stress testing

Pharmaceutical Technology On-Line APRIL 2000 1

he International Conference on Harmonization (ICH)guideline entitled ‘Stability Testing of New Drug Sub-stances and Products’ (Q1A) requires that stress testingbe carried out to elucidate the inherent stability charac-

teristics of the active substance (1). It suggests that the degra-dation products that are formed under a variety of conditionsshould be identified and degradation pathways established. Itis stated that the testing should include the effect of tempera-ture, humidity where appropriate; oxidation, photolysis andsusceptibility to hydrolysis across a wide range of pH values.

In the guideline, the study of the effect of temperature is sug-gested to be done in 10 �C increments above the acceleratedtemperature test condition (e.g. 50 �C, 60 �C, etc.) and that ofthe humidity at a level of 75 percent or greater. No details are,however, provided for the study of oxidation, photolysis andhydrolysis at different pH values. In the absence of guidance,difficulties are faced by practitioners to decide on the stress con-ditions to be employed for a new drug at the time of initiationof forced decomposition studies.

In this write-up the authors propose a classification systemfor categorization of drugs based on their inherent stability be-haviour. Flow charts have been drawn that are suggested to befollowed for determining the stress test conditions for hydro-lysis (under neutral, acid and alkaline conditions), oxidation,and photolysis.

The variable behavior of decomposition of drugs andthe dilemmasIt is well known that drugs vary very widely in their suscepti-bility to decomposition conditions. They follow different re-action pathways and show at times a very large variability inthe rate of decay. A fall-out of this variable behavior is that itbecomes difficult to decide on the stress conditions to be em-ployed for a new drug at the time of initiation of forced de-composition studies. Other dilemmas faced by practitioners arei) To what extreme of conditions one should go if a previouslytested condition does not give sufficient degradation, goodenough for degradation products to be isolated in quantity suit-able for characterisation and structure elucidation? ii) Are thereany limits where one should stop and carry no further studies?and iii) What sort of reagents or agents need to be employed

Guidance on Conduct of

Stress Tests to DetermineInherent Stability of DrugsSaranjit Singh* and Monika Bakshi

Saranjit Singh, PhD, is an associateprofessor and Monika Bakshi is doctoralstudent at the National Institute ofPharmaceutical Education and Research(NIPER), Sector 67, SAS Nagar 160 062,India, tel. +91 172 673848, fax +91 172677185, e-mail ([email protected])

*To whom all correspondence should be addressed.

TThe authors in this write-up provide guidanceon the conduct of stress tests for the deter-mination of inherent stability of drugs underhydrolytic, oxidative and photolytic con-ditions. A classification system is proposedand decision trees are drawn to guide on howto start and where to end in the search of theright type of stress conditions for a new drug.The predictability of the pathways of degra-dation of the drug, based on the functionalgroups present and overall structure, isdiscussed. Other considerations like benefit ofharsh conditions for the isolation of degra-dation products and the advantage ofcatalytic activity of metal ions are alsotouched upon. In addition, useful informationis provided on practical conduct of stressstudies.

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for creating a particular stress condition? Here the questionsare generally put in the form — For study of oxidation, whichamong the various oxidising agents is the best to use ? Does itmake difference if hydrochloric acid is substituted with sulfu-ric acid to maintain high acidity conditions ? etc. A general ques-tion quite often asked is — Are there any flow charts or deci-sion trees which can be followed as standards for all the drugsso that right stress conditions can be determined in minimumpossible trials?

The quest for answersUnfortunately, the practical aspects related to stress testing areneither addressed by regulatory guidelines, nor by any otherdocument. Therefore, in an endeavor to provide the practi-tioners with the answers to the volley of these questions andto examine the possibility of developing flow charts, a criticalstudy was done of the reports in literature for the stress con-ditions used for determining inherent stability of new drug

molecules. Most useful information was found in the mono-graphs given in the volumes of Analytical Profiles of Drug Sub-stances (2). The monographs carry a stability section whichrecords the inherent stability behavior of the drug and the con-ditions employed to determine the same. Examples of drugswhere complete information with respect to strength of re-actant, temperature of study, time period of exposure and ex-tent of decomposition was available were picked up. The men-tion of use of stress conditions was also found in some reportsin literature on establishment of stability-indicating assays.The total information was tabulated for different types of re-activities, viz., hydrolysis in acid, alkaline and neutral condi-tions, oxidation and photolysis. In each table, the drugs werelisted in an order of exaggeration of the strength of the reactant.The information, in a brief form, is given in Tables I–V.

The stress conditions used for the study of decomposition inacid conditions revealed that hydrochloric acid at a strength of0.1 N was mostly used. There were a few reports that indicated

Table I: Selected examples of stress conditions used for hydrolysis of drugs in acid.Reaction

Drug Strength Conditions Time Remarks Ref.

Dipyridamole 0.1 N HCl 40 �C 1 week Negligible degradation 19Hydrochlorthiazide 0.1 N HCl — 16 h Degradation at the 20

rate of 0.1% hEthacrynic acid 0.1 N HCl 65 �C 21 days 28.4% drug left 2*Trifluoperazine 0.1 N HCl 94 �C 95 h Total degradation 21Ketoprofen 0.1 N acid 98 �C 30 min No decomposition 2Morphine 0.1 N HCl Refluxing 2 h No degradation 2Metoprolol tartarate 0.1 N HCl Refluxing 20 h No chemical change 2Retinoic acid 0.1 N HCl Refluxing 5 min 65% recovery of drug 3Maprotiline HCl 0.1 N HCl Refluxing 94 h No degradation 2Timolol maleate 0.1 N HCl 105 �C 2 months Considerable degradation 2Nabilone 0.1 N acid 110 �C 1 week Stable 2Sodium levothyroxine 0.1 N HCl — 44 h 92.8% recovery of drug 22HI-6 pH 2.54 HCl 70 �C 100 h Around 50% degradation 23Suprofen 1 N HCl 50 �C 72 h Quantitative recovery 4Esmolol HCl 1 N HCl Boiling 1 h Total degradation 24Benperidol 1 N HCl 100 �C — Gradual decomposition 2Norfloxacin 2 N HCl 100 �C — Decarboxylated degradate 2

formedBetaxolol 2.5 N HCl Boiling 20 min No drug left 25Clioquinol 4 N HCl Refluxing 2 days 34% drug left after 2 days 2Sertraline HCl 5 N HCl Refluxing 3 h No significant degradation 2Lidocaine 6.5 N HCl 108 �C 24 h 50% hydrolysis 2Chlorobutanol conc. HCl R.T. — 43.5% recovery 26Lidocaine 50% H2SO4 116 �C 5 h 3% decomposition 2Spironolactone 0.1 N H2SO4 Boiling — Boiling till volume reduced 2

to one-third led to no decomposition

Mefenamic acid 0.5 N H2SO4 Refluxing 48 h No decomposition 27Omeprazole 1 N H2SO4 Boiling 5 min Total decomposition 28Cephalexin 1 N H2SO4 Boiling 5 min No degradation 29Acyclovir 1 N H2SO4 Boiling 10 min Potency reduced to 88% 2Ranitidine 1 N H2SO4 Boiling 20 min 15% loss in potency 2

*The references marked 2 are from Analytical Profiles of Drug Substances. It is suggested that one should look into the cumulative index ofdrugs in the latest volume of the profiles and search for the name of the drug to reach the monograph.

