindolol, 1248Pinellia Tuber, 1711Pioglitazone Hydrochloride, 1249

Tablets, 1250Pipemidic Acid Hydrate, 1251Piperacillin

Hydrate, 1252Sodium, 1254Sodium for Injection, 1255

PiperazineAdipate, 1256Phosphate Hydrate, 1256Phosphate Tablets, 1257

Pirarubicin, 1258Pirenoxine, 1259Pirenzepine Hydrochloride Hydrate,

1260Piroxicam, 1261Pivmecillinam Hydrochloride, 1262

Tablets, 1263Plantago

Herb, 1711Seed, 1711

PlatycodonFluidextract, 1712Root, 1712Root, Powdered, 1712

Pogostemon Herb, 1713Polyethylene Glycol

400, 10501500, 10514000, 10526000, 105220000, 1053Ointment, 1053

Polygala Root, 1713Powdered, 1714

Polygonatum Rhizome, 1714Polygonum Root, 1714Polymixin B Sulfate, 1263Polyoxyethylene Lauryl Alcohol Ether,

1026Polyoxyl 40 Stearate, 1264Polyporus Sclerotium, 1715

Powdered, 1715Polysorbate 80, 1265Polyvidone, 1273Polyvinylpyrrolidone, 1273Poria Sclerotium, 1715

Powdered, 1716Potash Soap, 1265Potassium

Bromide, 1265Canrenoate, 1266Carbonate, 1267

Chloride, 1267Clavulanate, 1268Guaiacolsulfonate, 1269Hydroxide, 1270Iodide, 1270Permanganate, 1271Sulfate, 1272

Potato Starch, 1272Povidone, 1273

-Iodine, 1275Powder

Ascorbic Acid, 398Chlordiazepoxide, 613Chlorpheniramine and Calcium,

621Chlorpheniramine Maleate, 623Codeine Phosphate, 1z, 671Codeine Phosphate, 10z, 672Compound Diastase and Sodium Bi-

carbonate, 706Compound Phellodendron, for

Cataplasm, 1709Compound Rhubarb and Senna,

1725Compound Scopolia Extract and Di-

astase, 1744Compound Vitamin B, 1567Diastase and Sodium Bicarbonate,

706Dihydrocodeine Phosphate, 1z,

724Dihydrocodeine Phosphate, 10z,

724Diluted Opium, 1184Diphenhydramine and

Bromovalerylurea, 736Diphenylhydantoin, 1242Ephedrine Hydrochloride, 783Ephedrine Hydrochloride, 10z,

783Famotidine, 825Gentian and Sodium Bicarbonate,

1645Hydralazine Hydrochloride, 924Kainic Acid and Santonin, 999dl-Methylephedrine Hydrochloride,

1101dl-Methylephedrine Hydrochloride,

10z, 1101Mosapride Citrate, 1134Nicergoline, 1155Nux Vomica Extract, 1696Opium Ipecac, 1698Phellodendron, Albumin Tannate

and Bismuth Subnitrate, 1709Phenobarbital, 1235Phenobarbital, 10z, 1235Phenytoin, 1242Reserpine, 1339Reserpine, 0.1z, 1339Riboflavin, 1342Salicylated Alum, 1371Scopolia Extract and Carbon, 1744


Powder (continued)Scopolia Extract and Ethyl

Aminobenzoate, 1744Scopolia Extract, 1743Scopolia Extract, Papaverine and

Ethyl Aminobenzoate, 1745Swertia and Sodium Bicarbonate,

1765Thiamine Chloride Hydrochloride,

1485Vitamin B1 Hydrochloride, 1485Vitamin B2, 1342Vitamin C, 398Zinc Oxide Starch, 1586

PowderedAcacia, 1593Agar, 1595Alisma Rhizome, 1596Aloe, 1597Amomum Seed, 1599Atractylodes Lancea Rhizome,

1605Atractylodes Rhizome, 1606Calumba, 1615Capsicum, 1616Cellulose, 602Cinnamon Bark, 1623Clove, 1625Cnidium Rhizome, 1627Coix Seed, 1627Coptis Rhizome, 1629Corydalis Tuber, 1632Cyperus Rhizome, 1633Dioscorea Rhizome, 1636Fennel, 1639Gambir, 1641Gardenia Fruit, 1642Gentian, 1644Geranium Herb, 1645Ginger, 1646Ginseng, 1647Glycyrrhiza, 1650Ipecac, 1667Japanese Angelica Root, 1669Japanese Gentian, 1669Japanese Valerian, 1670Longgu, 1684Magnolia Bark, 1687Moutan Bark, 1691Opium, 1183Oyster Shell, 1701Panax Japonicus Rhizome, 1702Peach Kernel, 1703Peony Root, 1705Phellodendron Bark, 1708Picrasma Wood, 1710Platycodon Root, 1712Polygala Root, 1714Polyporus Sclerotium, 1715Poria Sclerotium, 1716Processed Aconite Root, 1718Rhubarb, 1724Rose Fruit, 1728

Scutellaria Root, 1748Senega, 1749Senna Leaf, 1751Smilax Rhizome, 1762Sophora Root, 1763Sweet Hydrangea Leaf, 1763Swertia Herb, 1765Tragacanth, 1767Turmeric, 1769Zanthoxylum Fruit, 1771

Pranoprofen, 1276Pravastatin Sodium, 1277

Fine Granules, 1278Solution, 1279Tablets, 1281

Prazepam, 1282Tablets, 1283

Prazosin Hydrochloride, 1284Precipitated Calcium Carbonate, 495


Prednisolone, 1285Acetate, 1287Sodium Phosphate, 1288Sodium Succinate for Injection,

1290Succinate, 1289Tablets, 1286

Primidone, 1291Probenecid, 1292

Tablets, 1292Probucol, 1293


Procainamide Hydrochloride, 1296Injection, 1296Tablets, 1297

Procaine Hydrochloride, 1298Injection, 1299

Procarbazine Hydrochloride, 1299Procaterol Hydrochloride Hydrate,

1300Processed

Aconite Root, 1716Aconite Root, Powdered, 1718Ginger, 1719

Prochlorperazine Maleate, 1301Tablets, 1302

Progesterone, 1303Injection, 1304

Proglumide, 1304L-Proline, 1305Promethazine Hydrochloride, 1306Propafenone Hydrochloride, 1307

Tablets, 1308Propantheline Bromide, 1309Propiverine Hydrochloride, 1310

Tablets, 1311Propranolol Hydrochloride, 1312

Tablets, 1313Propyl Parahydroxybenzoate, 1314Propylene Glycol, 1315Propylthiouracil, 1315

Tablets, 1316Propyphenazone, 989Prostaglandin

E1, 349E1 a-Cyclodextrin Clathrate Com-

pound, 352F2a, 735

Protamine Sulfate, 1316Injection, 1317

Prothionamide, 1318Protirelin, 1318

Tartrate Hydrate, 1319Prunella Spike, 1720Pueraria Root, 1720Pullulan, 1320Purified

Dehydrocholic Acid, 695Gelatin, 880Lanolin, 1023Shellac, 1383Sodium Hyaluronate, 1401Water, 1572Water in Containers, 1572Water in Containers, Sterile, 1573

Pyrantel Pamoate, 1321Pyrazinamide, 1322Pyridostigmine Bromide, 1322Pyridoxine Hydrochloride, 1323

Injection, 1324Pyroxylin, 1324Pyrrolnitrin, 1325

Q

Quercus Bark, 1721Quick Lime, 501Quinapril Hydrochloride, 1325

Tablets, 1326Quinidine Sulfate Hydrate, 1328Quinine

Ethyl Carbonate, 1329Hydrochloride Hydrate, 1330Sulfate Hydrate, 1331

R

Rabeprazole Sodium, 1332Ranitidine Hydrochloride, 1333Rape Seed Oil, 1335Rebamipide, 1335

Tablets, 1336Red Ginseng, 1721Rehmannia Root, 1722Reserpine, 1337

Injection, 1338Powder, 1339Powder, 0.1z, 1339Tablets, 1339

RetinolAcetate, 1340Palmitate, 1341

Rhubarb, 1723and Senna Powder, Compound,


1725Powdered, 1724

Riboflavin, 1341Butyrate, 1343Phosphate, 1344Phosphate Injection, 1345Powder, 1342Sodium Phosphate, 1344Sodium Phosphate Injection, 1345

Ribostamycin Sulfate, 1345Rice Starch, 1346Rifampicin, 1347

Capsules, 1348Rikkunshito Extract, 1725Ringer's Solution, 1350Risperidone, 1351

Fine Granules, 1351Oral Solution, 1353Tablets, 1354

Ritodrine Hydrochloride, 1355Tablets, 1357

Rokitamycin, 1358Tablets, 1359

Rose Fruit, 1727Powdered, 1728

Rosin, 1728Roxatidine Acetate Hydrochloride,

1360Extended-release Capsules, 1361Extended-release Tablets, 1362for Injection, 1363

Roxithromycin, 1364Royal Jelly, 1728Ryokeijutsukanto Extract, 1729

S

Saccharated Pepsin, 1366Saccharin, 1366

Sodium, 1367Sodium Hydrate, 1367

Safflower, 1731Saffron, 1731Saibokuto Extract, 1732Saikokeishito Extract, 1734Saireito Extract, 1737Salazosulfapyridine, 1368Salbutamol Sulfate, 1369Salicylated Alum Powder, 1371Salicylic Acid, 1370

Adhesive Plaster, 1372Spirit, 1372Spirit, Compound, 1372

Santonin, 1373Saponated Cresol Solution, 681Saposhnikovia Root and Rhizome,

1740Sappan Wood, 1740Sarpogrelate Hydrochloride, 1374


Saussurea Root, 1740Schisandra Fruit, 1741

Schizonepeta Spike, 1741Scopolamine

Butylbromide, 1378Hydrobromide Hydrate, 1379

ScopoliaExtract, 1742Extract and Carbon Powder, 1744Extract and Diastase Powder, Com-

pound, 1744Extract and Ethyl Aminobenzoate

Powder, 1744Extract and Tannic Acid Supposito-

ries, 1746Extract Powder, 1743Extract, Papaverine and Ethyl

Aminobenzoate Powder, 1745Rhizome, 1741

Scutellaria Root, 1747Powdered, 1748

Senega, 1749Powdered, 1749Syrup, 1749

Senna Leaf, 1750Powdered, 1751

L-Serine, 1380Serrapeptase, 1380Serum Gonadotrophin, 901

for Injection, 903Sesame, 1752

Oil, 1381Sevoflurane, 1382Shakuyakukanzoto Extract, 1752Shellac

Purified, 1383White, 1384

Shimbuto Extract, 1753Shosaikoto Extract, 1756Shoseiryuto Extract, 1758Siccanin, 1384Silver

Nitrate, 1386Nitrate Ophthalmic Solution, 1387Protein, 1387Protein Solution, 1387

SimpleOintment, 1388Syrup, 1388

Simvastatin, 1388Sinomenium Stem and Rhizome,

1761Slaked Lime, 500Smilax Rhizome, 1762

Powdered, 1762Sodium

Acetate Hydrate, 1390Aurothiomalate, 1391Benzoate, 1392Bicarbonate, 1392Bicarbonate and Bitter Tincture Mix-

ture, 1762Bicarbonate Injection, 1393Bisulfite, 1393Borate, 1394

Bromide, 1394Carbonate Hydrate, 1395Chloride, 1396Chloride Injection, 0.9z, 1397Chloride Injection, 10z, 1397Chromate (51Cr) Injection, 1398Citrate Hydrate, 1398Citrate Injection for Transfusion,

