9

CHAPTER CHAPTER CHAPTER CHAPTER ----IIII

INTRODUCTITON TO STABILITY INDICATING

METHODS

AND

GENERAL METHODLOGY OF DEVELOPMENT

AND VALIDATION OF ASSAY AND IMPURITY

METHODS

10

Section (i)

1.1 Introduction

Pharmaceutical is defined as medicinal drug. Pharmaceutical is a chemical entity of

therapeutic interest and appropriate term is active pharmaceutical ingredient and also called as drug

substance. The drug product is prepared by formulating a drug substance with inert ingredients

(excipients) to make it suitable for administration to patients [1].The drug substance can be

formulated into various drug products based on desired dosage form and target site. The commonly

used dosage forms are tablets and capsules that are administered orally.

Inspite of progress made in pharmaceutical research, the drug product development process

remains both time-consuming and expensive. The drug product development process typically

involves pre-formulation, prototype formulation development, in-vitro in-vivo study, bio/clinical

study, scale up and regulatory approvals to bring the product to market.The analytical techniques

and methods are fundamentally important to enabling all phases of the process. Advances in

Analytical techniques can lead to both processes and product innovations. The scientific principles

behind analytical techniques are complex and advanced analytical instruments and applications are

needed at every step of development process.

1.2 Importance of stability-Indicating analytical Methods in pharmaceutical analysis

The quality of analytical data generated on stability samples is essential to the successful

completion of stability studies and to the ability to draw appropriate conclusions regarding the

stability of the product. The purpose of stability studies is to monitor possible changes to a product

11

or material over time and at different storage conditions [2, 3]. It is expected that only those

methods that are truly stability indicating should be used.

International conference on Harmonization (ICH) guideline clearly states that not just one

method that is stability indicating, but that stability can only be inferred by a combination of

analytical methods looking at the identity, purity and potency of the drug [4].

A major challenge in developing a stability indicating methods is the access to suitable

degraded samples to aid in method development. Ideally these degraded samples would be real-time

stability samples that contain all relevant degradants which form under normal storage conditions.

This is unrealistic for several reasons like development time lines, and how stability is affected by

batch characteristics such as process parameters, quality of excipients and environmental factors

such as humidity or temperature. That is why pharmaceutical chemists have to rely on forced

degradation samples to develop Stability-indicating methods. The ability of forced degradation

studies to forecast real-time degradation has been object of several studies. Formal stability

assessment of pharmaceuticals is typically done at three distinct times, namely, development,

registration and commercialization. These studies are to support the safety and efficacy of drug

product during development [5-9]. Atregistration, to ascertain the quality and shelf-life of the

marketed product and finally during commercialization phase to ensure the quality of the production

and to support site or other changes to the product.Stability information on both drug substance and

drug product is required as part of the registration dossier and serves to confirm the shelf-life and to

determine appropriate storage conditions [10-14].

In pharmaceutical industry analyst in R&D plays a major role in drug product development to

assure a new drug product meets the established standards like purity, efficacy, safety & quality and

to meet the purported quality throughout its shelf life. The analytical methods are generally

developed in analytical R&D department and transferred to quality control laboratory. After drug

12

product is approved by regulatory authorities, assuming that all batches of drug product are made to

the specific standards become the responsibility of pharmaceutical analyst in QC and QA

departments. Beyond that any changes in process and ingredients have to be continually monitored

to assure the quality and safety of the drug product. The Validated stability indicating methods can

detect the changes over time in the chemical and physical properties of the drug substance and drug

products, so that the contents of active ingredient, degradation products and other components of

interest can be estimated accurately without interference.

1.2.1 Requirements of stability testing and development of stability-indicating analytical

methods:

The purpose of stability testing is to provide evidence on how the quality of a drug

substance or drug product varies with time under the influence of a variety of environmental

factors such as temperature, humidity and light. These studies will help to determine

recommended storage conditions, re-test periods and shelf life.

The ICH guideline primarily addresses the information required in Registration

Applications for new molecular entities and associated drug products. This guideline does not

currently seek to cover the information required for abbreviated or abridged applications,

variations, clinical trial applications, etc. The choice of test conditions defined in the guideline

is based on the analysis of the effects of climatic conditions in the three areas of the EU, Japan

and USA. The mean kinetic temperature in any region of the world can be derived from

climatic data.

Information on the stability of the drug substance is an integral part of the systematic

approach to stability evaluation. The goal of stability program depends on the stage of

development of the drug product. At very beginning of product development, it is necessary

13

to understand the inherent stability of the drug substance and its interaction with the proposed

excipients. At this stage, the effect of pH, moisture, air (oxygen), and light on the stability of

the drug substance is also studied. The accelerated testing on drug substance and drug product

provides the information to the intrinsic stability of the molecule/formulation and may

establish the likely degradation pathways. The formulation group also has the responsibility

for recommending to the toxicology group about the stability of drug substance in the vehicle

used in the animal trials. On the analytical side, the analytical research group supports the pre-

formulation stability program, which ultimately responsible for developing and validating the

stability-indicating assays that, will be included in New Drug Application [NDA].

In the preclinical formulation stage, the selection of a stable drug product formula is

the primary goal. The temporary preclinical formula is included in the Investigational New

Drug application [IND]. The goal of the stability program in the clinical trial stage is to

ascertain that the drug product batches tested in the clinical trials are stable, and these data

will be subsequently included in the NDA. At the NDA approval stage, the validated stability-

indicating analytical method will be transferred to the quality control group, to ascertain that it

works well in the hands of those who have to monitor the stability of the marketed product.

The marketed product stability program fulfils the commitment of part of the NDA and also

ensures that the marketed drug products are stable (potent) until the expiry date stamped on

the product label. Usually, the first three marketed batches and at least one batch per year are

subjected to stability program.

14

1.3. Type of Analytical Techniques employed in literature for the development of

Stability Indicating analytical methods:

An analytical technique is a method that is used to determine the concentration of

chemical compound or chemical element. There are wide variety of techniques used for

analysis from simple weighting (gravimetric analysis) to titrations (titrimetric) to very

advanced techniques using highly specialized instrumentation. The most common techniques

used in analytical chemistry are the following:

i) Titrimetry : Based on the quantity of reagent needed to react with the analyte.

ii) Electroanalytical methods :Potentiometry and voltammetry

iii) Spectroscopy: Based on the different interaction of the analyte along with electromagnetic radiation

iv) Chromatography : The analyte is separated from the rest of the sample so that it may be measured without interference from other compounds;

v) Microscopy , Bio analysis and Radio analytical chemistry.

There are many more techniques that have specialized applications. Inspite of having all the

above techniques, High performance liquid chromatography is powerful tool as a separation

technique and widely used for quantification of drugs and its impurities.