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the use of 1 N HCl and even higher normalities (Table I). Therewere a few instances where sulfuric acid in varying normalitieshad been used. In a few other cases, mention was only found of‘acid’ without defining the type used. Large variations were alsoseen in the reaction (temperature) conditions and periods ofstudy. The temperature range varied between 40 �C and 110 �C.Examples can be seen in Table I where drugs were kept around100 �C or under boiling conditions for periods ranging from afew minutes to as long as even 2 months. The extent of de-composition also varied a lot, for example, a 35% loss of retinoicacid was observed on refluxing in 0.1 N HCl for just 5 min (3),while there are reports where no decomposition of the drugwas seen after refluxing in 0.1 N acid for even one week(nabilone).

It is evident from Table II that stress conditions used for thehydrolysis of drugs under alkaline conditions run parallel tothose used for acid conditions. Sodium hydroxide is mostlyused, at strength of 0.1 N and 1 N. Potassium hydroxide figures

only in few cases. Just like acidic degradation, lot of variationis observed in time and temperature of exposure of drugs to al-kali. Depending on the inherent stability characteristics, somedrugs show no degradation even after refluxing in 0.1 N NaOHfor one week (nabilone) while others like trifluoperazine un-dergo complete degradation when they are kept in 0.1 N alkalifor 24 h at a very low temperature of 30 �C. Similar behavior isseen when the drugs are treated with 1 N NaOH. In some cases,boiling with 1 N NaOH for 5–20 min led to extensive degra-dation or almost total loss of potency (acyclovir, ranitidine,etc.), while on the other hand boiling for 15 h did not cause anychange. Similarly, suprofen (4) exhibited 92.8% recovery evenafter heating in 1 N NaOH at 50 �C for 72 h, while retinoic aciddegraded by 70% after refluxing in 0.1 N KOH for a short pe-riod of 30 min (3).

Not many reports could be found that gave stress conditionsfor neutral pH. As may be seen from the examples given in TableIII, no significant degradation was obtained whether the tem-

Table II: Selected examples of stress conditions used for hydrolysis of drugs in alkali.Reaction


Trifluoperazine 0.1 N NaOH 30 �C 24 h Total decomposition 2Dipyridamole 0.1 N NaOH 40 �C 1 week Negligible degradation 19Tolnaftate 0.1 N NaOH 50 �C 24 h Negligible degradation 2Ethacrynic acid 0.1 N NaOH 65 �C 21 days Total decomposition 2Mefenamic acid 0.1 N NaOH Refluxing 48 h Very less decomposition 27Clioquinol 0.1 N NaOH Refluxing 2 days 32% drug left 2Morphine 0.1 N NaOH Refluxing 2 h No degradation 2Metoprolol tartarate 0.1 N NaOH Refluxing 20 h No chemical change 2Maprotiline HCl 0.1 N NaOH Refluxing 94 h Slight degradation 2Sodium levothyroxine 0.1 N NaOH — 87.5 h 90.6% recovery 22Spironolactone 0.1 N NaOH Boiling — Boiling till volume 2

reduced to one-third led to total decomposition

Nabilone 0.1 N base Refluxing 1 week Stable 2Morphine 0.125 N NaOH, Refluxing 4 h Around 50% degradation 2

passage of airXylometazoline HCl 0.5 N NaOH Refluxing 30 min No hydrolysis 2Polythiazide 1 N NaOH 35 �C 1 h Total degradation 2Meperidine HCl 1 N NaOH 50 �C 48 h Zero potency 30Suprofen 1 N NaOH 50 �C 72 h 92.8% recovery 4Omeprazole 1 N NaOH Boiling 5 min No degradation 28Cephalexin 1 N NaOH Boiling 5 min Extensive degradation 29Acyclovir 1 N NaOH Boiling 10 min Potency decreased to 95% 2Ranitidine 1 N NaOH Boiling 20 min Drug degraded by 84.4% 2Metronidazole 1 N NaOH Boiling 25 min Total decomposition 31Esmolol HCl 1 N NaOH Boiling 1 h Total degradation 24Norfloxacin 1 N NaOH 100 �C 15 h No change 2Benperidel 1 N NaOH 100 �C — Gradual decomposition 2Sertraline HCl 5 N NaOH Refluxing 3 h No significant degradation 2Morphine 5 N NaOH Refluxing 4 h Total degradation 2Dipivefrin HCl pH 9 — 1 day Complete degradation 2

Chlorobutanol Conc. NH4OH — — 40% recovery 26Retinoic acid 0.1 N KOH Refluxing 30 min 30% drug left 3



perature was 35 �C or refluxing conditions were used. The test-ing evidently is generally done in water. The slow rate of de-composition in neutral conditions is understandable becausereactions at neutral pH are non-catalytic and hence very longperiods under exaggerated temperature conditions may be re-quired to get sufficient quantities of degradation products.

Table IV depicts the stress conditions employed for the studyof oxidation. Evidently, hydrogen peroxide seems to be muchmore popular for the purpose than any other oxidising agent.The strength of H2O2 used varies between 1% to 30%. In somedrugs extensive degradation is seen when exposed to 3% H2O2

for very short time periods at room temperature (e.g., raniti-dine HCl and cimetidine HCl). In other cases, exposure to highconcentrations of H2O2 even under extreme conditions doesnot cause any significant degradation (e.g., sertraline HCl, zileu-ton, etc.). The behavior is on expected lines, as some drugs arein fact oxidisable, while there are others that are not. The latterare not expected to show any change even in the presence ofhigh dose of oxidising agents.