1398Cromoglicate, 1399Fusidate, 1401Hydrogen Carbonate, 1392Hydrogen Sulfite, 1393Hydroxide, 1403Iodide, 1404Iodide (123I) Capsules, 1404Iodide (131I) Capsules, 1404Iodide (131I) Solution, 1404Iodohippurate (131I) Injection, 1405Iotalamate Injection, 1405L-Lactate Solution, 1406Lauryl Sulfate, 1407Metabisulfite, 1411Pertechnetate (99mTc) Injection,

1408Picosulfate Hydrate, 1409Polystyrene Sulfonate, 1409Prasterone Sulfate Hydrate, 1411Pyrosulfite, 1411Risedronate Hydrate, 1412Risedronate Tablets, 1414Salicylate, 1415Starch Glycolate, 1415Thiosulfate Hydrate, 1417Thiosulfate Injection, 1417Valproate, 1418Valproate Syrup, 1419Valproate Tablets, 1419

SolutionAdrenaline, 332Alum, 358Benzalkonium Chloride, 429Benzethonium Chloride, 431Butenafine Hydrochloride, 484Chlorhexidine Gluconate, 616Compound Thianthol and Salicylic

Acid, 1488Cresol, 680Dental Sodium Hypochlorite, 387Diagnostic Sodium Citrate, 1399Epinephrine, 332Glycerin and Potash, 894Isotonic Salt, 1397Isotonic Sodium Chloride, 1397Ketoconazole, 1008Merbromin, 1085Mercurochrome, 1085Naphazoline and Chlorpheniramine,

1146Pravastatin Sodium, 1279Ringer's, 1350Risperidone Oral, 1353Saponated Cresol, 681


Solution (continued)Silver Protein, 1387Sodium Iodide (131I), 1404Sodium L-Lactate, 1406D-Sorbitol, 1421Terbinafine Hydrochloride, 1474Tolnaftate, 1514

Sophora Root, 1762Powdered, 1763

Sorbitan Sesquioleate, 1420D-Sorbitol, 1421

Solution, 1421Soybean Oil, 1422Spectinomycin Hydrochloride Hydrate,

1423Spiramycin Acetate, 1424Spirit

Capsicum and Salicylic Acid, 1618Compound Methyl Salicylate, 1107Compound Salicylic Acid, 1372Foeniculated Ammonia, 1640Iodine, Salicylic Acid and Phenol,

969Salicylic Acid, 1372

Spironolactone, 1425Tablets, 1425

SprayButenafine Hydrochloride, 485

StarchCorn, 678Potato, 1272Rice, 1346Wheat, 1574

Stearic Acid, 1426Stearyl Alcohol, 1427Sterile

Purified Water in Containers, 1573Water for Injection in Containers,

1573Streptomycin Sulfate, 1427

for Injection, 1428Sucralfate Hydrate, 1429Sucrose, 1430Sulbactam Sodium, 1433Sulbenicillin Sodium, 1434Sulfadiazine Silver, 1435Sulfafurazole, 1438Sulfamethizole, 1436Sulfamethoxazole, 1436Sulfamonomethoxine Hydrate, 1437Sulfasalazine, 1368Sulfisomezole, 1436Sulfisoxazole, 1438Sulfobromophthalein Sodium, 1438

Injection, 1439Sulfur, 1440

and Camphor Lotion, 1440Salicylic Acid and Thianthol Oint-

ment, 1441Sulindac, 1441Sulpiride, 1442

Capsules, 1442Tablets, 1443

SulpyrineHydrate, 1444Injection, 1444

Sultamicillin Tosilate Hydrate, 1445Sultiame, 1447Suppositories

Bisacodyl, 460Indometacin, 962Scopolia Extract and Tannic Acid,

1746Suxamethonium Chloride

for Injection, 1448Hydrate, 1447Injection, 1449

Sweet Hydrangea Leaf, 1763Powdered, 1763

Swertiaand Sodium Bicarbonate Powder,

1765Herb, 1764Herb, Powdered, 1765

SyntheticAluminum Silicate, 356Camphor, 510

SyrupAciclovir, 326Aciclovir for, 325Amphotericin B, 382Cefadroxil for, 535Cefalexin for, 539Cefatrizine Propylene Glycolate for,

542Cefroxadine for, 584Faropenem Sodium for, 828Ipecac, 1667Monosodium Trichloroethyl Phos-

phate, 1532Orange Peel, 1612Pemirolast Potassium for, 1219Senega, 1749Simple, 1388Sodium Valproate, 1419Triclofos Sodium, 1532

T

TabletsAcemetacin, 317Acetylsalicylic Acid, 400Ajmaline, 334Alacepril, 336Alendronate Sodium, 342Allopurinol, 344Alminoprofen, 346Amiodarone Hydrochloride, 366Amitriptyline Hydrochloride, 367Amlexanox, 370Amlodipine Besilate, 372Amosulalol Hydrochloride, 376Amphotericin B, 382Aspirin, 400Atorvastatin Calcium, 405Azathioprine, 409

Baclofen, 418Benidipine Hydrochloride, 425Beraprost Sodium, 439Betahistine Mesilate, 443Betamethasone, 446Bezafibrate Sustained Release, 456Bisoprolol Fumarate, 463Bucillamine, 473Buformin Hydrochloride Enteric-

coated, 476Buformin Hydrochloride, 478Cadralazine, 488Candesartan Cilexetil, 512Carvedilol, 526Cefcapene Pivoxil Hydrochloride,

551Cefditoren Pivoxil, 556Cefteram Pivoxil, 591Cetirizine Hydrochloride, 607Chlordiazepoxide, 614Chlorphenesin Carbamate, 620Chlorpheniramine Maleate, 624Chlorpromazine Hydrochloride,

627Chlorpropamide, 629Cibenzoline Succinate, 632Cilazapril, 638Cilostazol, 640Clarithromycin, 647Clomifene Citrate, 660Codeine Phosphate, 672Diazepam, 707Dichlorphenamide, 712Diclofenamide, 712Diethylcarbamazine Citrate, 714Digitoxin, 718Digoxin, 721Dimenhydrinate, 732Diphenylhydantoin, 1243Distigmine Bromide, 741Donepezil Hydrochloride, 747Doxazosin Mesilate, 752Dydrogesterone, 762Ebastine Orally Disintegrating, 764Ebastine, 765Emorfazone, 774Enalapril Maleate, 777Ephedrine Hydrochloride, 784Ergometrine Maleate, 789Erythromycin Enteric-Coated, 792Estriol, 799Etacrynic Acid, 801Ethinylestradiol, 806Etidronate Disodium, 814Etilefrine Hydrochloride, 815Etizolam, 819Famotidine, 826Faropenem Sodium, 829Flecainide Acetate, 838Flutoprazepam, 858Fluvoxamine Maleate, 860Folic Acid, 863Furosemide, 873


Glimepiride, 886Griseofulvin, 905Haloperidol, 911Hydralazine Hydrochloride, 924Imidapril Hydrochloride, 950Imipramine Hydrochloride, 955Indapamide, 957Ipriflavone, 976Irsogladine Maleate, 978Isoniazid, 986Isosorbide Dinitrate, 991Isoxsuprine Hydrochloride, 993Josamycin, 996Labetalol Hydrochloride, 1015Lanatoside C, 1021Levothyroxine Sodium, 1033Liothyronine Sodium, 1040Lisinopril, 1042Manidipine Hydrochloride, 1062Mefruside, 1072Metformin Hydrochloride, 1091Methyldopa, 1099Methylergometrine Maleate, 1102Methyltestosterone, 1108Metoclopramide, 1112Metoprolol Tartrate, 1113Metronidazole, 1115Minocycline Hydrochloride, 1125Mizoribine, 1128Morphine Hydrochloride, 1132Mosapride Citrate, 1136Nabumetone, 1141Nateglinide, 1149Nicergoline, 1156Nicomol, 1159Nilvadipine, 1164Nitrendipine, 1167Nitroglycerin, 1169Norgestrel and Ethinylestradiol,

1177Pemirolast Potassium, 1220Perphenazine Maleate, 1228Perphenazine, 1227Phenytoin, 1243Pioglitazone Hydrochloride, 1250Piperazine Phosphate, 1257Pivmecillinam Hydrochloride, 1263Pravastatin Sodium, 1281Prazepam, 1283Precipitated Calcium Carbonate,

496Prednisolone, 1286Probenecid, 1292Probucol, 1295Procainamide Hydrochloride, 1297Prochlorperazine Maleate, 1302Propafenone Hydrochloride, 1308Propiverine Hydrochloride, 1311Propranolol Hydrochloride, 1313Propylthiouracil, 1316Quinapril Hydrochloride, 1326Rebamipide, 1336Reserpine, 1339

Risperidone, 1354Ritodrine Hydrochloride, 1357Rokitamycin, 1359Roxatidine Acetate Hydrochloride

Extended-release, 1362Sarpogrelate Hydrochloride, 1377Sodium Risedronate, 1414Sodium Valproate, 1419Spironolactone, 1425Sulpiride, 1443Tamsulosin Hydrochloride Exten-

ded-release, 1455Temocapril Hydrochloride, 1470Thiamazole, 1482Tiapride Hydrochloride, 1495Tiaramide Hydrochloride, 1496Tipepidine Hibenzate, 1502Tolbutamide, 1513Tosufloxacin Tosilate, 1517Tranexamic Acid, 1521Trichlormethiazide, 1528Trihexyphenidyl Hydrochloride,

1533Trimetazidine Hydrochloride, 1536Trimethadione, 1538Troxipide, 1542Ursodeoxycholic Acid, 1555Verapamil Hydrochloride, 1562Voglibose, 1568Warfarin Potassium, 1571Zaltoprofen, 1582Zolpidem Tartrate, 1591

Tacrolimus Hydrate, 1449Talampicillin Hydrochloride, 1450Talc, 1451Tamoxifen Citrate, 1453Tamsulosin Hydrochloride, 1454

Extended-release Tablets, 1455Tannic Acid, 1456Tartaric Acid, 1456Taurine, 1457Tazobactam, 1457Teceleukin

for Injection (Genetical Recombina-tion), 1465

(Genetical Recombination), 1459Tegafur, 1465Teicoplanin, 1466Temocapril Hydrochloride, 1469

Tablets, 1470Teprenone, 1471Terbinafine Hydrochloride, 1472

Cream, 1473Solution, 1474Spray, 1475

Terbutaline Sulfate, 1475Testosterone

Enanthate, 1476Enanthate Injection, 1477Propionate, 1477Propionate Injection, 1478

Tetracaine Hydrochloride, 1479Tetracycline Hydrochloride, 1480

Thallium (201Tl) Chloride Injection,1481

Theophylline, 1481Thiamazole, 1482

Tablets, 1482Thiamine Chloride Hydrochloride,

1483Injection, 1484Powder, 1485

Thiamine Nitrate, 1485Thiamylal Sodium, 1486

for Injection, 1487Thianthol, 1488

and Salicylic Acid Solution, Com-pound, 1488

Thiopental Sodium, 1489for Injection, 1490

Thioridazine Hydrochloride, 1491Thiotepa, 1491L-Threonine, 1492Thrombin, 1492Thymol, 1493Tiapride Hydrochloride, 1494

Tablets, 1495Tiaramide Hydrochloride, 1496

Tablets, 1496Ticlopidine Hydrochloride, 1497Timepidium Bromide Hydrate, 1498Timolol Maleate, 1499Tincture

Bitter, 1613Capsicum, 1617Iodine, 966Iodine, Dilute, 967Nux Vomica, 1697Opium, 1184Orange Peel, 1612

Tinidazole, 1500Tipepidine Hibenzate, 1500

Tablets, 1502Titanium Oxide, 1503Tizanidine Hydrochloride, 1504Toad Venom, 1766Tobramycin, 1505