1.3.1. Importance of HPLC/UPLC in pharmaceutical analysis

HPLC/UPLC gives stead fast quantitative precision and accuracy with a linear dynamic choice

adequate to permit for the estimation of the active pharmaceutical ingredients and impurities in the

same chromatogram using different kind of detectors, and can be employed on fully computerized

instrumentation. HPLC/UPLC gives very good reproducibility and is valid to a wide collection of

compound types by careful selection of column chemistry [15, 16].

15

1.3.2. HPLC Instrumentation

The basic parts of the HPLC set up are:

a) Solvent delivery systems b) Sample injection system c) Columns and ovens

d) Detectors e) Data acquisition system

a) Solvent Delivery systems:

The first component of a modern liquid chromatograph is a reservoir of solvent(s) or buffer

solution(s) or the combination of both to be delivered to the pump. Basic requirements for a LC

pumping system are pressure capability to at least several thousand psi, and good solvent

compatibility. The ideal pumping system for LC should also provide accurate,precise, pulse-free

solvent delivery over a wide range of flow rates, be easy to change to a new solvent, be generally

convenient to use and maintain, be able to draw from a large external reservoir and be easily

adapted to gradient elution. The modern pumping system utilizes a pump(s) to achieve the gradient

of two or more solvents.

b) Sample Injection system:

The purpose of the injection system is to introduce the sample on to the pressurized column

as a sharp column plug, without loss in efficiency.

c) Columns and Ovens:

A good understanding of a chemical and physical interaction that takes place between the

sample, mobile phase and stationary phase is important, if a chromatographer is to quickly make the

correct mobile and stationary phase selection.

16

d) Detectors:

The function of the detector is to provide an electrical signal to the recorder and data system

from which the qualitative and quantitative analysis are made. The most common LC detectors are RI

and UV/PDA, followed by the Fluorescence, ELSD, Conductivity and the electrochemical detectors.

The UV detector is a photometer with a micro sample cell. This is very much similar to the

one used in conventional UV-Visible spectrophotometer, excepting for the cell design. The features

available in modern spectrophotometers have been incorporated into the UV detectors used in LC

like scanning, ratio recording etc.

The Photo Diode Array Detector [DAD] is the most used detector in LC today [17].

The DAD gives a three dimensional view of chromatogram (Intensity Vs Time) and Spectra

(Intensity Vs Wavelength) simultaneously. It can be called as Spectro-chromatogram. The

detailed analysis of the data reveals more information on the complexity of co elution and

helps in identifying the merged peaks and gives information on peak purity.In UV detectors,

the light source is typically a deuterium lamp, which provides acceptable light intensity from

190 to 400 nm. When measurements at visible wavelengths ( 400 to 700 nm) are required, a

higher energy tungsten-halide lamp is often used. However most HPLC applications are

carried out using wavelengths below 400 nm. Light from the lamp passes through a UV-

transmitting flow cell connected to the column and impinges on a diode that measures the

light intensity.

e) Data acquisition system :

Data Systems process the detector output and integrate it to form a meaningful

chromatogram. Modem Integration system does more than just that. They do processing of

17

the chromatogram, calculations, statistical analysis, data back-up and storage. Data Systems

also control various parameters of the system.

HPLC systems can be modular or integrated, and use either isocratic or gradient solvent

delivery. Modular systems, as the term implies, consist of separate modules connected in such a way

as to function as a single unit. Modular systems can provide a degree of flexibility to exchange

different components in and out of the system, sometimes necessary for maintenance purposes or

experimental requirements. In regulated laboratories this flexibility may not be viewed as an

advantage however due to compliance issues with instrument validation/qualification. In integrated

systems, the individual components can share electrical, communication and fluid connections and

control, and can operate in ways that provide better solvent and sample management than modular

systems. Modern integrated systems are holistically designed to take advantage of managing both

the sample and the solvent in ways that can significantly decrease injection cycle time and provide

increased precision and accuracy while still providing flexibility in detection choices. HPLC system

scan be modular or integrated, and use either isocratic or gradient solvent delivery.

Section (ii)

1.1 Over view of HPLC method development for Pharmaceuticals

High-pressure or high-performance liquid chromatography is predominantly used in

the pharmaceutical industryfor evaluations of a large variety of samples. It is the method

ofchoice for checking purity of drugs, monitoring changes in synthetic procedures or scale up,

evaluating new formulations, and carrying out quality control/assurance of the final drug

product.

It is important to pay special attention to the following during method development:

18

1. Methods that would resolve all potential known impurities and known and unknown

degradation products.

2. Instrument qualification and validation to meet regulatory requirements.

3. Validation of HPLC methods before they are utilized routinely.

Before starting the method development, it is more important to know about the sample and

Objective of development should be defined. The following could be a guiding objectives for

developing a stability indicating Assay and impurities method for drug products.

For Impurities method :

i). Separation of all impurities from each other and from Active components.

ii). Separation of all impurities from components of drug product matrix (placebo).

iii). Simple sample preparation procedures by choosing appropriate diluents.

iv). Shortest possible run times without compromising the robustness of the method.

For Assay method :

i). Separation of Active components from all impurities and components of placebo.

ii). Simple sample preparation procedures by choosing appropriate diluents.

iii). Shortest possible run times without compromising the robustness of the method.

1.1.1LC method development:

There are many different approaches to method development usedin LC, from simple "trial-

and-error", to more complex multivariateapproaches involving experimental design and/or

chemometrics.In general, gradient systems are preferred over isocratic systems formethod

development because of their multi-solvent capability. Using auto blend, different organic solvent

19

proportions, buffer strength, and pH can be generated by using the solvent manager to

proportionately mix the appropriate stock solutions to obtain the final mobile phase conditions. Auto

blending of this type can be used as an advantage during method development [18].

One of the primary drivers for the growth and continued use ofHPLC has been the evolution

of packing materials for superiorseparations of complex mixtures. With the new advancesin column

stationary phases with particle size less than 2 µm, not only is there a significant gain in efficiency;

butthe efficiency does not diminish at increased flow rates or linear velocities. By using smaller

particles, speed and peak capacity (number ofpeaks resolved per unit time) can be extended to new

limits. Liquid chromatographic systems also evolved to handle sub 2um columns and in effect the

high back pressures associated with them. High Performance systems are transforming into ultra

performance systems. UPLC takes full advantage of chromatographic principlesto run separations

using columns packed with smaller particles, and/orhigher flow rates for increased speed, with

superior resolution andsensitivity [19-25].