Table V emphasises the fact that there is a lot of variation inthe manner in which stress photostability testing is done on dif-ferent drugs. Mostly the drugs areexposed to short/long wavelengthUV light, or fluorescent light ofvarying illumination (400–1580foot candles). Efforts are usuallymade to maintain the temperaturearound room temperature (R.T.).The period of exposure ranges froma few hours to several months, de-pending upon the light source in-tensity. Like oxidation, based onwhether a drug is photolytic or not,

varied type of decomposition behavior is seen. For example,cimetidine HCl and maprotiline HCl do not undergo any changewhen exposed to photolytic stress for several weeks, while pho-tosensitive retinoic acid degrades on exposure to UV light forvery short periods. The photolability studies are done on drugsin either solid form or solution. It is apparent from the tablethat studies are performed in water or in acidic and alkaline so-lutions and also on drug dissolved in either methanol or ace-tonitrile.

The classification systemDepending upon the information obtained as above, and basedon the previous personal experience on degradation chemistryof drugs (5–8), an exercise was carried out to see whether it waspossible to broadly classify the drugs into specific categoriesand define stress conditions for each of them. The following sixclasses could be identified:● Class I: Extremely labile● Class II: Very labile ● Class III: Labile● Class IV: Stable

Table III: Selected examples of stress conditions used for hydrolysis of drugs underneutral conditions.

ReactionDrug Medium Conditions Time Remarks Ref.

Celiprolol Water 37 �C 2 weeks Drug 2remains stable

Dipyridamole Water 40 �C 1 week Negligible 19degradation

Sertraline HCl Water Refluxing 3 h No significant 2degradation

Table IV: Selected examples of stress conditions used for oxidation of drugs.Reaction


Dipyridamole 1% H2O2 40 �C 1 week Some degradation 19Loperamide 1.5% peroxide — Immediately 3.2% cis N-oxide and 2.4% 2

trans N-oxide of drug formedCimetidine HCl 3% H2O2 R.T. Short period Sulfoxide formation 2Ranitidine HCl 3% H2O2 R.T. 20 min 37.8% loss in potency 2Didanosine 3% H2O2 37 �C 6 h 40% potency 2Suprofen 3% H2O2 50 �C 72 h 97.4% recovery 4Morphine 3% H2O2 Refluxing 30 min Extensive degradation 2Zileuton 5% H2O2 — 1 week 1% degradation 2Sertraline HCl 10% H2O2 Refluxing 6 h No significant degradation 2Esmolol HCl 30% H2O2 — 1 h �50% degradation 24Mefenamic acid 30% H2O2 — 24 h Extensive decomposition 27Chlorobutanol 30% H2O2 — — 24.8% recovery 26Zileuton 0.4% Sodium — Immediately 50% degradation 2

hypochloriteSodium Tert. butyl — 1 h 37.5% drug left 22levothyroxine hydroperoxideCimetidine HCl Exposure to O2 50 �C Short period Sulfoxide formation 2




Table V: Selected examples of stress conditions used for photolysis of drugs.Distance/

Drug Source Temperature Time Remarks Ref.

Nifedipine Visible/UV light — 30 min Drug on TLC plates 2converted to nitrosoderivative within 5–30 min;no intact drug left after 5 h

Trifluoperazine UV light (254 nm) 14 cm — — 21HClHI-6 UV light (254 nm) 30 cm — — 23Retinoic acid UV light (254 nm) 20 cm 2 h 50% recovery of drug 3Sodium Long wavelength — 48 h 101.4% recovery 22levothyroxine UV lightSodium Short wavelength — 144 h 98.9% recovery 22levothyroxine UV lightTimolol Intense UV radiation — 2 h Aqueous solutions (pH 5) 2maleate suffer 2% loss in potencyPyridoxine HCl UV light — — Destroyed by UV radiation 2

in neutral or alkaline solutionsbut not in acidic media

Verapamil UV light — 2 h Compound dissolved in 2methanol showed degradation by 52%

Cimetidine HCl UV light — Several months No decomposition 2Imipenem UV light — 24 h Brown surface discoloration 2Piroxicam 300–830 nm 30 �C 72 h 99.6% drug left 2Zileuton High intensity UV light — 4 h Total degradation in 2

acetonitrile solutions butless degradation inmethanolic solutions

Zileuton Fluorescent light — 2 weeks 35% and 55% degradation 2(600 and 1500 f.c.*) respectively in acetonitrile

solutionImipenem Fluorescent light — Several weeks No effect 2

(1000 f.c.)Aztreonam Fluorescent light 33–35 �C 1 week 6% conversion to E isomer 2

(400 and 900 f.c.)Azathioprine Fluorescent or — 4 weeks Dark orange surface 2

UV light developedMaprotiline HCl 600 f.c. — 12 weeks Stable 2Clioquinol 600 f.c. — 6 days 30% drug left in 2

acetonitrile solutionChloroxazone Artificial light — 4 weeks No degradation 2

(1000 f.c.)Nabilone 200 W high pressure — 2 days Irradiation of drug in 2

Vycor-filtered Hg arc ethyl alcohol yieldscis and trans diols

Didanosine High intensity light 30 �C 8 weeks Potency � 96% 2(1000 f.c.)

Terfenadine Intense fluorescent light 27 �C 8 weeks Drug remains stable 2Loperamide 17000 lux (1580 f.c.) — 7 days No degradation 2Nifedipine Halogen lamp (1000 W) — Conversion to nitroso 2

derivative begins after 6 hand is completed after months

*f.c. denotes foot candles.



● Class V: Very stable● Class VI: Practically stable

The proposed stress conditions for the hydrolysis of each ofthe six classes of drugs under acid and alkaline conditions aregiven in Table VI. The term ‘sufficient decomposition’ is takenin the broadest sense. It may mean 80–100% decomposition ifthe objective is isolation of the degradation products, or be-tween 20–80% decomposition when the objective is to estab-lish the degradation pathways. In the latter case, the reaction isusually monitored up to two to three half-lives and the rise andfall of each degradation product is checked. The monitoring ofreaction is also an essential prerequisite for the establishmentof stability-indicating assays in which it is required that evenintermediate degradation products, if any, should not interferein the analysis of drug at any stage of reaction.

For hydrolysis under neutral conditions (Table VII), the drugsare classified into the same six categories. However, the timeperiod of exposure is suggested to be longer as the drugs areknown to hydrolyse much slower in neutral conditions than incatalytic acid and alkaline conditions.

The classification of drugs for oxidative decomposition isgiven in Table VIII. Hydrogen peroxide is recommended as theoxidising agent at different concentrations.

For photoreactions, unlike the above classification for hydro-lytic and oxidative decompositions, the distribution of drugs islimited only to two categories — photostable and photolabile(Table IX). Here the exposure conditions are based on thosesuggested by ICH. If drugs show sufficient or total decompo-sition under the ICH recommended total exposure of 1.2 � 106

lux hours (9), the drug is photolabile. Otherwise, if no changesare seen, the total exposure should be increased to 6.0 � 106

lux hours (10). Drugs stable to this high exposure can be safelydeclared as photostable.

