Guidance on Conduct of
Stress Tests to Determine
Inherent Stability of Drugs
Saranjit Singh* and Monika Bakshi
The authors in this write-up provide guidance
on the conduct of stress tests for the deter-
mination of inherent stability of drugs under
hydrolytic, oxidative and photolytic con-
ditions. A classification system is proposed
and decision trees are drawn to guide on how
to start and where to end in the search of the
right type of stress conditions for a new drug.
The predictability of the pathways of degra-
dation of the drug, based on the functional
groups present and overall structure, is
discussed. Other considerations like benefit of
harsh conditions for the isolation of degra-
dation products and the advantage of
catalytic activity of metal ions are also
touched upon. In addition, useful information
is provided on practical conduct of stress
studies.
Saranjit Singh, PhD, is an associate
professor and Monika Bakshi is doctoral
student at the National Institute of
Pharmaceutical Education and Research
(NIPER), Sector 67, SAS Nagar 160 062,
India, tel. +91 172 673848, fax +91 172
677185, e-mail (niper@chd.nic.in)
*To whom all correspondence should be addressed.
T
he International Conference on Harmonization (ICH)
guideline entitled ‘Stability Testing of New Drug Sub-
stances and Products’ (Q1A) requires that stress testing
be 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 conditions
should be identified and degradation pathways established. It
is stated that the testing should include the effect of tempera-
ture, humidity where appropriate; oxidation, photolysis and
susceptibility 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 accelerated
temperature test condition (e.g. 50 C, 60 C, etc.) and that of
the humidity at a level of 75 percent or greater. No details are,
however, provided for the study of oxidation, photolysis and
hydrolysis 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 initiation
of forced decomposition studies.
In this write-up the authors propose a classification system
for categorization of drugs based on their inherent stability be-
haviour. Flow charts have been drawn that are suggested to be
followed 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 and
the dilemmas
It 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 in
the rate of decay. A fall-out of this variable behavior is that it
becomes 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 are
i) To what extreme of conditions one should go if a previously
tested condition does not give sufficient degradation, good
enough for degradation products to be isolated in quantity suit-
able for characterisation and structure elucidation? ii) Are there
any limits where one should stop and carry no further studies?
and iii) What sort of reagents or agents need to be employed
Pharmaceutical Technology On-Line APRIL 2000 1
 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 19
Hydrochlorthiazide 0.1 N HCl — 16 h Degradation at the 20
rate of 0.1% h
Ethacrynic 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 21
Ketoprofen 0.1 N acid 98 C 30 min No decomposition 2
Morphine 0.1 N HCl Refluxing 2h No degradation 2
Metoprolol tartarate 0.1 N HCl Refluxing 20 h No chemical change 2
Retinoic acid 0.1 N HCl Refluxing 5 min 65% recovery of drug 3
Maprotiline HCl 0.1 N HCl Refluxing 94 h No degradation 2
Timolol maleate 0.1 N HCl 105 C 2 months Considerable degradation 2
Nabilone 0.1 N acid 110 C 1 week Stable 2
Sodium levothyroxine 0.1 N HCl — 44 h 92.8% recovery of drug 22
HI-6 pH 2.54 HCl 70 C 100 h Around 50% degradation 23
Suprofen 1 N HCl 50 C 72 h Quantitative recovery 4
Esmolol HCl 1 N HCl Boiling 1h Total degradation 24
Benperidol 1 N HCl 100 C — Gradual decomposition 2
Norfloxacin 2 N HCl 100 C — Decarboxylated degradate 2
formed
Betaxolol 2.5 N HCl Boiling 20 min No drug left 25
Clioquinol 4 N HCl Refluxing 2 days 34% drug left after 2 days 2
Sertraline HCl 5 N HCl Refluxing 3h No significant degradation 2
Lidocaine 6.5 N HCl 108 C 24 h 50% hydrolysis 2
Chlorobutanol conc. HCl R.T. — 43.5% recovery 26
Lidocaine 50% H2SO4 116 C 5h 3% decomposition 2
Spironolactone 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 27
Omeprazole 1 N H2SO4 Boiling 5 min Total decomposition 28
Cephalexin 1 N H2SO4 Boiling 5 min No degradation 29
Acyclovir 1 N H2SO4 Boiling 10 min Potency reduced to 88% 2
Ranitidine 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 of
drugs in the latest volume of the profiles and search for the name of the drug to reach the monograph.