1.1.1.1: Detection Sensitivity and Selectivity

Most Pharmaceutical compounds are organic compounds which contains one or more

chromophoric functional groups. Therefore, in most cases HPLC/UPLC method development is

carried out with ultraviolet detection using either a variable wavelength (VWD) or a diodearray

detector (DAD). When there is a possibility of Non-chromophoric impurities or components that are

needed to be estimated, alternate detectors like RI, ELSD to be used. Alternately a gas

chromatographic system can be employed if the compounds are reasonably volatile. Where very low

level contents needs to be estimated like below 1ppm, a Mass detector can also be employed.Where

compounds having good fluorescence properties, FLD detectors can also be used.

20

Choice of detector and wavelength of determination is selected based on nature of sample.

For many samples, good analytical results will be obtained only by careful selection of the

wavelength used for detection. If analyte standards are available, their UV spectra can be measured

prior to an LC method development. Alternatively DAD permits the acquisition of UV spectra for all

sample components during method development. The wave length chosen for UV detection must

provide acceptable absorbance by the various analytes in the sample, combined with acceptable light

transmittance by the mobile phase. For some samples it may also be important to select a

wavelength at which sample interferences have minimal absorption.

1.1.1.2 Selection of Mobile phase

Mobile phase selection is most critical parameter as it promotes solute-stationary phase

interactions. The important consideration is that it should not damage the column packing. Hence

use of strong acids, strong bases and halides solutions should be avoided. The purity of chemical

being used also an important consideration. Collect and review the literature for method related

information already reported on the molecule. Collect information if available on solubility profile.

Solubility of Drug in different solvents and at different pH conditions will help to choose the

appropriate buffer and solvents to be used in mobile phase. Collect the structuresof the molecule

and the impurities likely to be present and degradation products.Compare the structures of

impurities and degradation products with the structure of drug substances and arrive at the polarity

whether they are less polar or more polar than the compound of interest. This will help to assess to

understand about the most probable elution order. Depending on the closeness of the structures, it

is also possible to predict which are likely to give challenge in separations. For example, impurities

having similar structures which only differs in position of the functional groups, which only differs by

one methyl group which does not alter the polarity significantly.

21

Collect analytical profile (Physico-chemical properties, Eg: pKa, melting point, degradation

pathways, etc). Buffer pH to be used in mobile phase is based on the pKa of the analyte, which is

based on the structure of the molecule. Assess whether the compound is basic, acidic or neutral. If

compound is acidic, a mobile phase which contains an acidic buffer is generally used. For a basic

compound, use a buffer with low pH or basicpH. For a neutral compound, neutral mobile phase is

generally suitable. select a pH, which is ± 2 from the pKa values. Buffer strength of about 10 mM is

advisable for initial experiments.

Buffer strengths can be increased, when a peak tailing is observed. Increase beyond 50

mMsometimes may not be feasible, due to possible solubility problems of the salt in the

organic portion of the mobile phase

1.1.1.3 Selection of Buffers and pH

Buffer solution plays a vital role in HPLC mobile phase to achieve reproducible

chromatographic results. Buffer imparts constant ionic strength to the mobile phase. Therefore

buffer solution in aqueous portion of mobile phase is recommended for reverse phase

chromatography. Buffering increases ruggedness of the method. pH of buffer solution to be selected

based on nature of analyte like acidic, basic or neutral and its pKa value. Acidic pH range is

appropriate for acidic compounds, low or basic pH is recommended for basic compounds and neutral

pH for neutral compounds. When pH increases acid samples become ionized, where as pH decreases

base samples become ionized (Fig 1.1).

22

Fig 1.1 The Effect of pH on the Retention of Acids and Bases

In reverse phase chromatography, if the analyte is less or non polar, it retains in column

stationary phase for longer period. Where asanalyte is ionized it attains polarity and then it will

retain in the column stationary phase for shorter period.

To develop a robust method, pH of the buffer should select at 2 units away from pKa of

sample/analyte. It is derived from the Equation: pH = pKa + log([A-]/[HA]) [Henderson-Hasselback].

When pH of buffer solution is close to pKa of the analyte, theanalyte will be in a partly ionized state,

the analyte will partly in its weak acid or base form and partially in its conjugate form (Fig 1.2). This

will lead to distortion of analyte peak and/or poor peak repeatability. When pH of mobile phase

selected 2 units away from analytepKa, analyte will be in more than 99% single state. pH of the

buffer shall be near a pH equal to the pKa of the buffer. In General, maximum buffers work duly well

within one pH unit of its pKa.

23

Fig 1.2 Representative Diagram for Henderson-Hasselback Equation

1.1.1.4 Role of Organic modifiers inMobile phase :

Mobile phase strength in Reverse phase chromatography depends on both % buffer rand

type of organic solvent. Three commonly used solvents in reverse phase chromatography (RPC) are

acetonitrile, methanol and tetrahydrofuran.

Acetonitrile is the best organic modifier (Because of favorable UV transmittance and Low

viscosity). Methanol is the second best organic solvent and Third organic solvent is THF. These three

solvents are widely used to control selectivity and separations. The following is the order of polarity

and strength of solvents, which can be used as a general guidance while making decisions to achieve

separations.

Order of Polarity: Methanol>Acetonitrile >Ethanol>THF>Propanol

Order of Solvent Strength: Propanol>THF> Ethanol >Acetonitrile>Methanol.

THF has some disadvantages, higher UV absorbance, reactivity with oxygen and slower

column equilibration. But sometimes it gives very unique selectivity for closely eluting peaks.

Therefore these solvents are used for solvent type selectivity. Intermediate selectivity (if

24

needed for a particular sample) can be obtained by blending appropriate amounts of each of

these solvents.

Any other foregoing solvents might be used with water for PRC except methylene chloride which is

not water miscible. Acetonitrile and water mixtures can be used with UV detection at low

wavelengths which may be necessary for some compounds and also have lower viscosities, resulting

in higher plate numbers and lower column pressures. The below graph can be used as a general

guidance when solvent combinations are modified to arrive at equal elution strengths.

1.1.1.5Role of additives in Mobile phase :

The additives such as ionpair reagents and surfactants sometimes will help to enhance

sample solubility. The addition of additives like Triethylamine and acetic acid will improve the peak

tailing for basic and acidic samples respectively. The only additives that will work practically below

200nm in concentrations above 1mM are phosphoric acid and aliphatic amines.The addition of amine

modifiers to the mobile phase can affect the separation of basic samples and results in much

improved peak shapes. The retention of basic compounds will decrease at the concentration of

amine additive is increased due to blockage of ionized silanols by the amine. If an amine is added to

the mobile phase, its concentration should belarge enough to suppress silanol effects as much as

possible. The use of amine modifiers to affect selectivity also depends on the presence of ionized

silanols, and these tend to vary from column to column of the same type. For this reason, varying the

amine concentration is not a first choice for the control of selectivity.