The decision treesFigures 1–4 give the decision trees for investigating differenttypes of stress conditions for a new drug substance. The gene-ral approach taken in the construction of these flow charts isthat the new drug is assumed to be labile in nature and, ac-cordingly, it is subjected to stress conditions given for labile sub-stances in Tables VI–IX. Dependent upon the results, decisionis taken on whether to increase or decrease the strength of thereaction conditions. The increase or decrease, if required, isdone step-wise and those stress conditions are accepted wher-ever a sufficient decomposition is obtained.

Figure 1: Flow chart for performing stress studies for hydrolytic degradation under acid and alkali conditions.




Figure 1 suggests that in order to study hydrolytic degrada-tion (under acidic and alkaline conditions) of a new drug, whosestability behavior is not known, one can start by refluxing thedrug in 0.1N HCl/NaOH for 8 h, considering that the drug islabile. If a reasonable degradation is observed on subjecting thedrug to this treatment, no further studies need to be carriedout. In case no degradation is seen, drug should be subjectedto refluxing in 1N acid/alkali for 12 h. For a drug which canwithstand even these conditions, more extreme conditions ofacidity or alkalinity such as refluxing in 2 N HCl/NaOH for 24 h may be tried. The reaction should be monitored, and ifstill satisfactory change is not obtained, the drug should be re-fluxed in 5 N HCl/NaOH for up to 24 h. The drug may be de-clared to be “practically stable’’ if no hydrolytic products are

formed on subjecting the drug to this harsh condition. Goingto the other side of starting condition, if a total degradation isseen after refluxing in 0.1 N HCl/NaOH for 8 h, the strength ofacid/alkali can be decreased to 0.01 N along with decrease oftemperature to 40 �C while keeping the time as same 8 h. A drugshowing complete degradation even in these mild conditionsshould be treated with 0.01 N HCl/NaOH for 2 h at 25 �C andif still complete degradation is taking place, drug is extremelylabile and has to be tested under very mild conditions of tem-perature and pH.

Stress testing under neutral conditions can be started by re-fluxing the drug in water for 12 h (Fig. 2). Refluxing time shouldbe increased to 1 day in case no degradation is seen. It shouldbe increased further to 2 days if no change is observed. In case

of negligible degradation, the drug maybe refluxed for a period of 5 days. If stillfound stable, the drug may be declarednondegrading in neutral conditions.For this study, it may be advisable thata sufficient volume of solution shouldbe taken for the reaction initially, so thatthe time period can be continually in-creased, as required, and there is noneed to restart the reaction afresh. Fora drug undergoing complete degra-dation on refluxing in water for 12 h,both time and temperature of exposuremay be decreased to 8 h and 40 �C, re-spectively. More mild conditions, likekeeping the drug in water up to 2 h at25 �C, should be tried if no intact drugis left after exposure to above men-tioned conditions.

For determining the susceptibility ofthe drug to oxidative decomposition,testing may be started by keeping thedrug in 3% H2O2 for 6 h at room tem-perature (Fig. 3). The period of reac-tion should be increased to 24 h in casethere is no sufficient degradation. Stillif there is no change, the reactionshould be conducted in 10% H2O2 for24 h. For a drug which does not oxidiseeven under these conditions, more ex-treme conditions of 30% H2O2 for 24 h may be tried. The drug may be de-clared to be “practically stable’’ if noproducts are formed on subjecting thedrug to this condition. In an event ofdecomposition of whole drug under thestarting conditions, the strength ofH2O2 should be decreased from 3% to1% and the reaction may be monitoredfor a period sufficient to yield the de-sired percent of decomposition. Thedrugs undergoing complete degrada-tion even under these conditions are

Table VIII: Classification system for oxidative degradation.Strength of Time of Extent of

Category of drug hydrogen peroxide exposure Temperature decomposition

Practically stable 30% 48 h R.T. NoneVery stable 10% 24 h R.T. SufficientStable 3% 24 h R.T SufficientLabile 3% 6 h R.T. SufficientVery labile 1% 3 h R.T. SufficientExtremely labile 1% 30 min R.T. Sufficient

Table IX: Classification system for photolytic degradation.Category of drug Total exposure Temperature Extent of decomposition

Photolabile 1.2 � 106 lux h R.T. Sufficient or totalPhotostable 6.0 � 106 lux h R.T. None

Table VII: Classification system for hydrolysis underneutral conditions.

Extent ofCategory of drug Time of exposure Temperature decomposition

Practically stable 5 days Refluxing NoneVery stable 2 days Refluxing SufficientStable 1 day Refluxing SufficientLabile 12 h Refluxing SufficientVery labile 8 h 40 �C SufficientExtremely labile 2 h 25 �C Sufficient

Table VI: Classification system for acidic or alkaline hydrolysis.Strength of Time of Extent of

Category of drug acid/alkali exposure Temperature decomposition

Practically stable 5 N 2 days Refluxing NoneVery stable 2 N 1 day Refluxing SufficientStable 1 N 12 h Refluxing SufficientLabile 0.1 N 8 h Refluxing SufficientVery labile 0.01 N 8 h 40 �C SufficientExtremely labile 0.01 N 2 h 25 �C Sufficient



highly prone and should be tested in very dilute oxidising agentwith an exposure for very short duration.

In order to get an idea about photostability (Fig. 4), the drugsubstance should be initially subjected to an illumination up to1.2 � 106 lux hours which is the ICH recommended exposureand the reaction should be monitored periodically. The expo-sure may be increased by 5 folds in case there is negligible degra-dation. The drug may be declared photostable if the increase inexposure to 6.0 � 106 lux hours has no effect on the stability ofthe drug.

Postulation of the intrinsic stability behaviorfrom structureWhat is suggested above is applicable, in general, to any drugmolecule. However, following of all the steps given in the flowcharts can be avoided, when the new drug molecule is ‘close’in structure to the compound(s) whose inherent stability isalready known. In that event, one can go directly to the stepthat applies to the ‘like’ structure(s) and thus bypass the oth-ers in-between.

An idea on the degradation pathway likely to be followed by

the new drug can be obtained from the study of functionalgroups in the structure. Drugs with specific functional groupsundergo typical type of reactions. For example, esters and �-lactams are hydrolysed when subjected to decomposition. Thi-ols undergo oxidation. Likewise, drugs with specific functionalgroups undergo photolysis. The types of functional groups re-sponsible for decomposition of drugs by the three major routesare listed in Tables X–XII. Examples of drugs are also included,wherever possible.