for creating a particular stress condition? Here the questions
are generally put in the form — For study of oxidation, which
among the various oxidising agents is the best to use ? Does it
make 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 drugs
so that right stress conditions can be determined in minimum
possible trials?
The quest for answers
Unfortunately, the practical aspects related to stress testing are
neither addressed by regulatory guidelines, nor by any other
document. Therefore, in an endeavor to provide the practi-
tioners with the answers to the volley of these questions and
to examine the possibility of developing flow charts, a critical
study was done of the reports in literature for the stress con-
ditions used for determining inherent stability of new drug
2 Pharmaceutical Technology On-Line APRIL 2000
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 which
records the inherent stability behavior of the drug and the con-
ditions employed to determine the same. Examples of drugs
where 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 reports
in 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 were
listed 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 in
acid conditions revealed that hydrochloric acid at a strength of
0.1 N was mostly used. There were a few reports that indicated
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 Table II: Selected examples of stress conditions used for hydrolysis of drugs in alkali.
Reaction
Drug Strength Conditions Time Remarks Ref.
Trifluoperazine 0.1 N NaOH 30 C 24 h Total decomposition 2
Dipyridamole 0.1 N NaOH 40 C 1 week Negligible degradation 19
Tolnaftate 0.1 N NaOH 50 C 24 h Negligible degradation 2
Ethacrynic acid 0.1 N NaOH 65 C 21 days Total decomposition 2
Mefenamic acid 0.1 N NaOH Refluxing 48 h Very less decomposition 27
Clioquinol 0.1 N NaOH Refluxing 2 days 32% drug left 2
Morphine 0.1 N NaOH Refluxing 2h No degradation 2
Metoprolol tartarate 0.1 N NaOH Refluxing 20 h No chemical change 2
Maprotiline HCl 0.1 N NaOH Refluxing 94 h Slight degradation 2
Sodium levothyroxine 0.1 N NaOH — 87.5 h 90.6% recovery 22
Spironolactone 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 2
Morphine 0.125 N NaOH, Refluxing 4h Around 50% degradation 2
passage of air
Xylometazoline HCl 0.5 N NaOH Refluxing 30 min No hydrolysis 2
Polythiazide 1 N NaOH 35 C 1h Total degradation 2
Meperidine HCl 1 N NaOH 50 C 48 h Zero potency 30
Suprofen 1 N NaOH 50 C 72 h 92.8% recovery 4
Omeprazole 1 N NaOH Boiling 5 min No degradation 28
Cephalexin 1 N NaOH Boiling 5 min Extensive degradation 29
Acyclovir 1 N NaOH Boiling 10 min Potency decreased to 95% 2
Ranitidine 1 N NaOH Boiling 20 min Drug degraded by 84.4% 2
Metronidazole 1 N NaOH Boiling 25 min Total decomposition 31
Esmolol HCl 1 N NaOH Boiling 1h Total degradation 24
Norfloxacin 1 N NaOH 100 C 15 h No change 2
Benperidel 1 N NaOH 100 C — Gradual decomposition 2
Sertraline HCl 5 N NaOH Refluxing 3h No significant degradation 2
Morphine 5 N NaOH Refluxing 4h Total degradation 2
Dipivefrin HCl pH 9 — 1 day Complete degradation 2
Chlorobutanol Conc. NH4OH — — 40% recovery 26
Retinoic acid 0.1 N KOH Refluxing 30 min 30% drug left 3

the use of 1 N HCl and even higher normalities (Table I). There
were a few instances where sulfuric acid in varying normalities
had been used. In a few other cases, mention was only found of
‘acid’ without defining the type used. Large variations were also
seen in the reaction (temperature) conditions and periods of
study. The temperature range varied between 40 C and 110 C.
Examples can be seen in Table I where drugs were kept around
100 C or under boiling conditions for periods ranging from a
few minutes to as long as even 2 months. The extent of de-
composition also varied a lot, for example, a 35% loss of retinoic
acid was observed on refluxing in 0.1 N HCl for just 5 min (3),
while there are reports where no decomposition of the drug
was seen after refluxing in 0.1 N acid for even one week
(nabilone).
It is evident from Table II that stress conditions used for the
hydrolysis of drugs under alkaline conditions run parallel to
those used for acid conditions. Sodium hydroxide is mostly
used, at strength of 0.1 N and 1 N. Potassium hydroxide figures
only in few cases. Just like acidic degradation, lot of variation
is observed in time and temperature of exposure of drugs to al-
kali. Depending on the inherent stability characteristics, some
drugs show no degradation even after refluxing in 0.1 N NaOH
for one week (nabilone) while others like trifluoperazine un-
dergo complete degradation when they are kept in 0.1 N alkali
for 24 h at a very low temperature of 30 C. Similar behavior is
seen 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 any
change. Similarly, suprofen (4) exhibited 92.8% recovery even
after heating in 1 N NaOH at 50 C for 72 h, while retinoic acid
degraded 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 conditions
for neutral pH. As may be seen from the examples given in Table
III, no significant degradation was obtained whether the tem-
Pharmaceutical Technology On-Line APRIL 2000 3
perature was 35 C or refluxing conditions were used. The test- varied type of decomposition behavior is seen. For example,
ing evidently is generally done in water. The slow rate of de- cimetidine HCl and maprotiline HCl do not undergo any change
composition in neutral conditions is understandable because when exposed to photolytic stress for several weeks, while pho-
reactions at neutral pH are non-catalytic and hence very long tosensitive retinoic acid degrades on exposure to UV light for
periods under exaggerated temperature conditions may be re- very short periods. The photolability studies are done on drugs
quired to get sufficient quantities of degradation products. in either solid form or solution. It is apparent from the table
Table IV depicts the stress conditions employed for the study that studies are performed in water or in acidic and alkaline so-
of oxidation. Evidently, hydrogen peroxide seems to be much lutions and also on drug dissolved in either methanol or ace-
more popular for the purpose than any other oxidising agent. tonitrile.
The strength of H2O2 used varies between 1% to 30%. In some
drugs extensive degradation is seen when exposed to 3% H2O2 The classification system
for very short time periods at room temperature (e.g., raniti- Depending upon the information obtained as above, and based
dine HCl and cimetidine HCl). In other cases, exposure to high on the previous personal experience on degradation chemistry
concentrations of H2O2 even under extreme conditions does of drugs (5–8), an exercise was carried out to see whether it was
not cause any significant degradation (e.g., sertraline HCl, zileu- possible to broadly classify the drugs into specific categories
ton, etc.). The behavior is on expected lines, as some drugs are and define stress conditions for each of them. The following six
in fact oxidisable, while there are others that are not. The latter classes could be identified:
are not expected to show any change even in the presence of ● Class I: Extremely labile
high dose of oxidising agents. ● Class II: Very labile