Triethylamine (TEA) or diethyl amine (DEA) can be added to the mobile phase to

control peak tailing for bases. TEA /DEA acts as a competing base and minimizes solute-

silanol interactions. Whenever TEA or modifier is used to reduce the tailing, use columns

25

with very low level of silanols. Using 0.1% TFA as the aqueous modifier results in a more

transparent mobile phase and still reduces the tailing.

Ion pair reagents can be used when necessary, when a closely related compounds

separation is required. If a mixture of ionic and non-ionic analytes to be separated, start by

optimizing the method for non-ionic compounds. Then select the appropriate ion-pair reagent

to provide the necessary selectivity. It is preferred to dedicate HPLC column for a specific

analysis, whenever an ion pair reagent is used in mobile phase. Alternately, a specific

cleaning Procedure for the method shall be chosen which will clean up to the possible extent.

Alkyl sulphonates are good first choice for basic compounds. Quaternary amines are useful

for acidic compounds. Use of ion-pair reagents in the concentration range of 0.0005M to

0.02M is generally recommended. Sodium per chlorate also sometimes used as an ion-pair

reagent to get the required selectivity for acidic components.

1.1.1.6Column Selection

Columns with specific bonding phase can be chosen based on the polarity of the

molecule. A wide variety of columns are available covering a wide range of polarity by cross-

linking the Si-OH groups with alkyl chains likeC4, C6, C8, C18 and nitrile groups (CN), phenyl

groups (-C6H6) and amino groups (-NH2) etc for liquid chromatography. Silica based columns with

the increasing order of polarity are as follows:

<--------Non-polar--------moderately polar------------Polar----------->

C18 < C8 < C6/C4 < Phenyl < Amino <Cyano< Silica

The following factors needs to be considered while selecting a column for the separation

purposes.

a) Column Dimensions: Higher the column length, better are the separations.

26

b) Particle Shape : Spherical particles are preferred compared to irregular.

c) Particle Size: lower the size of the particles, better are the separations.

d) Surface Area : Higher surface are generally leads better selectivity.

e)Pore Size : 60-120 Å is generally used for Reverse phase chromatography.

f)Bonding Type : Either monomeric or Polymeric.

g)Carbon Load : Higher carbon loading generally leads to better selectivity. But based on the nature

of the mobile phase and nature of molecules to be separated, the effect could be different some

times.

1.1.1.7Selection of Elution mode:

If isocratic elution is possible, this means conditions for 0.5<k<20; If the k range exceeds

these limits, gradient elution is necessary. When an initial chromatogram suggests a wide

retention range (0.5>k>20) for an ionic sample, the use of ion pair reagent often permits

isocratic separation with 0.5<k<20. Similar changes in retention change can also be achieved by

a change in pH, to ionize late eluting compounds (for reduced retention) or reduce the

ionization of early eluting sample components (for late elution). Gradient elution methods are

widely being used due to availability of advanced liquid chromatographic instruments and need

for achieve robust separations. It is a good idea to start method development with the following

two default gradient programmes :

Programme 1 Programme 2

27

Time Buffer Organic phase Time Buffer Organic phase

0.01 50 50 0.01 95 5

60 5 95 60 5 95

70 5 95 70 5 95

71 50 50 71 50 50

75 50 50 75 50 50

Understand elution pattern of all the peaks. Based on the elution with these gradient

programmes, further optimization can be done.

1.1.1.8Flow rate and column temperature

Initial flow rate of 1.0 ml-1min or 1.5 ml-1min and column temperature as ambient

(25 – 30°C) is preferable.

1.1.1.9Diluent selection

Select a diluent in which impurities and degradation products and the analyte are soluble. It is

advisable to check first suitability of mobile phase as diluent. All the analytes should be

completely soluble and solution should be stable reasonably atleast until its injection into LC.

For Finished dosage forms, diluents shall be selected in such a way that the analyte(s) should

be extracted > 95% for impurities and >98% for Assay. Diluent should be compatible with the

mobile phase to obtain the good peak shape.

1.1.1.10Selection of test concentration and injection volume

Select a test concentration and injection volume to get an appropriate response for

theanalytes for an assay method. For impurities method, test concentration and injection

volume shall be based on the sensitivity required for the method. For impurities method,

28

LOQ of all impurities and analyte shall be less than or equal to the reporting threshold

(RT).

For drug products the ICH reporting thresholds are as follows.

Maximum daily dose : <1 g ------------ RT is less than or equal to 0.1%.

Maximum daily dose : >1g -------------RT is less than or equal to 0.05%.

1.1.1.11Degradation studies

Stressing the active pharmaceutical ingredient in solutions and in solid-state form produce the

sample that have the degradation products most possible to form under sensible storage conditions,

which is in turn used to develop the stability indicating analytical methods [26]. In a simple way, the

aim of the stability indicating method is to attain base to base resolution of all possible degradation

impurities with no co-elution.Degradation studies or stress testing is conducted in order to

investigate the likely degradation products, which in turn helps to establish the degradation

pathways and the intrinsic stability of the drug molecule and also to provide foundation for

developing a suitable stability indicating method. Stress testing the drug molecule under particular

stress condition generate samples containing degradation products. Use these samples to develop

suitable analytical methods. The degradation impurities formed through stressed samples are called

as “potential” degradation products. These impurities may or may not be generated under realistic

storage conditions. Stress drug product, and placebo separately to understand the peaks due to

placebo components, if any. Four major forced degradation studies are

(i) ThermolyticDegradation (ii) Hydrolytic degradation,

(iii) Oxidative degradation(iv) Photolytic degradation.

29

Preferably, have the degradation in the range of 1 % to 20%, in order to reflect the true

degradation pathways. Beyond 20%, sometimes secondary degradations will occur, which

may not occur in actual conditions of use.

Some general conditions used in conducting forced degradation studies for drug substances

are mentioned in the below Table 1.1.

Table1.1 General conditions used in conducting forced degradation

Stress condition Time of stressing

Solid state/ about 70-80 ºC 7 – 10 days

Solid state / 70-80 ºC / 75% RH 7 days

Solid state / simulated sunlight 3 x 1.2 million lux

0.1 to 2 N HCl either at RT or at about 70 ºC 24-48 hours

0.1 to 1 N NaOH either at RT or at about 70 ºC 24-48 hours

H2O2 (0.3 to 6%) at RT or at about 70 ºC 24-48 hours

Solution in water 24-48 hours

After the satisfactory separations are achieved, evaluate the peak purity of the analyte

peak using PDA detector. Conduct Mass balance study by assaying the stressed samples

apart from quantifying the impurities. Sum of Assay and impurities obtained in stressed

30

samples is presented as mass balance. A mass balance of >95% is always considered

satisfactory. If the mass balance is less than the required criteria, investigation to be done

inorder to correct or to justify.