A major percentage of new drugs are just modifications ofexisting drugs. In general, a new drug which is a variant of thealready known homologous series would show a close inherentstability behavior to the existing drug(s), provided there is noinfluence of modifying groups or major change at the site ofreaction. For example, there are over 40 penicillins in the mar-ket today and with all having an intact labile beta-lactam group,they are expected to show a similar degradation pattern. A studywas recently done in our laboratories on establishment of quan-titative structure-reactivity relationships among 16 penicillins(11) and it was found that degradation rate constants of mostof them clustered around one point and that of the remaining

Figure 2: Flow chart for performing stress studies for hydrolytic degradation under neutral conditions.




were also not very different. Similar is the case expected ofcephalosporins or other homologous series of compounds wherethe parent part of the molecules remains unchanged.

There are some functional group categories where drugs fol-low varied stability behavior. For example, some amides undergo hydrolysis very rapidly while others decompose slowly.Moreover, there are several examples where the drugs are un-stable in acid conditions but are stable in alkaline conditions,or vice versa. Therefore, a true idea on the severity of condi-tions required for use on new drugs can be obtained from achart drawn in a manner in which drugs are listed functionalgroup wise and further divided into the six classes defined ear-lier. Such a classification is required to be all comprehensive.Unfortunately, a classification of this type does not exist till

date. Preparing such a classification means an extensive searchof the literature for stability information, including deep studyof reports on decomposition kinetics of a large number of com-pounds. It may be considered as a subject matter of a futurereport.

Other general considerationsAlternate stress conditions for each class. Figure 1 suggests testingto be initiated by refluxing the drug in 0.1 N acid or alkali for8 h. There exists a possibility of using alternate conditions inwhich the acid strength is increased and the same is compen-sated by reducing the period of reaction. In sum, there may betwo or more possibilities for each reaction condition where thesame extent of decomposition can be obtained. In such situa-

Table XI: Functional group based classification of drugs (with examples) that undergo different types of oxidative reactions.*Functional group Degradation reaction Drug examples

Thiols Formation of S-oxides Dimercaprol, Captopril,and disulfides Cysteine, 6-mercaptopurine

Thioethers Formation of S-oxides Cimetidine, RanitidineBicyclic or tricyclic Formation of dimers Naphthol, Morphinephenolic compounds�-ketols Formation of glyoxals SteroidsPolyhydroxybenzenes Formation of quinones Epinephrine, HydroxyquinoneThiazines Formation of S-oxides PhenothiazinesUnsaturated compounds Formation of hydroperoxides Amphotericin B, Fatty acids

e.g. oleic acid, linoleic acid, etc.N-isopropyl Oxidation of N-isopropyl Practolol, Propranolol, Sotalolethanolamine derivatives ethanolamine grouplIndole derivatives Oxidation of N–H group Sumatriptan

in the indole moiety

*The list is only representative and not comprehensive.

Table X: Functional group based classification of drugs (with examples) that undergo different types of hydrolytic reactions.*

Functional group Degradation reaction Drug examples

Esters Lysis of ester group Atropine, Aspirin, Procaine,to acid and alcohol Nitroglycerin

Amides Lysis of amide group Chloramphenicol, Nicotinamide,to acid and amine Procainamide, Salicylamide

Lactams Lysis of lactam ring to Penicillins, Cephalosporins,open chain acid and alcohol Benzodiazepines

Lactones Lysis of lactone ring to Pilocarpine, Spironolactoneopen chain acid and alcohol

Imides Lysis of imide ring to Glutethimide, Ethosuximide,open chain products Barbiturates (di-imides)

Alkyl chlorides Conversion to Chloramphenicolcorresponding alcohols

Azomethines Bond breakage Nitrofurantoin

*The list is only representative and not comprehensive. Based on information in reference 32.



tion, should one prefer using a harsher condition spending lessertime or use the normal conditions, as suggested in either tablesor flow charts? The answer should always be in favor of the lat-ter. This is for the following reasons: i) there may be a changein mechanism of reaction when a harsh condition is used, andii) there is a practical problem in neutralising or diluting everysample, when it contains a high concentration of reactants, e.g.,acid or base, before an injection can be made on the HPLC col-umn. Both these reasons are strong enough to suggest that asnormal as possible conditions should be used for causing thedecomposition of the drug.

The role of harsh conditions for the isolation of products. Accord-ingly, while following the flow charts, one is expected to stop atthe step where sufficient degradation is obtained. If this hap-pens at the first one or two steps, then the drug generally is notto be subjected to harsh conditions of acid and alkali. However,in the experience of the authors, doing so is sometime advan-tageous, particularly if the end objective is the isolation of thedegradation products in pure form. In a very interesting case,a drug was refluxed in our laboratories in increasing concen-tration of NaOH (0.01 N, 0.1 N and 1 N) for 8 hours. When theHPLC was done, a new peak was shown to appear. The heightof the new peak was very small in 0.01 N NaOH which increasedalmost equal to the drug peak in 1 N NaOH. For further con-version of the drug to product, the concentration of alkali wasincreased to 2 N and the solution was refluxed for 12 hours.Good crystals of the products were obtained after overnightstorage. In a subsequent experiment, the same product wasfound to be formed immediately when the drug was broughtin contact with 5 N NaOH at room temperature. It was, there-

fore, very easy to isolate this unknown degradation product inpure form.

In another case, a research group had reported in literaturethe degradation chemistry of a drug at pH 10 and it was indi-cated that an unknown product was formed along with the onesthat were identified. In our laboratories, it was of our interest toisolate the degradation products of this drug for supply as stan-dards. Therefore, while attempting to isolate the products, at-tention was also paid to the unknown degradation product. Thedrug was heated under the stressful condition of 2 N NaOH at80 �C up to 10 minutes and the solution was left overnight. Inthe morning some shiny crystals were seen floating in the sample. These crystals were separated and subjected to TLC stud-ies. The spot and peak were surprisingly found to match thosefor the unknown. A separate reaction in concentrated ammo-nia solution resulted in formation of another product in pureform. This was a previously reported degradation product butprocedure for its isolation or synthesis was not known and at-tempts to synthesize it had earlier failed in our laboratories.

It has thus become a practice with us to try harsh conditionsof acid and alkali for the complete conversion of the drugs toproducts.

Can advantage be taken of the catalytic activity of the metal ions?It is well established that metal ions catalyze oxidative reactions.The addition of metal ions can accelerate the oxidative reactionup to several thousand times, for example, copper is consideredto be an extremely effective catalyst with the rate of monovalentascorbic acid oxidation increasing by ten thousand times. Shouldthen degradation studies in presence of metal ions be a part ofstandard stress testing protocol?