Injection, 1506Tocopherol, 1506

Acetate, 1507Calcium Succinate, 1508Nicotinate, 1509

dl-a-Tocopherol, 1506Acetate, 1507Nicotinate, 1509

Todralazine Hydrochloride Hydrate,1510

Tofisopam, 1511Tolazamide, 1512Tolbutamide, 1513

Tablets, 1513Tolnaftate, 1514

Solution, 1514Tolperisone Hydrochloride, 1515Tosufloxacin Tosilate

Hydrate, 1516


Tablets, 1517Tragacanth, 1767

Powdered, 1767Tranexamic Acid, 1518

Capsules, 1519Injection, 1520Tablets, 1521

Trapidil, 1521Trehalose Hydrate, 1522Trepibutone, 1523Tretoquinol Hydrochloride, 1539Triamcinolone, 1524

Acetonide, 1525Triamterene, 1526Tribulus Fruit, 1767Trichlormethiazide, 1527

Tablets, 1528Trichomycin, 1530Trichosanthes Root, 1768Triclofos Sodium, 1531

Syrup, 1532Trihexyphenidyl Hydrochloride, 1533

Tablets, 1533Trimebutine Maleate, 1534Trimetazidine Hydrochloride, 1535

Tablets, 1536Trimethadione, 1538

Tablets, 1538Trimetoquinol Hydrochloride Hydrate,

1539Tropicamide, 1540Troxipide, 1541


L-Tryptophan, 1543Tulobuterol Hydrochloride, 1544Turmeric, 1768

Powdered, 1769Turpentine Oil, 1545L-Tyrosine, 1545

U

Ubenimex, 1546Capsules, 1547

Ubidecarenone, 1548Ulinastatin, 1549Uncaria Hook, 1770Urapidil, 1551Urea, 1552Urokinase, 1552Ursodeoxycholic Acid, 1554

Granules, 1554Tablets, 1555

Uva Ursi Fluidextract, 1609

V

VaccineBCG, Freeze-dried, (for Percutane-

ous Use), 421Cholera, 630

Diphtheria-Purified Pertussis-Teta-nus Combined, Adsorbed, 739

Hepatitis B, Adsorbed, 909Influenza HA, 963Inactivated Tissue Culture Rabies,

Freeze-dried, 1333Japanese Encephalitis, 995Japanese Encephalitis, Freeze-dried,

995Live Attenuated Measles, Freeze-

dried, 1065Live Attenuated Mumps, Freeze-

dried, 1137Live Attenuated Rubella, Freeze-

dried, 1365Live Oral Poliomyelitis, 1263Purified Pertussis, Adsorbed, 1230Smallpox, Freeze-dried, 1390Smallpox, Freeze-dried, Prepared in

Cell Culture, 1390Weil's Disease and Akiyami Com-

bined, 1574L-Valine, 1557Vancomycin Hydrochloride, 1558

for Injection, 1559Vasopressin Injection, 1560Verapamil Hydrochloride, 1561

Tablets, 1562Vinblastine Sulfate, 1563

for Injection, 1564Vincristine Sulfate, 1565Vitamin A

Acetate, 1340Capsules, 1566Oil, 1566Oil Capsules, 1566Palmitate, 1341

Vitamin BPowder, Compound, 1567

Vitamin B1

Hydrochloride, 1483Hydrochloride Injection, 1484Hydrochloride Powder, 1485Nitrate, 1485

Vitamin B2, 1341Phosphate Ester, 1344Phosphate Ester Injection, 1345Powder, 1342

Vitamin B6, 1323Injection, 1324

Vitamin B12, 684Injection, 685

Vitamin C, 397Injection, 398Powder, 398

Vitamin D2, 787Vitamin D3, 630Vitamin E, 1506

Acetate, 1507Calcium Succinate, 1598Nicotinate, 1509

Vitamin K1, 1244

Voglibose, 1567Tablets, 1568

W

Warfarin Potassium, 1569Tablets, 1571

Water, 1572for Injection, 1572for Injection in Containers, Sterile,

1573in Containers, Purified, 1572in Containers, Sterile Purified,

1573Purified, 1572

Weak Opium Alkaloids and Scopola-mine Injection, 1189

Weil's Disease and Akiyami CombinedVaccine, 1574

Wheat Starch, 1574White

Beeswax, 422Ointment, 1575Petrolatum, 1232Shellac, 1384Soft Sugar, 1432

Whole Human Blood, 1575Wine, 1575Wood Creosote, 1577

X

Xylitol, 1579Injection, 1580

Y

YellowBeeswax, 423Petrolatum, 1232

Z

Zaltoprofen, 1581Tablets, 1582

Zanthoxylum Fruit, 1771Powdered, 1771

Zedoary, 1771Zidovudine, 1583Zinc

Chloride, 1584Oxide, 1585Oxide Oil, 1585Oxide Ointment, 1586Oxide Starch Powder, 1586Sulfate Hydrate, 1586Sulfate Ophthalmic Solution, 1587

Zinostatin Stimalamer, 1588Zolpidem Tartrate, 1590

Tablets, 1591

23072307

INDEX IN LATIN NAME

A

Achyranthis Radix, 1594Adeps

Lanae Purificatus, 1023Suillus, 1024

Agar, 1594Pulveratum, 1595

Akebiae Caulis, 1595Alismatis Rhizoma, 1595

Rhizoma Pulveratum, 1596Aloe, 1596

Pulverata, 1597Alpiniae

Fructus, 1611Officinari Rhizoma, 1598

Amomi Semen, 1599Semen Pulveratum, 1599

AmylumMaydis, 678Oryzae, 1346Solani, 1272Tritici, 1574

Anemarrhenae Rhizoma, 1599Angelicae

Dahuricae Radix, 1599Radix, 1668Radix Pulverata, 1669

Apilac, 1728Araliae Cordatae Rhizoma, 1601Arctii Fructus, 1614Arecae Semen, 1602Armeniacae Semen, 1600Artemisiae Capillaris Flos, 1602Asiasari Radix, 1602Asparagi Tuber, 1603Astragali Radix, 1604Atractylodis

Lanceae Rhizoma, 1604Lanceae Rhizoma Pulveratum,

1605Rhizoma, 1605Rhizoma Pulveratum, 1606

AurantiiFructus Immaturus, 1665Nobilis Pericarpium, 1624Pericarpium, 1612

B

Belladonnae Radix, 1609Benincasae Semen, 1611Benzoinum, 1611Bezoar Bovis, 1700Bufonis Venenum, 1766

Bupleuri Radix, 1613

C

Calumbae Radix, 1615Radix Pulverata, 1615

Cannabis Fructus, 1661Capsici Fructus, 1615

Fructus Pulveratus, 1616Cardamomi Fructus, 1618Carthami Flos, 1731Caryophylli Flos, 1625

Flos Pulveratus, 1625Cassiae Semen, 1618Catalpae Fructus, 1619Cera

Alba, 422Carnauba, 522Flava, 423

Chrysanthemi Flos, 1622Cimicifugae Rhizoma, 1622Cinnamomi Cortex, 1623

Cortex Pulveratus, 1623Clematidis Radix, 1625Cnidii

Monnieris Fructus, 1626Rhizoma, 1626Rhizoma Pulveratum, 1627

Coicis Semen, 1627Semen Pulveratum, 1627

Condurango Cortex, 1628Coptidis Rhizoma, 1628

Rhizoma Pulveratum, 1629Corni Fructus, 1630Corydalis Tuber, 1631

Tuber Pulveratum, 1632Crataegi Fructus, 1632Crocus, 1731Curcumae Rhizoma, 1768

Rhizoma Purveratum, 1769Cyperi Rhizoma, 1633

Rhizoma Pulveratum, 1633

D

Digenea, 1635Dioscoreae Rhizoma, 1635

Rhizoma Pulveratum, 1636Dolichi Semen, 1636

E

Eleutherococci senticosi Rhizoma,1636

Ephedrae Herba, 1637Epimedii Herba, 1638

Eriobotryae Folium, 1685Eucommiae Cortex, 1638Euodiae Fructus, 1639

F

Fel Ursi, 1608Foeniculi Fructus, 1639

Fructus Pulveratus, 1639Forsythiae Fructus, 1640Fossilia Ossis Mastodi, 1684

Ossis Mastodi Pulveratus, 1685Fritillariae Bulbus, 1641

G

Gambir, 1641Pulveratum, 1641

Gardeniae Fructus, 1642Fructus Pulveratus, 1642

Gastrodiae Tuber, 1643Gentianae

Radix, 1644Radix Pulverata, 1644Scabrae Radix, 1669Scabrae Radix Pulverata, 1669

Geranii Herba, 1645Herba Pulverata, 1645

Ginseng Radix, 1646Radix Pulverata, 1647Radix Rubra, 1721

Glehniae Radix cum Rhizoma, 1649Glycyrrhizae Radix, 1649

Radix Pulverata, 1650Gummi Arabicum, 1593

Arabicum Pulveratum, 1593Gypsum Fibrosum, 1647

H

Houttuyniae Herba, 1665Hydrangeae Dulcis Folium, 1763

Dulcis Folium Pulveratum, 1763

I

Imperatae Rhizoma, 1665Ipecacuanhae Radix, 1666

Radix Pulverata, 1667

K

Kasseki, 1598Koi, 1681

23082308 JP XVIIndex in Latin name

L

Leonuri Herba, 1682Lilii Bulbus, 1682Linderae Radix, 1683Lithospermi Radix, 1683Longan Arillus, 1684Lonicerae Folium Cum Caulis, 1685Lycii

Cortex, 1686Fructus, 1686

M

Magnoliae Cortex, 1687Cortex Pulveratus, 1687Flos, 1688

Malloti Cortex, 1688Mel, 1664Menthae Herba, 1689Mori Cortex, 1693Moutan Cortex, 1690

Cortex Pulveratus, 1691Myristicae Semen, 1695

N

Nelumbis Semen, 1693Notopterygii Rhizoma, 1694Nupharis Rhizoma, 1694

O

OleumArachidis, 1218Aurantii, 1190Cacao, 487Camelliae, 508Caryophylli, 1626Cinnamomi, 1623Cocois, 670Eucalypti, 822Foeniculi, 1640Maydis, 678Menthae Japonicae, 1689Olivae, 1182Rapae, 1335Ricini, 527Sesami, 1381Sojae, 1422Terebinthinae, 1545

Ophiopogonis Tuber, 1698Opium Pulveratum , 1183Oryzae Fructus, 1613

Ostreae Testa, 1701Testa Pulverata, 1701

P

Paeoniae Radix, 1704Radix Pulverata, 1705

Panacis Japonici Rhizoma, 1702Japonici Rhizoma Pulveratum,

1702Perillae Herba, 1706Persicae Semen, 1702

Semen Pulveratum, 1702Peucedani Radix, 1706Pharbitidis Semen, 1707Phellodendri Cortex, 1707

Cortex Pulveratus, 1708Picrasmae Lignum, 1710

Lignum Pulveratum, 1710Pinelliae Tuber, 1711Plantaginis

Herba, 1711Semen, 1711

Platycodi Radix, 1712Radix Pulverata, 1712

Pogostemoni Herba, 1713Polygalae Radix, 1713

Radix Pulverata, 1714Polygonati Rhizoma, 1714Polygoni Multiflori Radix, 1714Polyporus, 1715

Pulveratus, 1715Poria, 1715

Pulveratum, 1716Processi Aconiti Radix, 1716

Aconiti Radix Pulverata, 1718Prunellae Spica, 1720Puerariae Radix, 1720

Q

Quercus Cortex, 1721

R

Rehmanniae Radix, 1722Resina Pini, 1728Rhei Rhizoma, 1723

Rhizoma Pulveratum, 1724Rosae Fructus, 1727

Fructus Pulveratus, 1728

S

Saposhnikoviae Radix, 1740

Sappan Lignum, 1740Saussureae Radix, 1740Schisandrae Fructus, 1741Schizonepetae Spica, 1741Scopoliae Rhizoma, 1741Scutellariae Radix, 1747