The following are the reasons for not achieving the mass balance.

(1). Degradation products are not eluted from the HPLC column,

(2). Degradation products are not detected by the detector used.

(3). Degradation products lost from the sample matrix, due to insolubility, volatility.

(4). Parent compound lost from the sample matrix, due to insolubility, volatility.

(5). Degradation products are co-eluted with the parent compound.

(6). Degradation products are not integrated due to poor chromatography.

(7). Inaccurate quantification due to differences in response factors.

1.1.1.12Finalisation of chromatographic conditions

Based on the interferences, and based on the separations required the experiments are

to be planned to achieve the following separation goals. A base to base separation between all

the impurities. A base to base separation between impurities and placebo peaks (if any). A

base-to-base separation between all the impurities and Principal analyte peak. A base-to-base

separation between placebo peak(s), if any and Principal analyte peak. Good peak shapes for

all the impurities. Ensure that the separations are affected by small changes in the set

conditions.

1.1.1.13 Relative response factors

31

Establish the relative response factors of all known impurities by linearity method against the

target analyte. Relative response factor is dividing the slope of impurity by slope of active

compound.

1.1.1.14Selection of system suitability or Performance calculations

Calculating one or more of the following values is necessary to access overall system and

method performance. (i). Relative retention times, (ii). Theoretical plates, (iii). Capacity factor, (iv).

Resolution, (v).tailing factor and (vi). % RSD of peak area of replicate standard injections.

1.1.2 Analytical method validation

Validation is, of course, a basic requirement to ensure quality and reliability of the results for

all analytical applications [27]. However, in comparison with analytical chemistry, in pharmaceutical

analysis, some special aspects and conditions exist that need to be taken into consideration. For

example, the analytical procedures (apart from pharmacopoeial monographs) are often in-house

developments and applications. Therefore, the degree of knowledge and expertise is initially much

larger compared with standard methods. The same can be assumed for the samples analysed. The

matrix (placebo) in pharmaceutical analysis is usually constant and well known and the ranges where

the sample under analysis can be expected are usually well defined and not very large.

32

The object of validation of an analytical procedure is to demonstrate that it is suitable for its

intended purpose , determined by means of well-documented experimental studies. Accuracy and

reliability of the analytical results is crucial for ensuring quality, safety and efficacy of

pharmaceuticals. In Table 1.2, the required validation characteristics[28-30] for the various types of

analytical procedures are shown.

Table 1.2 Validation characteristics normally evaluated for the different types of test procedures.

Type of Analytical Procedure

IDENTIFICATION TESTING FOR IMPURITIES ASSAY

Characteristics Quantitation Limit

Dissolution ( measurement only) Content / potency

Accuracy - + - +

Precision

Repeatability - + - +

Intermediate precision - + (1) - + (1)

Specificity (2) + + + +

Detection limit - - (3) + -

Quantitation limit - + - -

Linearity - + - +

33

Range - + - +

- Signifies that this characteristic is not normally evaluated. + Signifies that this characteristic is normally evaluated. (1) In cases where reproducibility has been performed, intermediate precision is not

needed. (2) Lack of specificity of one analytical procedure could be compensated by other

supporting analytical procedure(s). (3) May be needed in some cases.

1.1.2.1 Precision

“The precision of an analytical procedure expresses the closeness of agreement (degree of

scatter) between a series of measurements obtained from multiple sampling of the same

homogeneous sample under the prescribed conditions. Precision may be considered at three levels;

repeatability, intermediate precision and reproducibility.”.Precision should be obtained preferably

using authentic samples. As parameters, the standard deviation, the relative standard deviation

(coefficient of variation)and the confidence interval should be calculated for each level of precision.

Repeatability expresses the analytical variability under the same operating conditions over a short

interval of time (within-assay, intra-assay). Intermediate precision includes the influence of

additional random effects within laboratories, according to the intended use of the procedure, for

example, different days, analysts or equipment, etc.

1.1.2.2 Accuracy

The accuracy of an analytical procedure expresses the “closeness of agreement between the

value which is accepted either as a conventional true value or an accepted reference value and the

value found”.This is sometimes termed as trueness.

Accuracy can be demonstrated by the following approaches:

34

(a) Recovery of drug substance spiked to placebo or drug product (for

Drugproduct)

(b) Recovery of the impurity spiked to drug substance or drug product

(for impurities)

For the quantitative approaches, at least nine determinations across the specified range

should be obtained, for example, three replicates at threeconcentration levels each. The percentage

recovery or the difference betweenthe mean and the accepted true value together with the

confidence intervals are recommended.

1.1.2.3 Specificity

Specificity is the ability to assess unequivocally the analyte in the presence of components

which may be expected to be present. Typically these might include impurities, degradants, matrix,

etc. Lack of specificity of an individual procedure may be compensated by other supporting analytical

procedure(s).

With respect to identification, discrimination between closely related compounds likely to be

present should be demonstrated by positive and negative samples. In the case of chromatographic

assay and impurity tests, available impurities/degradants can be spiked at appropriate levels to the

corresponding matrix or else degraded samples can be used. For assay, it can be demonstrated that

the result is unaffected by the spiked material. Impurities should be separated individually and/or

from other matrix components. Specificity can also be demonstrated by verification of the result with

an independent analytical procedure.

35

In the case of chromatographic separation, resolution factors should beobtained for critical

separation. Tests for peak homogeneity, for example, bydiode array detection (DAD) or mass

spectrometry (MS) are recommended.There has been some controversial discussion about the

terminology for this validation characteristic. In contrast to the ICH, most other analytical

organisations define this as selectivity, whereas specificity is regarded in an absolute sense, as the

“ultimate degree of selectivity” (IUPAC) [31]. Despite this controversy, there is a broad agreement

that specificity/selectivity is the critical basis of each analytical procedure. Without a sufficient

selectivity, the other performance parameters are meaningless.

In contrast to chemical analysis, where each analytical procedure is regarded (and evaluated)

separately, in pharmaceutical analysis, a whole range of control tests is used to evaluate a batch.

Therefore, the performance of these individual analytical procedures can complement each other in

order to achieve the required overall level of selectivity. For example, an assay by means of a less

selective titration that will include impurities with the same functional groups, can be confirmed (or

corrected) by a selective impurity determination by LC.

Specificity is to be considered from the beginning of the method development,taking into

account the properties of both analyte and sample (matrix). The (sufficiently)selective determination

of the analyte can be achieved by appropriate sample preparation, separation, and/or detection.

Usually, a combination of several approaches will be developed.