Table XII: Functional group based classification of drugs (with examples) that undergo different types of photolytic reactions.*Functional group Degradation reaction Drug examples

Olefins Isomeric conversion Retinoic acid, Cinnamic acid, NitrofurazoneIsomeric conversion

and/or photocyclization Stilboestrol, Dienoesterol, ClomipheneFormation of epoxide Menadione, NorethisteroneFormation of hydroperoxide Cyclobarbitone2�–2� cycloaddition of singlet

oxygen Menaquinone-I, PhylloquinoneDimerisation mediated by

2�–2� cycloaddition ThymineAryl acetic acids Decarboxylation Flurbiprofen, Naproxen, Ketoprofen, ButibufenAromatic nitro Reduction to nitroso group and Nifedipine, Furnidipine, Nirendipine, compounds oxidation of the ring Nicardipine, Nimodipine, Nitrazepam,

ChloramphenicolAryl halo derivatives Dehalogenation Chlorpromazine (and other chlorosubstituted

phenothiazines), Amiodarone, Frusemide, Diclofenac, Perphenazine

N-alkyl derivatives N-dealkylation Diphenhydramine, Methotrexate, Folic acid, Chloroquine

Benzophenones Formation of radical derivatives Fenofibrate, Ketoprofen, DithranolN-Oxides Formation of oxaziridines Chlordiazepoxide, Methaqualone-N-oxide

*The list is only representative and not comprehensive. Based on information in reference 33.




In the author’s personal viewpoint, the answer to this ques-tion should be ‘yes’. Logically, high acid and alkaline conditionsare also used in stress studies due to their catalytic activity. Nodoubt sometimes they also lead to newer degradation pathways,but the same is also true with metal ions. For the latter, an in-teresting example can be cited here. A company was adding asmall amount of hydrochloric acid to a formulation to bringdown the pH to maintain the color of the drug which waswhitish in neutral form. This formulation was being packed ina metallic container. A browning of the formulation was ob-served in samples kept on stability. It was later found that thedrug had a nitro group and in the presence of hydrochloric acidand the metal species (contributed by packaging and/or excipi-ents), the nitro group was getting reduced to amino group. Thisamino compound was further unstable resulting in brown col-ored products. In the absence of metal ions, the drug did notfollow this decomposition route.

If one really looks into literature, very different type of reac-tions have been reported to occur in presence of metal ions. Towhat may be a surprise to some, metals ions even have been im-plicated in hydrolytic reactions (12,13). Thus it is suggested that

studies in presence of metal ions should also be included instress testing protocols.

Important practical aspects in conduct of stress testingDrug concentration. Till this point, no discussion has been doneon the drug concentration of the reaction solution during stresstesting. From the observation of the concentrations used in dif-ferent literature studies and from personal experience of the au-thor, it is recommended that the studies should be initiated at aconcentration of 1 mg/ml. If solubility is a limitation, varyingamounts of methanol may be used to get a clear solution. Byusing drug concentration of 1 mg/ml, it is usually possible to geteven minor decomposition products in the range of detection.

It is suggested that some degradation studies should also bedone at a concentration at which the drug is expected to be pres-ent in the final formulations. The reason for proposing this arethe examples of aminopenicillins and aminocephalosporinswhere a range of polymeric products have been found to beformed in commercial preparations of containing drug in highconcentrations (14). Incidentally, little or no polymerization isshown at lower drug concentrations.

Figure 3: Flow chart for performing stress studies for degradation under oxidative conditions.



Handling the reaction samples for chromatographic studies. Thereis another practical aspect of stress testing that generates in-quiries from practitioners. The question is generally put in theform — What is the best way to handle samples containing highconcentrations of acid, alkali or an oxidizing agent for inject-ing them into HPLC or loading on a TLC plate?

For this, one approach is diluting the sample enough so thatthe concentration of reagent falls within the tolerable range.For HPLC, the dilution can be done in the mobile phase, whileit can be a suitable solvent like methanol, ethanol, etc., for TLCstudies. The second approach involves neutralization of acidand alkali solutions to a tolerable pH. In the experience of theauthors, the dilution is a more easy method than neutraliza-tion. The former applies to acid/alkali solutions and even tosamples containing oxidizing agents. The problems withneutralization are that it becomes difficult to carry it out in aquantitative manner and, moreover, it generally leads to pre-cipitation of the dissolved ingredients of the sample. Both theseproblems do not exist when the dilution is done. The only con-trol required in dilution method is that the starting drug con-centration in solution should be sufficiently high so that it re-mains well above the detectable range after a dilution of100 times or even more. The starting drug concentration of1 mg/ml suggested in this paper is considered optimum in thisrespect also. However, if in any situation the response in dilutedsamples is not good, the problem can be simply handled by in-creasing the volume of injection or loading. The extent of in-crease, in any case, should be guided by the buffer capacity ofthe buffer used in the mobile phase.

Design of the studies. For every stress study, it is advised to

generate four samples and report the results of each. First is theblank solution stored under normal conditions, second is theblank subjected to stress in the same manner as the drug solu-tion, third is the zero time sample containing the drug whichis stored under normal conditions and fourth is the drug solu-tion subjected to stress treatment. A real assessment of changesis only made through the comparison of the results of all these.

Equipment for stress studies. Although there is nothing big todiscuss as far as equipment for stress studies is concerned, as itis a common thing for the chemists usually involved in the job,still a discussion on the options available and some of the pre-cautions may be relevant.

There are two components of the hardware for stress testing.One is the container in which the reaction is done and the sec-ond is equipment for creating the stress condition. Of course,either or both may vary, dependent upon whether the reactionbeing studied is hydrolytic, oxidative or photolytic.

For hydrolytic studies in dilute acid and alkali conditions attemperatures between 5 �C above room temperature up to 70 �C, the reactions can simply be carried out in containers likevolumetric flasks or stoppered culture tubes and stored in a waterbath set at the desired temperature. If precision water bath is notavailable, a simple student water bath can be converted to giveprecise control of temperature by tying the thermostat sensordirectly to the heater (15). A single odd reaction at a time canalso be carried out using the rotary film evaporator employinga round bottom (RB) or pear shaped flask of the optimum size.If it is found that there is a change in reaction mechanism athigh temperatures and the reaction needs to be carried out atlower temperatures for longer period running into days, it is ad-

Figure 4: Flow chart for performing stress studies for photolytic degradation.




visable to use dry block thermostatic device for maintaining thetemperature. Capped containers made of glass of suitable sizeto fit the pockets of the block can be used in such case. For re-actions above 80 �C and reflux conditions, one option is to usea boiling water bath equipped with a voltage regulator. Alterna-tively, an oil bath with a voltage regulator may be employed. Forrefluxing, a RB flask-condenser combination can be used, butampoules may be a better choice for use in oil baths. Anotherpossibility of carrying out reactions at varied temperatures is bymaking use of constant boiling systems. In this set-up, a suitablequantity of a single solvent or an azeotropic mixture of solventsis added to a RB flask and sealed ampoules containing differentreaction mixtures are dipped into the solvent in the flask. Therefluxing is done after attaching the flask to a condenser andmaking use of a heating mantle. Here the necessary precautionis that water supply must be continuously available otherwisethe solvent in the flask would evaporate and may lead to dan-gerous situation due to overheating of sealed ampoules. The useof recirculating chiller is suggested here. The major advantageof using constant boiling solvents is that one can achieve a pre-cise control of temperature even in absence of precision waterbaths. In addition, in the experience of authors, a large numberof reactions under different conditions can be done at one timeas simply the flask size and the mantle need to be optimised toaccommodate all the ampoules containing the intended reactionsolutions.