Radix Pulverata, 1748Senegae Radix, 1749

Radix Pulverata, 1749Sennae Folium, 1750

Folium Pulveratum, 1751Sesami Semen, 1752Sevum Bovinum, 422Sinomeni Caulis et Rhizoma, 1761Smilacis Rhizoma, 1762

Rhizoma Pulveratum, 1762Sophorae Radix, 1762

Radix Pulverata, 1763Strychni Semen, 1695Swertiae Herba, 1764

Herba Pulverata, 1765

T

Tinctura Amara, 1613Tragacantha, 1767

Pulverata, 1767Tribuli Fructus, 1767Trichosanthis Radix, 1768

U

Uncariae Uncis Cum Ramulus, 1770Uvae Ursi Folium, 1608

V

Valerianae Radix, 1670Radix Pulverata, 1670

Z

Zanthoxyli Fructus, 1771Fructus Pulveratus, 1771

Zedoariae Rhizoma, 1771Zingiberis

Processum Rhizoma, 1719Rhizoma, 1645Rhizoma Pulveratum, 1646

ZizyphiFructus, 1670Semen, 1671

23092309

INDEX IN JAPANESE

ア

亜鉛華デンプン 1586亜鉛華軟膏 1586アカメガシワ 1688アクチノマイシン D 331アクラルビシン塩酸塩 327アクリノール・亜鉛華軟膏 330アクリノール水和物 328アクリノール・チンク油 329アザチオプリン 408アザチオプリン錠 409亜酸化窒素 1170アシクロビル 323アシクロビルシロップ 326アシクロビル注射液 325アジスロマイシン水和物 412アジマリン 334アジマリン錠 334亜硝酸アミル 387アスコルビン酸 397アスコルビン酸散 398アスコルビン酸注射液 398アズトレオナム 413L-アスパラギン酸 399アスピリン 400アスピリンアルミニウム 401アスピリン錠 400アスポキシシリン水和物 402アセグルタミドアルミニウム 314アセタゾラミド 319アセチルシステイン 322アセトアミノフェン 318アセトヘキサミド 320アセブトロール塩酸塩 313アセメタシン 315アセメタシンカプセル 316アセメタシン錠 317アゼラスチン塩酸塩 410アゼラスチン塩酸塩顆粒 411アセンヤク 1641アセンヤク末 1641アテノロール 403アトルバスタチンカルシウム錠 405アトルバスタチンカルシウム水和物

404アドレナリン 331アドレナリン液 332アドレナリン注射液 332アトロピン硫酸塩水和物 407アトロピン硫酸塩注射液 407亜ヒ酸パスタ 396アプリンジン塩酸塩 388アプリンジン塩酸塩カプセル 389アフロクアロン 333

アヘンアルカロイド・アトロピン注射液 1187

アヘンアルカロイド・スコポラミン注射液 1188

アヘンアルカロイド塩酸塩 1185アヘンアルカロイド塩酸塩注射液

1186アヘン散 1184アヘンチンキ 1184アヘン・トコン散 1698アヘン末 1183アマチャ 1763アマチャ末 1763アマンタジン塩酸塩 358アミオダロン塩酸塩 364アミオダロン塩酸塩錠 366アミカシン硫酸塩 361アミカシン硫酸塩注射液 362アミドトリゾ酸 360アミドトリゾ酸ナトリウムメグルミン

注射液 1074アミトリプチリン塩酸塩 367アミトリプチリン塩酸塩錠 367アミノ安息香酸エチル 809アミノフィリン水和物 363アミノフィリン注射液 363アムホテリシン B 380アムホテリシン Bシロップ 382アムホテリシン B錠 382アムロジピンベシル酸塩 370アムロジピンベシル酸塩錠 372アモキサピン 377アモキシシリンカプセル 379アモキシシリン水和物 378アモスラロール塩酸塩 375アモスラロール塩酸塩錠 376アモバルビタール 373アラセプリル 335アラセプリル錠 336L-アラニン 337アラビアゴム 1593アラビアゴム末 1593アリメマジン酒石酸塩 343亜硫酸水素ナトリウム 1393アルガトロバン水和物 392L-アルギニン 394L-アルギニン塩酸塩 394L-アルギニン塩酸塩注射液 395アルジオキサ 339アルプラゾラム 347アルプレノロール塩酸塩 348アルプロスタジル 349アルプロスタジルアルファデクス

352アルプロスタジル注射液 350アルベカシン硫酸塩 390

アルベカシン硫酸塩注射液 392アルミノプロフェン 345アルミノプロフェン錠 346アレンドロン酸ナトリウム錠 342アレンドロン酸ナトリウム水和物

340アレンドロン酸ナトリウム注射液

341アロエ 1596アロエ末 1597アロチノロール塩酸塩 396アロプリノール 344アロプリノール錠 344安息香酸 432安息香酸ナトリウム 1392安息香酸ナトリウムカフェイン 491安息香酸ベンジル 434アンソッコウ 1611アンチピリン 388アンピシリン水和物 384アンピシリンナトリウム 385アンベノニウム塩化物 359アンモニア・ウイキョウ精 1640アンモニア水 373アンレキサノクス 368アンレキサノクス錠 370

イ

イオウ 1440イオウ・カンフルローション 1440イオウ・サリチル酸・チアントール軟膏

1441イオタラム酸 972イオタラム酸ナトリウム注射液 1405イオタラム酸メグルミン注射液 1073イオトロクス酸 973イオパミドール 971イクタモール 944イコサペント酸エチル 810イセパマイシン硫酸塩 980イセパマイシン硫酸塩注射液 981イソクスプリン塩酸塩 992イソクスプリン塩酸塩錠 993イソソルビド 989イソニアジド 985イソニアジド錠 986イソニアジド注射液 986イソフルラン 982l-イソプレナリン塩酸塩 987イソプロパノール 988イソプロピルアンチピリン 989L-イソロイシン 983イソロイシン・ロイシン・バリン顆粒

984イダルビシン塩酸塩 945

23102310 JP XVIIndex in Japanese

イドクスウリジン 947イドクスウリジン点眼液 947イトラコナゾール 994イフェンプロジル酒石酸塩 948イブジラスト 943イブプロフェン 944イプラトロピウム臭化物水和物 974イプリフラボン 975イプリフラボン錠 976イミダプリル塩酸塩 949イミダプリル塩酸塩錠 950イミプラミン塩酸塩 954イミプラミン塩酸塩錠 955イミペネム水和物 952イルソグラジンマレイン酸塩 976イルソグラジンマレイン酸塩細粒

977イルソグラジンマレイン酸塩錠 978イレイセン 1625インジゴカルミン 959インジゴカルミン注射液 959インダパミド 956インダパミド錠 957インチンコウ 1602インデノロール塩酸塩 958インドメタシン 960インドメタシンカプセル 961インドメタシン坐剤 962インフルエンザ HAワクチン 963インヨウカク 1638

ウ

ウイキョウ 1639ウイキョウ末 1639ウイキョウ油 1640ウコン 1768ウコン末 1769ウベニメクス 1546ウベニメクスカプセル 1547ウヤク 1683ウラピジル 1551ウリナスタチン 1549ウルソデオキシコール酸 1554ウルソデオキシコール酸顆粒 1554ウルソデオキシコール酸錠 1555ウロキナーゼ 1552ウワウルシ 1608ウワウルシ流エキス 1609

エ

エイジツ 1727エイジツ末 1728エーテル 805エカベトナトリウム水和物 767エカベトナトリウム顆粒 768液状フェノール 1237エコチオパートヨウ化物 769エスタゾラム 795エストラジオール安息香酸エステル

796エストラジオール安息香酸エステル水

性懸濁注射液 797エストラジオール安息香酸エステル注

射液 796エストリオール 798エストリオール錠 799エストリオール水性懸濁注射液 798エタクリン酸 800エタクリン酸錠 801エタノール 802エタンブトール塩酸塩 801エチオナミド 807エチゾラム 817エチゾラム細粒 817エチゾラム錠 819エチドロン酸二ナトリウム 813エチドロン酸二ナトリウム錠 814エチニルエストラジオール 806エチニルエストラジオール錠 806L-エチルシステイン塩酸塩 809エチルモルヒネ塩酸塩水和物 813エチレフリン塩酸塩 815エチレフリン塩酸塩錠 815エチレンジアミン 812エデト酸ナトリウム水和物 1400エテンザミド 804エトスクシミド 808エトドラク 820エトポシド 821エドロホニウム塩化物 770エドロホニウム塩化物注射液 770エナラプリルマレイン酸塩 775エナラプリルマレイン酸塩錠 777エノキサシン水和物 779エバスチン 763エバスチン口腔内崩壊錠 764エバスチン錠 765エピリゾール 785エピルビシン塩酸塩 786エフェドリン塩酸塩 782エフェドリン塩酸塩散10z 783エフェドリン塩酸塩錠 784エフェドリン塩酸塩注射液 783エペリゾン塩酸塩 781エモルファゾン 774エモルファゾン錠 774エリスロマイシン 791エリスロマイシンエチルコハク酸エス

テル 793エリスロマイシンステアリン酸塩

794エリスロマイシン腸溶錠 792エリスロマイシンラクトビオン酸塩

793エルカトニン 771エルゴカルシフェロール 787エルゴタミン酒石酸塩 790エルゴメトリンマレイン酸塩 788エルゴメトリンマレイン酸塩錠 789エルゴメトリンマレイン酸塩注射液

789塩化亜鉛 1584塩化インジウム（111In）注射液 960塩化カリウム 1267

塩化カルシウム水和物 497塩化カルシウム注射液 497塩化タリウム（201Tl）注射液 1481塩化ナトリウム 139610z塩化ナトリウム注射液 1397エンゴサク 1631エンゴサク末 1632塩酸 925塩酸リモナーデ 926エンビオマイシン硫酸塩 780エンフルラン 778

オ

オウギ 1604オウゴン 1747オウゴン末 1748黄色ワセリン 1232オウセイ 1714オウバク 1707オウバク・タンナルビン・ビスマス散

1709オウバク末 1708オウレン 1628黄連解毒湯エキス 1698オウレン末 1629オキサゾラム 1193オキサピウムヨウ化物 1191オキサプロジン 1192オキシコドン塩酸塩水和物 1196オキシテトラサイクリン塩酸塩 1201オキシトシン 1203オキシトシン注射液 1205オキシドール 1199オキシブプロカイン塩酸塩 1195オキシメトロン 1200オキセサゼイン 1194オクスプレノロール塩酸塩 1194オザグレルナトリウム 1206オフロキサシン 1181オメプラゾール 1182オリブ油 1182オルシプレナリン硫酸塩 1191オレンジ油 1190オンジ 1713オンジ末 1714

カ

カイニン酸・サントニン散 999カイニン酸水和物 998カオリン 1004カカオ脂 487加香ヒマシ油 528カゴソウ 1720カシュウ 1714ガジュツ 1771加水ラノリン 1022ガスえそウマ抗毒素 878カッコウ 1713カッコン 1720葛根湯エキス 1674カッセキ 1598

23112311JP XVI Index in Japanese

過テクネチウム酸ナトリウム（99mTc）注射液 1408

果糖 870果糖注射液 870カドララジン 487カドララジン錠 488カナマイシン一硫酸塩 1002カナマイシン硫酸塩 1003カノコソウ 1670カノコソウ末 1670カフェイン水和物 490カプセル 514カプトプリル 514ガベキサートメシル酸塩 875過マンガン酸カリウム 1271加味逍遙散エキス 1676カモスタットメシル酸塩 509b-ガラクトシダーゼ（アスペルギルス）