1.1.2.4 Detection and Quantitation Limit

Regulatory authorities require impurity profiling of drug substances and drug products as

part of the marketing authorization process. The safety requirements are linked to toxicological

studies for the active substance itself as well as the impurities of synthesis and degradation. Hence

36

there is a need to demonstrate that impurity profiles are within the ranges examined within the

toxicological studies and to limit any degradation products. The purpose of this section is to examine

the methods available for determining when an analyte is present (Detection Limit, DL) and for the

smallest amount of analyte that can be reliably measured (Quantitation Limit, QL).

“The detection limit of an individual analytical procedure is the lowest amount of analyte in

a sample which can be detected but not necessarily quantitated as an exact value. The quantitation

limit of an individual analytical procedure is the lowest concentration of analyte in a sample which

can be quantitatively determined with suitable precision and accuracy.” The methodology of Limit of

detection and Limit of quantification is given in the ICH guidance and hence does not need a

reproduction.

1.1.2.5Linearity

The linearity of an analytical procedure is its ability (within a given range) to obtain test

results, which are directly proportional to the concentration (amount) of analyte in the sample.

Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte

concentration or content. Ifthere is a linear relationship, test results should be evaluated by

appropriate statistical methods, for example, by calculation of a regression line by the method of

least squares. Insome cases, to obtain linearity between assays and sample concentrations, the test

data may need to be subjected to a mathematical transformation prior to the regression analysis.

Data from the regression line itself may be helpful to provide mathematical estimates of the degree

of linearity.

The correlation coefficient, y-intercept, slope of the regression line and residual sum of

squares should be submitted. A plot of the data should be included. In addition, an analysis of the

37

deviation of the actual data points from the regression line may also be helpful for evaluating

linearity. For the establishment of linearity, a minimum of 5 concentrations is recommended.

1.1.2.6Range

“The range of an analytical procedure is the interval between the upper and lower

concentration (amounts) of analyte in the sample (including these concentrations) for which it has

been demonstrated that the analytical procedure has a suitable level of precision, accuracy and

linearity.” The required range depends on the application intended for the analytical

Procedure. Table 1.3 provides the minimum working range of an analytical procedure is usually

derived from the results of the validation characteristics.

Table 1.3 Minimum ranges for different types of analytical procedures

1.1.2.7 Robustness

The robustness of an analytical procedure is a measure of its capacity to remain

38

unaffected by small, but deliberate variations in method parameters and provides an

indication of its reliability during normal usage.

If measurements are susceptible to variations in analytical conditions, the analytical

conditions should be suitably controlled or a precautionary statement should be included in

the procedure. One consequence of the evaluation of robustness should be that a series of

system suitability parameters (e.g., resolution test) is established to ensure that the validity of

the analytical procedure is maintained whenever used.

Examples of typical variations are

o Influence of variations of pH in a mobile phase,

o Influence of variations in mobile phase composition,

o Different columns (different lots and/or suppliers),

o Temperature,

o Flow rate.

1.2Scope and Objectives of research work:

Keeping in view the above discussion, author has examined the present state of

development of analytical methods for some of the widely used drug products. After the review of

available literature, author has undertaken research to develop stability indicating methods for four

most widely used drug products made up of drugs, namely, Rabeprazole, Omeprazole +

Domperidone, Telmisartan + Chlorthalidone and Metaprolol + Atorvastatin + Ramipril. These are

selected due to the reason that the author has found an opportunity for thedevelopment of new

analytical methods using liquid chromatography. The following is the brief account on the drug

products selected for this Research work.

39

1.2.1 RabeprazoleDR Tablets

Rabeprazole is an antiulcer drug in the class of proton pump inhibitors [32]. The drug

is unstable to heat, humidity, oxidation and acid hydrolysis conditions. Therefore, it is a

challenge for a pharmaceutical researcher to develop a stable formulation which retains the

drug potency during its shelf life. To facilitate the testing of the quality of Rabeprazole drug

product, it is essential to have suitable methods for potency and purity determination, which

will help to establish the shelf life of the drug product. During the development studies, it is

observed that few unknown degradants are formed upon stability. Review of current literature

showed that there is not enough information on the details about unknown degradants and

suitable validated methods which are capable of separating and quantifying all possible

impurities of Rabeprazole. When the current known methods are employed, all the known and

unknown degradants are found to be not well separated. Therefore, author has seen this as an

opportunity toresearch on identification of unknown degradants as well as a stability

indicating methods for assay and impurities.

1.2.2 Omeprazole + Domperidone capsules

Omeprazole is the first drug came to the market which belong to the class of proton

pump inhibitors. Omeprazole is one of the most widely prescribed drugs globally and is

available over-the-counter in some countries. Omeprazole monotherapy is used to treat

gastroesophageal reflux disease (GERD), gastric and duodenal ulceration, and gastritis [33].

Omprazole is also used in combination with other drugs for different kinds of therapeutic

treatments.

Domperidone, belongs to the prokinetic class of drugs. Prokinetic, is a type

of drug which enhances gastrointestinal motility by increasing the frequency

40

of contractions in the small intestine or making them stronger, but without disrupting their

rhythm. They are used to relieve gastrointestinal symptoms such as abdominal discomfort,

bloating, constipation, heart burn, nausea, and vomiting. Combination of PPI with a

prokineticwith a prokinetic drug is found to provide significant benefits to patients [34].

Combination of omeprazole with domperidone in GERD patients is found to be more superior

compared to omeprazole monotherapy.

Due to the above reason,a fixed dose combination of Omeprazole with domperidone is

now being used widely to have better patient compliance. Review of literature showed that

there are no published methods which can be used for determination of potency and purity of

this fixed dose combination. Author has seen an opportunity to develop a single analytical

method for estimating Assay and a single method for determination of impurities for this

combination product.

1.2.3 Telmisartan + Chlorthalidone Tablets

Telmisartan belongs to the class of Angiotensin II receptor antagonists, also known

as angiotensin receptor blockers (ARBs) or sartans. These are a group of pharmaceuticals that

modulate the renin-angiotensin-aldosterone system. Their main uses are in the treatment

of hypertension (high blood pressure),diabetic nephropathy (kidney damage due to diabetes)

and congestive heart failure. ARBs are used primarily for the treatment of hypertension where

the patient is intolerant of ACE inhibitor therapy [35].

These substances are AT1-receptor antagonists; that is, they block the activation

of angiotensin II AT1 receptors. Blockage of AT1 receptors directly causes vasodilation,

reduces secretion of vasopressin, and reduces production and secretion of aldosterone, among

other actions. The combined effect reduces blood pressure. The specific efficacy of each ARB

withinthis class depends upon a combination of three pharmacodynamic and pharmacokinetic

41

parameters. Efficacy requires three key PD/PK areas at an effective level; the parameters of

the three characteristics, namely.,Pressor inhibition, AT1 affinity and biological half life.