In general, while carrying out the stress reactions by the abovesaid approaches, one must use containers made of thick boro-silicate glass or laminated containers. When studies are beingcarried out in multiple ampoules at one time, care should betaken on labeling, so that the ampoules for different reactionconditions are identifiable till the end of the study. Labelingwith marker pens sometimes does not work if the reactions arebeing carried out in oil baths or boiling in solvent or water. Inour laboratories, we use with success ringing of ampoules withdifferent color wires. Different samples are differentiated by thenumber of rounds. Sometimes when the ampoule size is big-ger and the ampoules float in the liquid straight with neck up,flags can be attached to the neck for identification.

Another aspect that is important to be discussed here is thatstress studies should be discouraged from being conducted inmechanical convection ovens or heating chambers. This is im-portant, particularly, if the sealed containers like ampoules arebeing used as the containers. There have been incidents wherethe thermostat relay failure during the study resulted in shoot-ing up of the temperature and consequent explosion of thesealed ampoules, and the whole chamber was thrown away fromits place. This can be dangerous if a worker is close by. The cham-bers or ovens, if ever to be used, must be fitted with an addi-tional safety thermostat put in series with the original one (16).The additional thermostat can be set at 3–5 degrees higher thanthe temperature of study so that in an event of failure, the sec-ond thermostat can take over the control and prevent the tem-perature from rising up. It might also be useful if the chambersare fitted with failure alarms.

The oxidative stress studies are suggested to be done in Fig.3 under normal laboratory conditions only. Hence no specific

equipment is needed for the purpose. The studies should bedone in a leak proof stoppered containers. A caution is that theheadspace left above the solution during the study should besmall, for which, either the solution volume can be increasedor the size of the container can be selected so that it is sufficientjust to accommodate the total volume of reaction solution.

For photolytic reactions, as the advice is to follow ICH guide-lines (Fig. 4), hence any of the different kind of lamp sourcesdefined in the guideline might be used. The output of the lampshould meet D65/ID65 emission standards defined by ISO. Theguideline suggests use of artificial daylight fluorescent lampcombining visible and UV outputs, xenon or metal halide lamp.Photostability test equipment using these types of lamps andbased on the international standards are sold commercially. Ina typical xenon lamp equipment, the defined 1.2 � 106 lux hoursexposure is obtained in around 4 hours. In comparison, fluo-rescent and near UV lamps based equipment give an output of5000–10,000 lux and almost 5–10 days are required to get thetotal exposure. Hence, the more intense xenon or even metalhalide lamps are more useful for stress studies as results arerapidly obtained.

The author has at times been asked how an exposure of1.2 million lux hours is calculated. The answer is that if a lampgives a total light of 6250 lux and if the exposure is done forone hour, then it means an exposure of 6250 lux hours. Onefull day exposure means 150 kilo lux hours (6250 � 24). An 8 days exposure accordingly is equivalent to 1.2 million luxhours (6250 � 24 � 8).

Although not suggested in Fig. 4, advantage of direct sunlightexposure can also be taken for forcing decomposition of drug.In tropical climates, an exposure for 4 hours on a hot sunny daywould almost give same degradation as can be obtained with axenon lamp in the same period or in a fluorescent and UV lampsfitted photostability chamber in 8 days. The advantage of usingsun test equipment, however, should not be undermined as onegets an equal standard exposure round the year while sunlighttesting is highly dependent upon vagaries of nature.

Stress testing of biological or biotechnological productsStress studies are also required to be conducted on biotechno-logical and biological products. This is a requirement under theICH guideline Q5C (17). The stress studies are said to be usefuli) in determining if accidental exposures to conditions other thanthose proposed are deleterious to the product, ii) for evaluatingwhich specific test parameters may be the best indicators of prod-uct stability and iii) in revealing patterns of degradation. Theguideline emphasizes that the conditions of the stress study asenshrined in the tripartite stability guideline Q1A may not beappropriate for biotechnological/biological products. It is statedthat ‘conditions should be carefully selected on a case-by-casebasis’.

Accordingly, it is not claimed that the classification systemand decision trees, as discussed in this paper, would apply tobiotechnological/biological drugs and their products. Unlike,chemical drugs, the bio products are particularly sensitive tofactors such as temperature changes, oxidation, light, ionicstrength and shear. Their main routes of decomposition also



vary from chemical drugs. The bio products undergo reactionssuch as deamidation, oxidation, aggregation, proteolysis, etc.(17). The exact classification system for these products need tobe developed, by making a survey of the reports on intrinsicstability of these drugs. This exercise remains to be done.

ConclusionsThe determination of inherent stability of compounds usingstress testing, in exact terms, is not something new. It has beenpracticed since decades as evident from the reports in literature.The only difference is that it is now spelt out as a requirementin more clear terms in the ICH guidelines, which are being ac-cepted and followed as industry standards today the world over.

The information on stress testing, in general, forms part ofthe chemistry part of the dossier and remains closed till the in-novator itself decides to make it open. Sometimes studies aredone and reported by independent groups and they appear inliterature. Except that there is some laxity given on existing andpharmacopoeial drugs, like done by CPMP guideline QWP/556/96 (18), stress studies otherwise need to be done on all newdrugs.

As it mostly applies to new drugs, the activity related to stresstesting has been practiced mainly by those who were involvedin the process of new drug discovery, meaning that the activitywas restricted to countries in the developed world from wheremost new drugs originated till date. However, with the imple-mentation of GATT and WTO, the new drug discovery pro-grams are being initiated in more and more countries and hencethe activity of stress testing also is going to be wide spread.

From this aspect, it is hoped that the guidance provided inthis paper would be of general and wide interest. However, itmay be pertinent to add here that the opinions expressed arepurely personal to the authors and do not represent thinkingof the regulatory agencies.