876b-ガラクトシダーゼ（ペニシリウム）

876カリジノゲナーゼ 1000カリ石ケン 1265カルシトニン（サケ） 492カルテオロール塩酸塩 522カルナウバロウ 522カルバゾクロムスルホン酸ナトリウム

水和物 516カルバマゼピン 515カルビドパ水和物 517カルベジロール 525カルベジロール錠 526L-カルボシステイン 518カルメロース 519カルメロースカルシウム 519カルメロースナトリウム 520カルモナムナトリウム 523カルモフール 521カロコン 1768カンキョウ 1719カンゾウ 1649乾燥亜硫酸ナトリウム 1417カンゾウエキス 1651乾燥甲状腺 1494乾燥酵母 1580乾燥細胞培養痘そうワクチン 1390乾燥ジフテリアウマ抗毒素 738乾燥弱毒生おたふくかぜワクチン

1137乾燥弱毒生風しんワクチン 1365乾燥弱毒生麻しんワクチン 1065乾燥水酸化アルミニウムゲル 353乾燥水酸化アルミニウムゲル細粒

354カンゾウ粗エキス 1651乾燥組織培養不活化狂犬病ワクチン

1333乾燥炭酸ナトリウム 1395乾燥痘そうワクチン 1390乾燥日本脳炎ワクチン 995乾燥破傷風ウマ抗毒素 1479乾燥はぶウマ抗毒素 908乾燥 BCGワクチン 421

乾燥ボツリヌスウマ抗毒素 469カンゾウ末 1650乾燥まむしウマ抗毒素 1061乾燥硫酸アルミニウムカリウム 357カンデサルタンシレキセチル 511カンデサルタンシレキセチル錠

512カンテン 1594カンテン末 1595含糖ペプシン 1366d-カンフル 510dl-カンフル 510肝油 674カンレノ酸カリウム 1266

キ

希塩酸 925キキョウ 1712キキョウ末 1712キキョウ流エキス 1712キクカ 1622キササゲ 1619キジツ 1665キシリトール 1579キシリトール注射液 1580キタサマイシン 1010キタサマイシン酢酸エステル 1012キタサマイシン酒石酸塩 1013キナプリル塩酸塩 1325キナプリル塩酸塩錠 1326キニジン硫酸塩水和物 1328キニーネエチル炭酸エステル 1329キニーネ塩酸塩水和物 1330キニーネ硫酸塩水和物 1331牛脂 422吸水クリーム 313キョウカツ 1694キョウニン 1600キョウニン水 1601希ヨードチンキ 967金チオリンゴ酸ナトリウム 1391

ク

グアイフェネシン 906グアナベンズ酢酸塩 907グアネチジン硫酸塩 908グアヤコールスルホン酸カリウム

1269クエン酸ガリウム（67Ga）注射液 878クエン酸水和物 645クエン酸ナトリウム水和物 1398クコシ 1686クジン 1762クジン末 1763苦味重曹水 1762苦味チンキ 1613クラブラン酸カリウム 1268グラミシジン 903クラリスロマイシン 646クラリスロマイシン錠 647グリクラジド 883

グリシン 895グリセオフルビン 904グリセオフルビン錠 905グリセリン 892グリセリンカリ液 894クリノフィブラート 654グリベンクラミド 883グリメピリド 884グリメピリド錠 886クリンダマイシン塩酸塩 650クリンダマイシン塩酸塩カプセル

651クリンダマイシンリン酸エステル

652クリンダマイシンリン酸エステル注射

液 653グルコン酸カルシウム水和物 499グルタチオン 891L-グルタミン 890L-グルタミン酸 889クレゾール 680クレゾール水 680クレゾール石ケン液 681クレボプリドリンゴ酸塩 649クレマスチンフマル酸塩 650クロカプラミン塩酸塩水和物 656クロキサシリンナトリウム水和物

668クロキサゾラム 669クロコナゾール塩酸塩 681クロスカルメロースナトリウム 682クロチアゼパム 666クロトリマゾール 667クロナゼパム 661クロニジン塩酸塩 662クロフィブラート 657クロフィブラートカプセル 658クロフェダノール塩酸塩 657クロベタゾールプロピオン酸エステル

655クロペラスチン塩酸塩 663クロミフェンクエン酸塩 659クロミフェンクエン酸塩錠 660クロミプラミン塩酸塩 661クロム酸ナトリウム(51Cr)注射液

1398クロモグリク酸ナトリウム 1399クロラゼプ酸二カリウム 664クロラゼプ酸二カリウムカプセル

665クロラムフェニコール 610クロラムフェニコールコハク酸エステ

ルナトリウム 612クロラムフェニコールパルミチン酸エ

ステル 611クロルジアゼポキシド 613クロルジアゼポキシド散 613クロルジアゼポキシド錠 614クロルフェニラミン・カルシウム散

621クロルフェニラミンマレイン酸塩

622d-クロルフェニラミンマレイン酸塩


625クロルフェニラミンマレイン酸塩散

623クロルフェニラミンマレイン酸塩錠

624クロルフェニラミンマレイン酸塩注射

液 623クロルフェネシンカルバミン酸エステ

ル 619クロルフェネシンカルバミン酸エステ

ル錠 620クロルプロパミド 629クロルプロパミド錠 629クロルプロマジン塩酸塩 626クロルプロマジン塩酸塩錠 627クロルプロマジン塩酸塩注射液 627クロルヘキシジン塩酸塩 616クロルヘキシジングルコン酸塩液

616クロルマジノン酢酸エステル 617クロロブタノール 618

ケ

ケイガイ 1741経口生ポリオワクチン 1263ケイ酸マグネシウム 1056軽質無水ケイ酸 1385軽質流動パラフィン 1214桂枝茯苓丸エキス 1679ケイヒ 1623ケイヒ末 1623ケイヒ油 1623ケタミン塩酸塩 1005結晶セルロース 599血清性性腺刺激ホルモン 901ケツメイシ 1618ケトコナゾール 1005ケトコナゾール液 1008ケトコナゾールクリーム 1006ケトコナゾールローション 1007ケトチフェンフマル酸塩 1009ケトプロフェン 1008ケノデオキシコール酸 609ゲファルナート 878ケンゴシ 1707ゲンタマイシン硫酸塩 881ゲンタマイシン硫酸塩点眼液 882ゲンチアナ 1644ゲンチアナ・重曹散 1645ゲンチアナ末 1644ゲンノショウコ 1645ゲンノショウコ末 1645

コ

コウイ 1681コウカ 1731硬化油 934コウジン 1721合成ケイ酸アルミニウム 356コウブシ 1633コウブシ末 1633

コウベイ 1613コウボク 1687コウボク末 1687ゴオウ 1700コカイン塩酸塩 670ゴシツ 1594牛車腎気丸エキス 1652ゴシュユ 1639コデインリン酸塩散1z 671コデインリン酸塩散10z 672コデインリン酸塩錠 672コデインリン酸塩水和物 671ゴナドレリン酢酸塩 895ゴボウシ 1614ゴマ 1752ゴマ油 1381ゴミシ 1741コムギデンプン 1574コメデンプン 1346コリスチンメタンスルホン酸ナトリウ

ム 676コリスチン硫酸塩 677コルチゾン酢酸エステル 679コルヒチン 674コレカルシフェロール 630コレステロール 631コレラワクチン 630コロンボ 1615コロンボ末 1615コンズランゴ 1628コンズランゴ流エキス 1628

サ

サイクロセリン 687サイコ 1613柴胡桂枝湯エキス 1734サイシン 1602柴朴湯エキス 1732柴苓湯エキス 1737酢酸 319酢酸ナトリウム水和物 1390サッカリン 1366サッカリンナトリウム水和物 1367サフラン 1731サラシ粉 617サラシミツロウ 422サラゾスルファピリジン 1368サリチル酸 1370サリチル酸精 1372サリチル酸ナトリウム 1415サリチル酸絆創膏 1372サリチル酸メチル 1106サリチル・ミョウバン散 1371ザルトプロフェン 1581ザルトプロフェン錠 1582サルブタモール硫酸塩 1369サルポグレラート塩酸塩 1374サルポグレラート塩酸塩細粒 1375サルポグレラート塩酸塩錠 1377酸化亜鉛 1585酸化カルシウム 501酸化チタン 1503

酸化マグネシウム 1055サンキライ 1762サンキライ末 1762サンザシ 1632三酸化ヒ素 397サンシシ 1642サンシシ末 1642サンシュユ 1630サンショウ 1771サンショウ末 1771酸素 1199サンソウニン 1671サントニン 1373サンヤク 1635サンヤク末 1636

シ

ジアスターゼ 706ジアスターゼ・重曹散 706ジアゼパム 706ジアゼパム錠 707シアナミド 683シアノコバラミン 684シアノコバラミン注射液 685ジエチルカルバマジンクエン酸塩

713ジエチルカルバマジンクエン酸塩錠

714ジオウ 1722歯科用アンチホルミン 387歯科用トリオジンクパスタ 1540歯科用パラホルムパスタ 1215歯科用フェノール・カンフル 1237歯科用ヨード・グリセリン 968ジギトキシン 717ジギトキシン錠 718シクラシリン 634ジクロキサシリンナトリウム水和物

713シクロスポリン 634ジクロフェナクナトリウム 710ジクロフェナミド 711ジクロフェナミド錠 712シクロペントラート塩酸塩 686シクロホスファミド水和物 686シゴカ 1636ジゴキシン 719ジゴキシン錠 721ジゴキシン注射液 720ジコッピ 1686シコン 1683次硝酸ビスマス 462ジスチグミン臭化物 741ジスチグミン臭化物錠 741L-システイン 688L-システイン塩酸塩水和物 689シスプラチン 644ジスルフィラム 742ジソピラミド 740シタラビン 690シッカニン 1384シツリシ 1767


ジドブジン 1583ジドロゲステロン 762ジドロゲステロン錠 762シノキサシン 642シノキサシンカプセル 643ジノスタチンスチマラマー 1588ジノプロスト 735ジヒドロエルゴタミンメシル酸塩

725ジヒドロエルゴトキシンメシル酸塩

726ジヒドロコデインリン酸塩 723ジヒドロコデインリン酸塩散1z 724ジヒドロコデインリン酸塩散10z

724ジピリダモール 739ジフェニドール塩酸塩 715ジフェンヒドラミン 735ジフェンヒドラミン塩酸塩 737ジフェンヒドラミン・バレリル尿素散

736ジフェンヒドラミン・フェノール・亜鉛

華リニメント 737ジブカイン塩酸塩 709ジフテリアトキソイド 738ジフテリア破傷風混合トキソイド

739ジフルコルトロン吉草酸エステル

715シプロヘプタジン塩酸塩水和物 688ジベカシン硫酸塩 708ジベカシン硫酸塩点眼液 709シベンゾリンコハク酸塩 632シベンゾリンコハク酸塩錠 632シメチジン 641ジメモルファンリン酸塩 730ジメルカプロール 732ジメルカプロール注射液 733ジメンヒドリナート 731ジメンヒドリナート錠 732次没食子酸ビスマス 461ジモルホラミン 733ジモルホラミン注射液 734弱アヘンアルカロイド・スコポラミン

注射液 1189シャクヤク 1704芍薬甘草湯エキス 1752シャクヤク末 1705ジャショウシ 1626シャゼンシ 1711シャゼンソウ 1711臭化カリウム 1265臭化ナトリウム 1394十全大補湯エキス 1671ジュウヤク 1665シュクシャ 1599シュクシャ末 1599酒石酸 1456ショウキョウ 1645ショウキョウ末 1646小柴胡湯エキス 1756硝酸イソソルビド 990硝酸イソソルビド錠 991