Pressor inhibition relates to the degree of blockade or inhibition of the blood pressure-raising

("pressor") effect of angiotensin II. The specific AT1 affinity relates to how specifically

attracted the medicine is for the correct receptor. The third area needed to complete the

overall efficacy picture of an ARB is its biological half-life, time it takes for a substance to

lose half of its pharmacologic, physiologic activity. Telmisartan, is having 40% pressor

inhibition, 3000 fold AT1 affinity and 24 hours half-life.

Chlorthalidone is a diuretic drug used to treat hypertension. It is described as

a thiazide diuretic. Chlortalidone increases the excretion of sodium, chloride, and water into

the renal lumen by inhibiting sodium ion transport across the renal tubular epithelium. Its

primary site of action is in the cortical diluting segment of the ascending limb of the loop of

Henle. Thiazides and related compounds also decrease the glomerular filtration rate, which

further reduces the drug's efficacy in patients with renal impairment (e.g. renal insufficiency).

It initially lowers blood pressure by decreasing cardiac output and reducing plasma and

extracellular fluid volume. Eventually, cardiac output returns to normal, and plasma and

extracellular fluid volume return to slightly less than normal, but a reduction in peripheral

vascular resistance is maintained, thus resulting in an overall lower blood pressure. The

reduction in intravascular volume induces an elevation in plasma renin activity and

aldosterone secretion, further contributing to the potassium loss associated with thiazide

diuretic therapy [36].

Both ARBs and Diuretics are well established as antihypertensive

monotherapies.Chlorthalidone is prescribed in combination with beta blockers (eg : Atenolol)

42

and ARBs (Telmisartan, Olemesartanetc). Recent outcome studies suggest that ARBs have

beneficial effects beyond those expected from BP lowering alone. They appear more effective

in preventing stroke than β blockers. Therefore, a Fixed dose combination of sartans with

diuretics has gained wider prescription. Hence, author has chosen to study the available

literature on the analytical methods for analysis of fixed dose combination of Telmisartan and

Chlorthalidone. Review of Literature has revealed that there is no common methods available

for estimation of impurities of both Telmisartan and Chlorthalidone simultaneously. Author

has seen an opportunity to develop a single analytical method for estimating Assay and a

single method for determination of impurities for this combination product.

1.2.4 Metoprolol + Atorvastatin + Ramiprilcapsules

Metoprolol is a selective β1 receptor blocker used in treatment of several diseases of

the cardiovascularsystem including: hypertension, angina, acute myocardial infarction, supra-

ventricular tachycardia, ventricular tachycardia, congestive heart failure, and prevention

of migraine headaches. Due to Metoprolol's β1 selectivity, it tends to interfere less with

asthma drugs. Due to its selectivity in blocking the β1 receptors in the heart, Metoprolol is

also prescribed for use in performance anxiety, social anxiety disorder, and other anxiety

disorders [37].

Atorvastatin marketed by Pfizer as a calcium salt under the trade name Lipitor, is a

member of the drug class known as statins, used for lowering blood cholesterol. It also

stabilizes plaque and prevents strokes through anti-inflammation and other mechanisms.

Atorvastatin was first synthesized in 1985 by Bruce Roth of Parke-Davis Warner-Lambert

Company (since acquired by Pfizer). The best selling drug in pharmaceutical history, sales of

Lipitor since it was approved in 1996 exceed US$125 billion, and the drug has topped the list

of best-selling branded pharmaceuticals in the world for nearly a decade.

43

The primary usesof Atorvastatin is for the treatment of dyslipidemia and the

prevention of cardiovascular disease. It is recommended to be used only after other measures

such as diet, exercise, and weight reduction have not improved cholesterol levels.Like all

statins, atorvastatin works by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-

CoA) reductase, an enzyme found in liver tissue that plays a key role in production of

cholesterol in the body. Inhibition of the enzyme decreases de novo cholesterol synthesis,

increasing expression of low-density lipoprotein receptors (LDL receptors) on hepatocytes.

This increases LDL uptake by the hepatocytes, decreasing the amount of LDL-cholesterol in

the blood. Like other statins, atorvastatin also reduces blood levels of triglycerides and

slightly increases levels of HDL-cholesterol. Atorvastatin has gained significant popularity

among medical practitioners due to its ability to decrease LDL-cholesterol and increase HDL-

cholesterol and hence preventing cardiovascular diseases. Over a period of time, several fixed

dose combinations which contains atorvastatin gained popularity in treatment of diseases

which are interrelated [38].

Ramipril is an angiotensin-converting enzyme (ACE) inhibitor, used to treat high

blood pressure and congestive heart failure. ACE inhibitors, as the name suggests, inhibit the

actions of angiotensin converting enzyme (ACE),thereby lowering the production of

angiotension II and also decreasing the breakdown of bradykinin.Thedecrease in angiotensin

II results in relaxation of arteriole smooth muscle leading to a decrease intotal peripheral

resistance, reducing blood pressure as the blood is pumped through widened vessels [39].

The Triple drug combination is a fixed dose combination to address an unmet medical

need as 2nd line treatment of patients with both essential hypertension and

44

hypercholesterolemia who are not adequately controlled by combination of an anti-

hypertensive with lipid lowering agent. Review of Literature has revealed that there is no

common methods available for estimation of impurities of triple combination drug product

simultaneously. Author has seen an opportunity to develop a single analytical method for

estimating Assay and a single method for determination of impurities for this combination

product.

The present research study was focuses on the development of new stability-indicating

analytical methods for four pharmaceutical dosage forms which contains eight active

pharmaceutical ingredients. The research work also reveals the validation of the developed

LC methods as per ICH requirement and shows the fitness of developed methods to monitor

the stability study of the pharmaceutical dosage forms. The list of active pharmaceutical

compounds taken for research study was listed in the below Table 1.4

45

Table 1.4The Chemical names, structure of Active pharmaceutical ingredients (API)