References1. ICH,“Stability Testing of New Drug Substances and Products,” (Inter-

national Conference on Harmonization, Geneva, October 1993).2. K. Florey, Analytical Profiles of Drug Substances, Vol. 10–24 (Academic

Press, London).3. G. Caviglioli et al., “Stability Indicating HPLC Assay for Retinoic acid

in Hard Gelatine Capsules Containing Lactose and as Bulk Drug Sub-stance,” Drug Dev. Ind. Pharm. 20, 2395–2408 (1994).

4. A.L. Lagu et al., “High Performance Liquid Chromatographic Deter-mination of Suprofen in Drug Substance and Capsules,” J. Pharm. Sci.71, 85–88 (1982).

5. S.K. Baveja and S. Singh, “Thin-layer Chromatographic Examinationof the Degradation of Centbucridine in Aqueous Solutions,” J. Chro-matogr. 396, 337–344 (1986).

6. S. Singh and R.L. Gupta, “A Critical Study on Degradation of Aza-thioprine in Aqueous Solutions,” Int. J. Pharm. 42, 263–266 (1988).

7. S. Singh,“Dobupride — What Exactly is Its Degradation Behaviour?”J. Pharm. Biomed. Anal. 15, 1801–1803 (1997).

8. M. Grover, M. Gulati, and S. Singh, “Stability-Indicating Analysis ofIsoxazolyl Penicillins using Dual Wavelength High-Performance Liq-uid Chromatography,” J. Chromatogr. 708, 153–159 (1998).

9. ICH,“Stability Testing: Photostability Testing of New Drug Substancesand Products” (International Conference on Harmonization, Geneva,November 1996).

10. N.H. Anderson,“Photostability Testing: Design and Interpretation ofTests on Drug Substances and Dosage Forms,” in The Photostability of

Drugs and Drug Formulations, H.H. Tonnersons, Ed. (Taylor and Fran-cis, London, 1996), pp. 305–321.

11. M. Grover, “Computer-Aided Quantitative Structure-Stability Rela-tionship Studies on �-lactam Antibiotics,” Ph. D. Thesis (Panjab Uni-versity, Chandigarh, 1999).

12. M.F. Powell, “Stability of Lidocaine in Aqueous Solution: Effect ofTemperature, pH, Buffer, and Metal Ions on Amide Hydrolysis,” Pharm.Res. 4, 42–45 (1987).

13. A. Sher, et al.,“Complexation of Copper(II) ions with Ampicillin. Part1. Spectroscopic and Electrochemical Investigation of Interactionsunder Equilibrium Conditions,” Int. J. Pharm. 90, 181–186 (1993).

14. C. Larsen and H. Bundgaard, “Polymerization of Penicillins. V. Sepa-ration, Identification and Quantitative Determination of AntigenicPolymerization Products in Ampicillin Sodium Preparations by High-Performance Liquid Chromatography,” J. Chromatogr. 147, 143–150(1978).

15. S. Singh and A. Singh, “A Simple Way to Increase Temperature Con-trol Precision in Student Water/Oil Baths,” J. Chem. Edu. 65, 1095(1988).

16. S. Singh and A. Singh,“Thermostatic Ovens & Baths For Stability Stud-ies,” The Eastern Pharmacist 30, 356 (1987).

17. ICH, “Quality of Biotechnological Products: Stability Testing of Bio-technological/Biological Products,” (International Conference on Har-monization, Geneva, November 1995).

18. CPMP,“Note for Guidance on Stability Testing of Existing Active Sub-stances and Related Finished Products,” (The European Agency forthe Evaluation of Medicinal Products, London, April 1998).

19. J.H. Bridle and M.T. Brimble,“A Stability Indicating Method for Dipyri-damole,” Drug Dev. Ind. Pharm. 19, 371–381 (1993).

20. S.L. Daniel and A.J. Vanderwielen,“Stability Indicating Assay for Hydro-chlorthiazide,” J. Pharm. Sci. 70, 211–215 (1981).

21. E.M. Abdel-Moety and O.A. Al-Deeb, “Stability-Indicating LiquidChromatographic Determination of Trifluoperazine hydrochloride inBulk Form and Tablets,” Eur. J. Pharm. Sci. 5, 1–5 (1997).

22. R.L. Garnick et al.,“High-Performance Liquid Chromatographic Assayfor Sodium levothyroxine in Tablet Formulations: Content UniformityApplications,” J. Pharm. Sci. 73, 75–77 (1984).

23. D.-P. Wang, J.-D. Lee, and R.-A. Lin, “Stability of HI-6 in Solution,”Drug Dev. Ind. Pharm. 21, 509–516 (1995).

24. Y.-C. Lee, D.M. Baaske, and A.S. Alam, “High-Performance LiquidChromatographic Method for the Determination of Esmolol hydro-chloride,” J. Pharm. Sci. 73, 1660–1661 (1984).

25. D. Mahalaxmi et al., “Stability-Indicating HPLC Method for Betaxo-lol HCl and its Pharmaceutical Dosage Forms,” Drug Dev. Ind. Pharm.22, 1037–1039 (1996).

26. D.L. Dunn,“Analysis of Chlorobutanol in Ophthalmic Ointments andAqueous Solutions by High Performance Liquid Chromatography,” J.Pharm. Sci. 72, 277–280 (1983).

27. N. Maron and G. Wright,“Application of Photodiode Array UV Detec-tion in the Development of Stability-Indicating LC Methods: Determi-nation of Mefenamic acid,” J. Pharm. Biomed. Anal. 8, 101–105 (1990).

28. M. Mathew, V.D. Gupta, and R.E. Bailey,“Stability of Omeprazole So-lutions at Various pH Values as Determined by High-PerformanceLiquid Chromatography,” Drug Dev. Ind. Pharm. 21, 965–971 (1995).

29. V.D. Gupta and J. Parasrampuria,“Quantitation of Cephalexin in Phar-maceutical Dosage Forms Using High-Performance Liquid Chro-matography,” Drug Dev. Ind. Pharm. 13, 2231–2238 (1987).

30. V.D. Gupta, “Quantitation of Meperidine hydrochloride in Pharma-ceutical Dosage Forms by High Performance Liquid Chromatogra-phy,” J. Pharm. Sci. 72, 695–697 (1983).

31. V.D. Gupta,“Quantitation of Metronidazole in Pharmaceutical DosageForms Using High-Performance Liquid Chromatography,” J. Pharm.Sci. 73, 1331–1333 (1984).

32. K.A. Connors, G.L. Amidon, and V.J. Stella, Chemical Stability ofPharmaceuticals (J. Wiley & Sons, New York, 1986).

33. H.H. Tonnerson, Photostability Testing: Design and Interpretation ofTests on Drug Substances and Dosage Forms (Taylor and Francis, Lon-don, 1996).



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