硝酸銀 1386硝酸銀点眼液 1387常水 1572ショウズク 1618小青竜湯エキス 1758焼セッコウ 1655消毒用エタノール 804消毒用フェノール 1236消毒用フェノール水 1238ショウマ 1622ジョサマイシン 995ジョサマイシン錠 996ジョサマイシンプロピオン酸エステル

997シラザプリル錠 638シラザプリル水和物 637シラスタチンナトリウム 636ジラゼプ塩酸塩水和物 728ジルチアゼム塩酸塩 729シロスタゾール 639シロスタゾール錠 640シロップ用アシクロビル 325シロップ用セファトリジンプロピレン

グリコール 542シロップ用セファドロキシル 535シロップ用セファレキシン 539シロップ用セフロキサジン 584シロップ用ファロペネムナトリウム

828シロップ用ペミロラストカリウム

1219シンイ 1688親水クリーム 934親水ワセリン 1231診断用クエン酸ナトリウム液 1399シンバスタチン 1388真武湯エキス 1753

ス

水酸化カリウム 1270水酸化カルシウム 500水酸化ナトリウム 1403スキサメトニウム塩化物水和物 1447スキサメトニウム塩化物注射液 1449スクラルファート水和物 1429スコポラミン臭化水素酸塩水和物

1379ステアリルアルコール 1427ステアリン酸 1426ステアリン酸カルシウム 508ステアリン酸ポリオキシル 40 1264ステアリン酸マグネシウム 1057ストレプトマイシン硫酸塩 1427スピラマイシン酢酸エステル 1424スピロノラクトン 1425スピロノラクトン錠 1425スペクチノマイシン塩酸塩水和物

1423スリンダク 1441スルタミシリントシル酸塩水和物

1445スルチアム 1447

スルバクタムナトリウム 1433スルピリド 1442スルピリドカプセル 1442スルピリド錠 1443スルピリン水和物 1444スルピリン注射液 1444スルファジアジン銀 1435スルファメチゾール 1436スルファメトキサゾール 1436スルファモノメトキシン水和物 1437スルフイソキサゾール 1438スルベニシリンナトリウム 1434スルホブロモフタレインナトリウム

1438スルホブロモフタレインナトリウム注

射液 1439

セ

成人用沈降ジフテリアトキソイド738

精製水 1572精製水（容器入り） 1572精製ゼラチン 880精製セラック 1383精製デヒドロコール酸 695精製白糖 1430精製ヒアルロン酸ナトリウム 1401精製ラノリン 1023生理食塩液 1397石油ベンジン 1233セタノール 605セチリジン塩酸塩 606セチリジン塩酸塩錠 607セッコウ 1655セトラキサート塩酸塩 608セネガ 1749セネガシロップ 1749セネガ末 1749セファクロル 528セファクロルカプセル 529セファクロル細粒 531セファクロル複合顆粒 532セファゾリンナトリウム 543セファゾリンナトリウム水和物 545セファトリジンプロピレングリコール

541セファドロキシル 534セファドロキシルカプセル 535セファレキシン 536セファレキシンカプセル 537セファロチンナトリウム 540セフィキシムカプセル 561セフィキシム水和物 560セフェピム塩酸塩水和物 557セフォジジムナトリウム 566セフォゾプラン塩酸塩 576セフォタキシムナトリウム 569セフォチアム塩酸塩 575セフォチアムヘキセチル塩酸塩

572セフォテタン 570セフォペラゾンナトリウム 568


セフカペンピボキシル塩酸塩細粒550

セフカペンピボキシル塩酸塩錠551

セフカペンピボキシル塩酸塩水和物548

セフジトレンピボキシル 555セフジトレンピボキシル細粒 556セフジトレンピボキシル錠 556セフジニル 552セフジニルカプセル 553セフジニル細粒 554セフスロジンナトリウム 585セフタジジム水和物 586セフチゾキシムナトリウム 593セフチブテン水和物 592セフテラムピボキシル 589セフテラムピボキシル細粒 590セフテラムピボキシル錠 591セフトリアキソンナトリウム水和物

594セフピラミドナトリウム 578セフピロム硫酸塩 580セフブペラゾンナトリウム 546セフポドキシムプロキセチル 581セフミノクスナトリウム水和物 565セフメタゾールナトリウム 564セフメノキシム塩酸塩 562セフロキサジン水和物 582セフロキシムアキセチル 596セボフルラン 1382セラセフェート 598ゼラチン 879セラペプターゼ 1380L-セリン 1380セルモロイキン（遺伝子組換え） 602センキュウ 1626センキュウ末 1627ゼンコ 1706センコツ 1694センソ 1766センナ 1750センナ末 1751センブリ 1764センブリ・重曹散 1765センブリ末 1765

ソ

ソウジュツ 1604ソウジュツ末 1605ソウハクヒ 1693ソボク 1740ソヨウ 1706ソルビタンセスキオレイン酸エステル

1420ゾルピデム酒石酸塩 1590ゾルピデム酒石酸塩錠 1591D-ソルビトール 1421D-ソルビトール液 1421

タ

ダイオウ 1723大黄甘草湯エキス 1634ダイオウ末 1724ダイズ油 1422タイソウ 1670ダウノルビシン塩酸塩 692タウリン 1457タクシャ 1595タクシャ末 1596タクロリムス水和物 1449タゾバクタム 1457ダナゾール 691タムスロシン塩酸塩 1454タムスロシン塩酸塩徐放錠 1455タモキシフェンクエン酸塩 1453タランピシリン塩酸塩 1450タルク 1451炭酸カリウム 1267炭酸水素ナトリウム 1392炭酸水素ナトリウム注射液 1393炭酸ナトリウム水和物 1395炭酸マグネシウム 1054炭酸リチウム 1043単シロップ 1388ダントロレンナトリウム水和物 691単軟膏 1388タンニン酸 1456タンニン酸アルブミン 338タンニン酸ジフェンヒドラミン 738タンニン酸ベルベリン 441

チ

チアプリド塩酸塩 1494チアプリド塩酸塩錠 1495チアマゾール 1482チアマゾール錠 1482チアミラールナトリウム 1486チアミン塩化物塩酸塩 1483チアミン塩化物塩酸塩散 1485チアミン塩化物塩酸塩注射液 1484チアミン硝化物 1485チアラミド塩酸塩 1496チアラミド塩酸塩錠 1496チアントール 1488チオテパ 1491チオペンタールナトリウム 1489チオリダジン塩酸塩 1491チオ硫酸ナトリウム水和物 1417チオ硫酸ナトリウム注射液 1417チクセツニンジン 1702チクセツニンジン末 1702チクロピジン塩酸塩 1497チザニジン塩酸塩 1504窒素 1168チニダゾール 1500チペピジンヒベンズ酸塩 1500チペピジンヒベンズ酸塩錠 1502チメピジウム臭化物水和物 1498チモ 1599

チモール 1493チモロールマレイン酸塩 1499注射用アズトレオナム 414注射用アセチルコリン塩化物 322注射用アミカシン硫酸塩 362注射用アムホテリシン B 381注射用アモバルビタールナトリウム

374注射用アンピシリンナトリウム 386注射用イダルビシン塩酸塩 946注射用イミペネム･シラスタチンナト

リウム 953注射用オザグレルナトリウム 1207注射用血清性性腺刺激ホルモン 903注射用水 1572注射用水（容器入り） 1573注射用スキサメトニウム塩化物 1448注射用ストレプトマイシン硫酸塩

1428注射用セファゾリンナトリウム 544注射用セフェピム塩酸塩 559注射用セフォゾプラン塩酸塩 577注射用セフォチアム塩酸塩 576注射用セフタジジム 588注射用セフメタゾールナトリウム

565注射用チアミラールナトリウム 1487注射用チオペンタールナトリウム

1490注射用テセロイキン（遺伝子組換え）

1465注射用ドキソルビシン塩酸塩 756注射用バンコマイシン塩酸塩 1559注射用ヒト絨毛性性腺刺激ホルモン

899注射用ヒドララジン塩酸塩 923注射用ピペラシリンナトリウム 1255注射用ビンブラスチン硫酸塩 1564注射用ファモチジン 823注射用フェニトインナトリウム 1244注射用プレドニゾロンコハク酸エステ

ルナトリウム 1290注射用フロモキセフナトリウム 840注射用ペプロマイシン硫酸塩 1226注射用ベンジルペニシリンカリウム

437注射用ホスホマイシンナトリウム

868注射用マイトマイシン C 1127注射用ミノサイクリン塩酸塩 1124注射用メロペネム 1087注射用ロキサチジン酢酸エステル塩酸

塩 1363チョウジ 1625チョウジ末 1625チョウジ油 1626チョウトウコウ 1770釣藤散エキス 1619チョレイ 1715チョレイ末 1715L-チロシン 1545チンク油 1585沈降ジフテリア破傷風混合トキソイド


739沈降精製百日せきジフテリア破傷風混

合ワクチン 739沈降精製百日せきワクチン 1230沈降炭酸カルシウム 495沈降炭酸カルシウム細粒 495沈降炭酸カルシウム錠 496沈降破傷風トキソイド 1479沈降はぶトキソイド 908沈降 B型肝炎ワクチン 909チンピ 1624

ツ

ツバキ油 508ツロブテロール塩酸塩 1544

テ

テイコプラニン 1466低置換度ヒドロキシプロピルセルロー

ス 937テオフィリン 1481テガフール 1465デキサメタゾン 699デキストラン40 700デキストラン40注射液 701デキストラン70 702デキストラン硫酸エステルナトリウム

イオウ5 703デキストラン硫酸エステルナトリウム

イオウ18 704デキストリン 704デキストロメトルファン臭化水素酸塩

水和物 705テストステロンエナント酸エステル

1476テストステロンエナント酸エステル注

射液 1477テストステロンプロピオン酸エステル

1477テストステロンプロピオン酸エステル

注射液 1478デスラノシド 698デスラノシド注射液 698テセロイキン（遺伝子組換え） 1459テトラカイン塩酸塩 1479テトラサイクリン塩酸塩 1480デヒドロコール酸 694デヒドロコール酸注射液 696デフェロキサミンメシル酸塩 693テプレノン 1471デメチルクロルテトラサイクリン塩酸

塩 696テモカプリル塩酸塩 1469テモカプリル塩酸塩錠 1470テルビナフィン塩酸塩 1472テルビナフィン塩酸塩液 1474テルビナフィン塩酸塩クリーム 1473テルビナフィン塩酸塩スプレー 1475テルブタリン硫酸塩 1475テレビン油 1545天然ケイ酸アルミニウム 354

デンプングリコール酸ナトリウム1415

テンマ 1643テンモンドウ 1603

ト

トウガシ 1611トウガラシ 1615トウガラシ・サリチル酸精 1618トウガラシチンキ 1617トウガラシ末 1616トウキ 1668トウキ末 1669トウニン 1702トウニン末 1703トウヒ 1612トウヒシロップ 1612トウヒチンキ 1612トウモロコシデンプン 678トウモロコシ油 678ドキサゾシンメシル酸塩 751ドキサゾシンメシル酸塩錠 752ドキサプラム塩酸塩水和物 750ドキシサイクリン塩酸塩水和物 757ドキシフルリジン 753ドキシフルリジンカプセル 754ドキソルビシン塩酸塩 755ドクカツ 1601トコフェロール 1506トコフェロールコハク酸エステルカル

シウム 1508トコフェロール酢酸エステル 1507トコフェロールニコチン酸エステル

1509トコン 1666トコンシロップ 1667トコン末 1667トスフロキサシントシル酸塩錠 1517トスフロキサシントシル酸塩水和物

1516トチュウ 1638トドララジン塩酸塩水和物 1510ドネペジル塩酸塩 745ドネペジル塩酸塩細粒 746ドネペジル塩酸塩錠 747ドパミン塩酸塩 749ドパミン塩酸塩注射液 749トフィソパム 1511ドブタミン塩酸塩 743トブラマイシン 1505トブラマイシン注射液 1506トラガント 1767トラガント末 1767トラザミド 1512トラネキサム酸 1518トラネキサム酸カプセル 1519トラネキサム酸錠 1521トラネキサム酸注射液 1520トラピジル 1521トリアムシノロン 1524トリアムシノロンアセトニド 1525トリアムテレン 1526