S.No. API/ chemical names Structure Molecule Name

1 2-[[(4-(3-methoxypropoxy)-3-

methyl-2-

pyridinyl)methyl]sulfinyl]-1H-

benzimidazole

Rabeprazole

2 5-methoxy-2-[{(4-methoxy-3,5-

dimethyl-2-pyridinyl)methyl}sulfinyl]-

1H-benzimidazole

Omeprazole

5-chloro-1-[1-{3-(2,3-dihydro-2-

oxo-1H-benzimidazol-1-yl)propyl}-

4-piperidinyl]-1,3-dihydro-2H-

benzimidazol-2-one

Domperidone

3 2-(4-{[4-methyl-6-(1-methyl-1H-

1,3-benzodiazol-2-yl)-2-propyl-1H-

1,3-benzodiazol-1-

yl]methyl}phenyl)benzoic acid

Telmisartan

46

API/ chemical names Structure Molecule Name

2-chloro-5-(1-hydroxy-3-oxo-2,3-

dihydro-1H-isoindol-1-yl)benzene-

1-sulfonamide

Chlorthalidone

4 1-[4-(2-methoxyethyl)-phenoxy]-3-[(1-

methylethylamino]-2-propanol

Metoprolol Tartrate

1H-pyrrole-1-heptanoic acid, [R-

(R*,R*)]-2-(4-flurophenyl)-β , d-

dihydroxy-5-(1-methylethyl)-3-phenyl-

4-[(phenylamino) carbonyl]-calcium

salt

AtorvastatinCa

1S,5S,7S)-8 [(2S)-2-[[(1S)-1-

ethoxycarbonyl-3-phenyl

propyl]amino]-

propanoyl]-8azabicyclo[3.3.0] octane-

7-carboxylic acid

Ramipril

47

48

References

1. Ahuja S, Stephen S, Hand book of Modern Pharmaceutical Analysis, Separation Science

and Technology. Volume III.

2. ICH. Harmonized tripartite guidelines for stability testing for new drug substances and

productsQ1A(R2), 2003.

3. Sanjay B, Neha S, Stability Testing of Pharmaceutical Products, Journal of Applied

Pharmaceutical Science. 2012, 02, 129-138.

4. International Conference on Harmonization; http://www.ich.org/products/ guidelines.

5. George N, Forced Degradation as an Integral Part of HPLC Stability-Indicating Method

Development, Drug delivery Technology. 2010, 10, 5.

6. Simon RJ, Hicks, Forced Degradation to Develop Stability-indicating Methods, The journal of

pharmaceutical and Biopharmaceutical contract services. January 01 2012.

7. Alsante KM, Martin L, Baertschi S, “A Stress Testing Benchmarking Study”, Pharm Technol. 2003,

27, 60-72.

8. Klick S, Muijselaar PG, Waterval J, Eichinger T, Korn C, Gerding TK, Debets AJ, Sangervan de

Griend C, van den Beld C, Somsen GW, De Jong GJ, “Towards a Generic Approach for Stress

Testing of Drug Substances and Drug Products”, Pharm Technol. 2005, 29, 48-66.

9. Dan WR, Kevin LF, June FM, Karen MA, Todd DH, Michael GM, Available Guidance and Best

Practices for Conducting Forced Degradation Studies, Pharm Technol. 2002, 48-52.

10. FDA, Center for Drug Evaluation and Research, “Submitting Documentation for the Stability of

Human Drugs and Biologics”, Rockville, MD, February 1987.

11. ICH. Stability Testing: Photostability Testing of New Drug Substances and New Drug

ProductsQ1B, 1996.

12. ICH. Impurities in New Drug SubstancesQ3A(R2),2006.

49

13. ICH. Impurities in New Drug Products Q3B(R2),2006.

14. ICH, Guidance for Industry, M4Q: CTD — Quality, August 2001.

15. Leven S, High Performance Liquid Chromatography (HPLC) in the Pharmaceutical Analysis,

Pharmaceutical Sciences Encyclopedia. 2010, 1–34.

16. Kumar A, Saini G, Nair A, Sharma R, UPLC: a preeminent technique in pharmaceutical analysis,

Acta Pol Pharm. 2012, 69, 371-80.

17. Maron N, Wright G, Application of photodiode array UV detection in the development of

stability-indicating LC methods: determination of mefenamic acid, J Pharm Biomed

Anal. 1990, 8,101-105.

18. Lloyd R Snyder, Joseph J Kirkland, Joseph L Glajch, Practical HPLC Method Development, Second

Edition, 1997.

19. MacNair JE, Patel KD, Jorgenson JW, Ultra pressure reversed phase capillary Liquid

Chromatography: Isocratic and gradient elusion using columns packed with 1ìm particles, Anal

Chem. 1999, 71, 700–708.

20. Wu N, Lippert JA, Lee ML, "Practical aspects of ultrahigh pressure capillary liquid

chromatography, J Chromatogr. 2001, A911, 1–12.

21. Swartz M E, Ultra Performance Liquid Chromatography (UPLC): An Introduction, Separation

Science Re- Defined, LCGC Supplement.2005, 8-11.

22. Jerkovich AD, Mellors JS, Jorgenson JW, The use of micrometer-sized particles in ultrahigh

pressure liquid chromatography,LCGC. 2003, 21, 600–610.

23. MacNair JE, Lewis KC, Jorgenson JW, Ultrahigh-pressure reversed-phase liquid chromatography

in packed capillary columns,Anal Chem. 1997, 69, 983–989.

24. Van DeemterJJ, Zuiderweg EJ, Klinkenberg A, Longitudinal diffusion and resistance to mass

transfer as causes of non ideality in chromatography,ChemEng Sci. 1956, 5, 271–289.

50

25. Michael E Swartz, UPLC: An Introduction and Review Waters Corporation, Milford,

Massachusetts, USA, Journal Chromatography & Related Technologies.2005, 28, 1253–1263.

26. Baertschi SW, The Role of Stress Testing in Pharmaceutical Product Development,

presented at the American Association of Pharmaceutical Scientists Midwest Regional

Meeting, Chicago, IL, May 20, 1996.

27. EURACHEM: The fitness for purpose of analytical methods. A Laboratory

Guide to Method Validation and Related Topics. http://www. eurachem.ul.pt, 1998.

28. ICH.Harmonized Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology

Q2(R1),2005.

29. Carr GP, Wahlich JC, A practical approach to method validation in pharmaceutical analysis,J

Pharm Biomed Anal. 1990, 86, 613-618.

30. Jenke DR. Chromatographic method validation: a review of common practices and procedures II,

J LiqChromat. 1996, 19, 737-757.

31. Vessman J, Selectivity or specificity? Validation of analytical methods from the

perspective of an analytical chemist in the pharmaceutical industry,J. Pharm.

Biomed.Anal.1996, 14, 867–869.

32. http://en.wikipedia.org/wiki/Rabeprazole

33. http://en.wikipedia.org/wiki/Omeprazole

34. http://en.wikipedia.org/wiki/Domperidone

35. http://en.wikipedia.org/wiki/Telmisartan

36. http://en.wikipedia.org/wiki/Chlortalidone

37. http://en.wikipedia.org/wiki/Metoprolol

38. http://en.wikipedia.org/wiki/Atorvastatin

51

39. http://en.wikipedia.org/wiki/Ramipril