トリクロホスナトリウム 1531トリクロホスナトリウムシロップ

1532トリクロルメチアジド 1527トリクロルメチアジド錠 1528トリコマイシン 1530L-トリプトファン 1543トリヘキシフェニジル塩酸塩 1533トリヘキシフェニジル塩酸塩錠 1533トリメタジオン 1538トリメタジオン錠 1538トリメタジジン塩酸塩 1535トリメタジジン塩酸塩錠 1536トリメトキノール塩酸塩水和物 1539トリメブチンマレイン酸塩 1534トルナフタート 1514トルナフタート液 1514トルブタミド 1513トルブタミド錠 1513トルペリゾン塩酸塩 1515L-トレオニン 1492トレハロース水和物 1522トレピブトン 1523ドロキシドパ 759ドロキシドパカプセル 760ドロキシドパ細粒 761トロキシピド 1541トロキシピド細粒 1541トロキシピド錠 1542トロピカミド 1540ドロペリドール 758トロンビン 1492豚脂 1024ドンペリドン 744

ナ

ナイスタチン 1180ナタネ油 1335ナテグリニド 1148ナテグリニド錠 1149ナドロール 1142ナファゾリン塩酸塩 1145ナファゾリン・クロルフェニラミン液

1146ナファゾリン硝酸塩 1146ナファモスタットメシル酸塩 1143ナブメトン 1139ナブメトン錠 1141ナプロキセン 1147ナリジクス酸 1144ナロキソン塩酸塩 1145

ニ

ニガキ 1710ニガキ末 1710ニカルジピン塩酸塩 1152ニカルジピン塩酸塩注射液 1153ニクズク 1695ニコチン酸 1161ニコチン酸アミド 1160ニコチン酸注射液 1162


ニコモール 1158ニコモール錠 1159ニコランジル 1159ニザチジン 1171ニザチジンカプセル 1172二酸化炭素 518ニセリトロール 1157ニセルゴリン 1154ニセルゴリン散 1155ニセルゴリン錠 1156ニトラゼパム 1166ニトレンジピン 1166ニトレンジピン錠 1167ニトログリセリン錠 1169ニフェジピン 1162日本脳炎ワクチン 995乳酸 1016L-乳酸 1017乳酸カルシウム水和物 500L-乳酸ナトリウム液 1406乳糖水和物 1019尿素 1552ニルバジピン 1163ニルバジピン錠 1164ニンジン 1646ニンジン末 1647ニンドウ 1685

ネ

ネオスチグミンメチル硫酸塩 1151ネオスチグミンメチル硫酸塩注射液

1151

ノ

濃グリセリン 893濃ベンザルコニウム塩化物液 50 429ノスカピン 1179ノスカピン塩酸塩水和物 1180ノルアドレナリン 1173ノルアドレナリン注射液 1174ノルエチステロン 1175ノルゲストレル 1176ノルゲストレル・エチニルエストラジ

オール錠 1177ノルトリプチリン塩酸塩 1178ノルフロキサシン 1175

ハ

バイモ 1641バカンピシリン塩酸塩 415白色セラック 1384白色軟膏 1575白色ワセリン 1232白糖 1432バクモンドウ 1698麦門冬湯エキス 1606バクロフェン 417バクロフェン錠 418バシトラシン 416バソプレシン注射液 1560

八味地黄丸エキス 1656ハチミツ 1664ハッカ 1689ハッカ水 1690ハッカ油 1689パップ用複方オウバク散 1709パニペネム 1208パパベリン塩酸塩 1211パパベリン塩酸塩注射液 1212ハマボウフウ 1649バメタン硫酸塩 419パラアミノサリチル酸カルシウム顆粒

503パラアミノサリチル酸カルシウム水和

物 502パラオキシ安息香酸エチル 811パラオキシ安息香酸ブチル 486パラオキシ安息香酸プロピル 1314パラオキシ安息香酸メチル 1103パラフィン 1212パラホルムアルデヒド 1214L-バリン 1557パルナパリンナトリウム 1216バルビタール 419バルプロ酸ナトリウム 1418バルプロ酸ナトリウムシロップ 1419バルプロ酸ナトリウム錠 1419バレイショデンプン 1272ハロキサゾラム 913ハロタン 912ハロペリドール 909ハロペリドール細粒 910ハロペリドール錠 911パンクレアチン 1207パンクロニウム臭化物 1208ハンゲ 1711半夏厚朴湯エキス 1659バンコマイシン塩酸塩 1558パンテチン 1210パントテン酸カルシウム 501

ヒ

ピオグリタゾン塩酸塩 1249ピオグリタゾン塩酸塩錠 1250ビオチン 458ピコスルファートナトリウム水和物

1409ビサコジル 459ビサコジル坐剤 460L-ヒスチジン 920L-ヒスチジン塩酸塩水和物 920ビソプロロールフマル酸塩 462ビソプロロールフマル酸塩錠 463ビタミン A油 1566ビタミン A油カプセル 1566ヒトインスリン（遺伝子組換え） 963ヒト下垂体性性腺刺激ホルモン 899ヒト絨毛性性腺刺激ホルモン 897人全血液 1575人免疫グロブリン 923ヒドララジン塩酸塩 923ヒドララジン塩酸塩散 924

ヒドララジン塩酸塩錠 924ヒドロキシジン塩酸塩 938ヒドロキシジンパモ酸塩 938ヒドロキシプロピルセルロース 935ヒドロキソコバラミン酢酸塩 935ヒドロクロロチアジド 926ヒドロコタルニン塩酸塩水和物 933ヒドロコルチゾン 927ヒドロコルチゾンコハク酸エステル

932ヒドロコルチゾンコハク酸エステルナ

トリウム 931ヒドロコルチゾン酢酸エステル 928ヒドロコルチゾン・ジフェンヒドラミ

ン軟膏 929ヒドロコルチゾン酪酸エステル 929ヒドロコルチゾンリン酸エステルナト

リウム 930ピブメシリナム塩酸塩 1262ピブメシリナム塩酸塩錠 1263ヒプロメロース 940ヒプロメロースフタル酸エステル

942ピペミド酸水和物 1251ピペラシリン水和物 1252ピペラシリンナトリウム 1254ピペラジンアジピン酸塩 1256ピペラジンリン酸塩錠 1257ピペラジンリン酸塩水和物 1256ビペリデン塩酸塩 459ビホナゾール 457ヒマシ油 527ピマリシン 1246ヒメクロモン 939ピモジド 1247ビャクゴウ 1682ビャクシ 1599ビャクジュツ 1605ビャクジュツ末 1606氷酢酸 320ピラジナミド 1322ピラルビシン 1258ピランテルパモ酸塩 1321ピリドキシン塩酸塩 1323ピリドキシン塩酸塩注射液 1324ピリドスチグミン臭化物 1322ピレノキシン 1259ピレンゼピン塩酸塩水和物 1260ピロ亜硫酸ナトリウム 1411ピロカルピン塩酸塩 1245ピロキシカム 1261ピロキシリン 1324ピロールニトリン 1325ビワヨウ 1685ビンクリスチン硫酸塩 1565ピンドロール 1248ビンブラスチン硫酸塩 1563ビンロウジ 1602

フ

ファモチジン 822ファモチジン散 825


ファモチジン錠 826ファモチジン注射液 824ファロペネムナトリウム錠 829ファロペネムナトリウム水和物 827フィトナジオン 1244フェキソフェナジン塩酸塩 833フェニトイン 1242フェニトイン散 1242フェニトイン錠 1243L-フェニルアラニン 1240フェニルブタゾン 1240フェニレフリン塩酸塩 1241フェネチシリンカリウム 1233フェノバルビタール 1234フェノバルビタール散10z 1235フェノール 1236フェノール・亜鉛華リニメント 1237フェノール水 1238フェノールスルホンフタレイン 1238フェノールスルホンフタレイン注射液

1239フェルビナク 831フェンタニルクエン酸塩 832フェンブフェン 831複方アクリノール・チンク油 330複方オキシコドン・アトロピン注射液

1197複方オキシコドン注射液 1197複方サリチル酸精 1372複方サリチル酸メチル精 1107複方ジアスターゼ・重曹散 706複方ダイオウ・センナ散 1725複方チアントール・サリチル酸液

1488複方ビタミン B散 1567複方ヨード・グリセリン 967複方ロートエキス・ジアスターゼ散

1744ブクモロール塩酸塩 474ブクリョウ 1715ブクリョウ末 1716ブシ 1716フシジン酸ナトリウム 1401ブシ末 1718ブシラミン 472ブシラミン錠 473ブスルファン 482ブチルスコポラミン臭化物 1378ブテナフィン塩酸塩 483ブテナフィン塩酸塩液 484ブテナフィン塩酸塩クリーム 484ブテナフィン塩酸塩スプレー 485ブドウ酒 1575ブドウ糖 888ブドウ糖注射液 888ブトロピウム臭化物 485ブナゾシン塩酸塩 479ブフェトロール塩酸塩 475ブプラノロール塩酸塩 480ブプレノルフィン塩酸塩 481ブホルミン塩酸塩 476ブホルミン塩酸塩錠 478ブホルミン塩酸塩腸溶錠 476

ブメタニド 479フラジオマイシン硫酸塩 869プラステロン硫酸エステルナトリウム

水和物 1411プラゼパム 1282プラゼパム錠 1283プラゾシン塩酸塩 1284プラノプロフェン 1276プラバスタチンナトリウム 1277プラバスタチンナトリウム液 1279プラバスタチンナトリウム細粒 1278プラバスタチンナトリウム錠 1281フラビンアデニンジヌクレオチドナト

リウム 834フラボキサート塩酸塩 836プリミドン 1291フルオキシメステロン 852フルオシノニド 848フルオシノロンアセトニド 847フルオレセインナトリウム 849フルオロウラシル 851フルオロメトロン 850フルコナゾール 843フルジアゼパム 845フルシトシン 844フルスルチアミン塩酸塩 874フルタミド 856フルトプラゼパム 857フルトプラゼパム錠 858フルドロコルチゾン酢酸エステル

845フルニトラゼパム 846フルフェナジンエナント酸エステル

853フルボキサミンマレイン酸塩 859フルボキサミンマレイン酸塩錠 860フルラゼパム 853フルラゼパム塩酸塩 855フルラゼパムカプセル 854プルラン 1320フルルビプロフェン 855ブレオマイシン塩酸塩 465ブレオマイシン硫酸塩 467フレカイニド酢酸塩 836フレカイニド酢酸塩錠 838プレドニゾロン 1285プレドニゾロンコハク酸エステル

1289プレドニゾロン酢酸エステル 1287プレドニゾロン錠 1286プレドニゾロンリン酸エステルナトリ

ウム 1288プロカインアミド塩酸塩 1296プロカインアミド塩酸塩錠 1297プロカインアミド塩酸塩注射液 1296プロカイン塩酸塩 1298プロカイン塩酸塩注射液 1299プロカテロール塩酸塩水和物 1300プロカルバジン塩酸塩 1299プログルミド 1304プロクロルペラジンマレイン酸塩

1301プロクロルペラジンマレイン酸塩錠

1302プロゲステロン 1303プロゲステロン注射液 1304フロセミド 871フロセミド錠 873フロセミド注射液 872プロタミン硫酸塩 1316プロタミン硫酸塩注射液 1317プロチオナミド 1318プロチレリン 1318プロチレリン酒石酸塩水和物 1319プロテイン銀 1387プロテイン銀液 1387プロパフェノン塩酸塩 1307プロパフェノン塩酸塩錠 1308プロパンテリン臭化物 1309プロピベリン塩酸塩 1310プロピベリン塩酸塩錠 1311プロピルチオウラシル 1315プロピルチオウラシル錠 1316プロピレングリコール 1315プロブコール 1293プロブコール細粒 1294プロブコール錠 1295プロプラノロール塩酸塩 1312プロプラノロール塩酸塩錠 1313フロプロピオン 841フロプロピオンカプセル 842プロベネシド 1292プロベネシド錠 1292ブロマゼパム 469ブロムヘキシン塩酸塩 470プロメタジン塩酸塩 1306フロモキセフナトリウム 839ブロモクリプチンメシル酸塩 471ブロモバレリル尿素 471L-プロリン 1305粉末セルロース 602

ヘ

ベカナマイシン硫酸塩 423ベクロメタゾンプロピオン酸エステル

421ベザフィブラート 455ベザフィブラート徐放錠 456ベタキソロール塩酸塩 454ベタネコール塩化物 455ベタヒスチンメシル酸塩 443ベタヒスチンメシル酸塩錠 443ベタミプロン 453ベタメタゾン 445ベタメタゾン吉草酸エステル 449ベタメタゾン吉草酸エステル･ゲンタ

マイシン硫酸塩クリーム 450ベタメタゾン吉草酸エステル･ゲンタ

マイシン硫酸塩軟膏 452ベタメタゾンジプロピオン酸エステル

447ベタメタゾン錠 446ベタメタゾンリン酸エステルナトリウ

ム 448ペチジン塩酸塩 1230


ペチジン塩酸塩注射液 1231ベニジピン塩酸塩 424ベニジピン塩酸塩錠 425ヘパリンカルシウム 914ヘパリンナトリウム 916ヘパリンナトリウム注射液 919ペプロマイシン硫酸塩 1224ペミロラストカリウム 1218ペミロラストカリウム錠 1220ベラドンナエキス 1610ベラドンナコン 1609ベラパミル塩酸塩 1561ベラパミル塩酸塩錠 1562ベラプロストナトリウム 438ベラプロストナトリウム錠 439ペルフェナジン 1226ペルフェナジン錠 1227ペルフェナジンマレイン酸塩 1228ペルフェナジンマレイン酸塩錠 1228ベルベリン塩化物水和物 440ベンザルコニウム塩化物 428ベンザルコニウム塩化物液 429ベンジルアルコール 432ベンジルペニシリンカリウム 436ベンジルペニシリンベンザチン水和物

434ヘンズ 1636ベンズブロマロン 430ベンゼトニウム塩化物 431ベンゼトニウム塩化物液 431ベンセラジド塩酸塩 427ペンタゾシン 1221ペントキシベリンクエン酸塩 1223ベントナイト 427ペントバルビタールカルシウム 1222ペンブトロール硫酸塩 1221

ホ

ボウイ 1761ボウコン 1665ホウ砂 1394ホウ酸 468抱水クロラール 610ボウフウ 1740ボクソク 1721ボグリボース 1567ボグリボース錠 1568ホスホマイシンカルシウム水和物

865ホスホマイシンナトリウム 867ボタンピ 1690ボタンピ末 1691補中益気湯エキス 1661ポビドン 1273ポビドンヨード 1275ホマトロピン臭化水素酸塩 921ホミカ 1695ホミカエキス 1696ホミカエキス散 1696ホミカチンキ 1697ホモクロルシクリジン塩酸塩 922ポリスチレンスルホン酸カルシウム

506ポリスチレンスルホン酸ナトリウム

1409ポリソルベート 80 1265ホリナートカルシウム 498ポリミキシンB硫酸塩 1263ホルマリン 864ホルマリン水 864ホルモテロールフマル酸塩水和物

865ボレイ 1701ボレイ末 1701

マ

マイトマイシン C 1126マオウ 1636マーキュロクロム 1084マーキュロクロム液 1085マクリ 1635マクロゴール400 1050マクロゴール1500 1051マクロゴール4000 1052マクロゴール6000 1052マクロゴール20000 1053マクロゴール軟膏 1053マシニン 1661麻酔用エーテル 805マニジピン塩酸塩 1061マニジピン塩酸塩錠 1062マプロチリン塩酸塩 1065マルトース水和物 1060D-マンニトール 1063D-マンニトール注射液 1064

ミ

ミグレニン 1122ミクロノマイシン硫酸塩 1119ミコナゾール 1118ミコナゾール硝酸塩 1118ミゾリビン 1127ミゾリビン錠 1128ミツロウ 423ミデカマイシン 1120ミデカマイシン酢酸エステル 1121ミノサイクリン塩酸塩 1123ミノサイクリン塩酸塩錠 1125ミョウバン水 358

ム

無コウイ大建中湯エキス 1691無水アンピシリン 383無水エタノール 803無水カフェイン 489無水クエン酸 645無水乳糖 1018無水リン酸水素カルシウム 504ムピロシンカルシウム水和物 1137ムピロシンカルシウム軟膏 1139

メ

メキシレチン塩酸塩 1117メキタジン 1083メグルミン 1073メクロフェノキサート塩酸塩 1065メコバラミン 1066メストラノール 1087メダゼパム 1067メタンフェタミン塩酸塩 1091L-メチオニン 1092メチクラン 1109メチラポン 1116dl-メチルエフェドリン塩酸塩 1100dl-メチルエフェドリン塩酸塩散10z

1101メチルエルゴメトリンマレイン酸塩

1101メチルエルゴメトリンマレイン酸塩錠

1102メチルジゴキシン 1110メチルセルロース 1096メチルテストステロン 1107メチルテストステロン錠 1108メチルドパ錠 1099メチルドパ水和物 1098メチルプレドニゾロン 1104メチルプレドニゾロンコハク酸エステ

ル 1104メチルベナクチジウム臭化物 1096メチルロザニリン塩化物 1105滅菌精製水（容器入り） 1573メテノロンエナント酸エステル 1089メテノロンエナント酸エステル注射液

1089メテノロン酢酸エステル 1088メトキサレン 1095メトクロプラミド 1111メトクロプラミド錠 1112メトトレキサート 1093メトトレキサートカプセル 1093メトプロロール酒石酸塩 1113メトプロロール酒石酸塩錠 1113メトホルミン塩酸塩 1090メトホルミン塩酸塩錠 1091メトロニダゾール 1114メトロニダゾール錠 1115メナテトレノン 1077メピチオスタン 1080メピバカイン塩酸塩 1081メピバカイン塩酸塩注射液 1082メフェナム酸 1069メフルシド 1071メフルシド錠 1072メフロキン塩酸塩 1070メペンゾラート臭化物 1079メルカプトプリン水和物 1083メルファラン 1076メロペネム水和物 1085dl-メントール 1078l-メントール 1079


モ

木クレオソート 1577モクツウ 1595モサプリドクエン酸塩散 1134モサプリドクエン酸塩錠 1136モサプリドクエン酸塩水和物 1133モッコウ 1740モノステアリン酸アルミニウム 356モノステアリン酸グリセリン 894モルヒネ・アトロピン注射液 1130モルヒネ塩酸塩錠 1132モルヒネ塩酸塩水和物 1131モルヒネ塩酸塩注射液 1131

ヤ

ヤクチ 1611ヤクモソウ 1682薬用石ケン 1069薬用炭 1068ヤシ油 670

ユ

ユウタン 1608ユーカリ油 822輸血用クエン酸ナトリウム注射液

1398ユビデカレノン 1548

ヨ

ヨウ化カリウム 1270ヨウ化ナトリウム 1404ヨウ化ナトリウム（123I）カプセル

1404ヨウ化ナトリウム（131I）液 1404ヨウ化ナトリウム（131I）カプセル

1404ヨウ化人血清アルブミン（131I）注射液

966ヨウ化ヒプル酸ナトリウム（131I）注射

液 1405葉酸 861葉酸錠 863葉酸注射液 862ヨウ素 966ヨクイニン 1627ヨクイニン末 1627ヨーダミド 965ヨーダミドナトリウムメグルミン注射

液 1075ヨード・サリチル酸・フェノール精

969ヨードチンキ 966ヨードホルム 971

ラ

ラウリル硫酸ナトリウム 1407

ラウロマクロゴール 1026ラクツロース 1020ラタモキセフナトリウム 1024ラッカセイ油 1218ラナトシド C 1021ラナトシド C錠 1021ラニチジン塩酸塩 1333ラベタロール塩酸塩 1014ラベタロール塩酸塩錠 1015ラベプラゾールナトリウム 1332

リ

リオチロニンナトリウム 1039リオチロニンナトリウム錠 1040リシノプリル錠 1042リシノプリル水和物 1041L-リシン塩酸塩 1049L-リシン酢酸塩 1048リスペリドン 1351リスペリドン細粒 1351リスペリドン錠 1354リスペリドン内服液 1353リセドロン酸ナトリウム錠 1414リセドロン酸ナトリウム水和物 1412リゾチーム塩酸塩 1050六君子湯エキス 1725リドカイン 1034リドカイン注射液 1035リトドリン塩酸塩 1355リトドリン塩酸塩錠 1357リファンピシン 1347リファンピシンカプセル 1348リボスタマイシン硫酸塩 1345リボフラビン 1341リボフラビン散 1342リボフラビン酪酸エステル 1343リボフラビンリン酸エステルナトリウ

ム 1344リボフラビンリン酸エステルナトリウ

ム注射液 1345リマプロストアルファデクス 1036リュウガンニク 1684リュウコツ 1684リュウコツ末 1684硫酸亜鉛水和物 1586硫酸亜鉛点眼液 1587硫酸アルミニウムカリウム水和物

356硫酸カリウム 1272硫酸鉄水和物 832硫酸バリウム 420硫酸マグネシウム水 1059硫酸マグネシウム水和物 1058硫酸マグネシウム注射液 1059リュウタン 1669リュウタン末 1669流動パラフィン 1213リョウキョウ 1598苓桂朮甘湯エキス 1729リンゲル液 1350リンコマイシン塩酸塩水和物 1037

リンコマイシン塩酸塩注射液 1038リン酸水素カルシウム水和物 504リン酸水素ナトリウム水和物 1408リン酸二水素カルシウム水和物 505

レ

レセルピン 1337レセルピン散0.1z 1339レセルピン錠 1339レセルピン注射液 1338レチノール酢酸エステル 1340レチノールパルミチン酸エステル

1341レナンピシリン塩酸塩 1026レバミピド 1335レバミピド錠 1336レバロルファン酒石酸塩 1029レバロルファン酒石酸塩注射液 1029レボチロキシンナトリウム錠 1033レボチロキシンナトリウム水和物

1032レボドパ 1030レボフロキサシン水和物 1031レボメプロマジンマレイン酸塩 1032レンギョウ 1640レンニク 1693

ロ

L-ロイシン 1028ロキサチジン酢酸エステル塩酸塩

1360ロキサチジン酢酸エステル塩酸塩徐放

カプセル 1361ロキサチジン酢酸エステル塩酸塩徐放

錠 1362ロキシスロマイシン 1364ロキソプロフェンナトリウム水和物

1047ロキタマイシン 1358ロキタマイシン錠 1359ロサルタンカリウム 1045ロジン 1728ロートエキス 1742ロートエキス・アネスタミン散 1744ロートエキス・カーボン散 1744ロートエキス・タンニン坐剤 1746ロートエキス・パパベリン・アネスタミ

ン散 1745ロートエキス散 1743ロートコン 1741ローヤルゼリー 1728ロラゼパム 1045

ワ

ワイル病秋やみ混合ワクチン 1574ワルファリンカリウム 1569ワルファリンカリウム錠 1571

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