Table V emphasises the fact that there is a lot of variation in ● Class III: Labile
the manner in which stress photostability testing is done on dif- ● Class IV: Stable
ferent drugs. Mostly the drugs are
exposed to short/long wavelength Table III: Selected examples of stress conditions used for hydrolysis of drugs under
UV light, or fluorescent light of neutral conditions.
varying illumination (400–1580
foot candles). Efforts are usually Reaction
made to maintain the temperature Drug Medium Conditions Time Remarks Ref.
around room temperature (R.T.). Celiprolol Water 37 C 2 weeks Drug 2
The period of exposure ranges from remains stable
a few hours to several months, de- Dipyridamole Water 40 C 1 week Negligible 19
pending upon the light source in- degradation
tensity. Like oxidation, based on Sertraline HCl Water Refluxing 3h No significant 2
whether a drug is photolytic or not, degradation

Table IV: Selected examples of stress conditions used for oxidation of drugs.
Reaction
Drug Strength Conditions Time Remarks Ref.
Dipyridamole 1% H2O2 40 C 1 week Some degradation 19
Loperamide 1.5% peroxide — Immediately 3.2% cis N-oxide and 2.4% 2
trans N-oxide of drug formed
Cimetidine HCl 3% H2O2 R.T. Short period Sulfoxide formation 2
Ranitidine HCl 3% H2O2 R.T. 20 min 37.8% loss in potency 2
Didanosine 3% H2O2 37 C 6h 40% potency 2
Suprofen 3% H2O2 50 C 72 h 97.4% recovery 4
Morphine 3% H2O2 Refluxing 30 min Extensive degradation 2
Zileuton 5% H2O2 — 1 week 1% degradation 2
Sertraline HCl 10% H2O2 Refluxing 6h No significant degradation 2
Esmolol HCl 30% H2O2 — 1h 50% degradation 24
Mefenamic acid 30% H2O2 — 24 h Extensive decomposition 27
Chlorobutanol 30% H2O2 — — 24.8% recovery 26
Zileuton 0.4% Sodium — Immediately 50% degradation 2
hypochlorite
Sodium Tert. butyl — 1h 37.5% drug left 22
levothyroxine hydroperoxide
Cimetidine HCl Exposure to O2 50 C Short period Sulfoxide formation 2

4 Pharmaceutical Technology On-Line APRIL 2000 www.phar maportal.com

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 2
converted to nitroso
derivative within 5–30 min;
no intact drug left after 5 h
Trifluoperazine UV light (254 nm) 14 cm — — 21
HCl
HI-6 UV light (254 nm) 30 cm — — 23
Retinoic acid UV light (254 nm) 20 cm 2h 50% recovery of drug 3
Sodium Long wavelength — 48 h 101.4% recovery 22
levothyroxine UV light
Sodium Short wavelength — 144 h 98.9% recovery 22
levothyroxine UV light
Timolol Intense UV radiation — 2h Aqueous solutions (pH 5) 2
maleate suffer 2% loss in potency
Pyridoxine HCl UV light — — Destroyed by UV radiation 2
in neutral or alkaline solutions
but not in acidic media
Verapamil UV light — 2h Compound dissolved in 2
methanol showed
degradation by 52%
Cimetidine HCl UV light — Several months No decomposition 2
Imipenem UV light — 24 h Brown surface discoloration 2
Piroxicam 300–830 nm 30 C 72 h 99.6% drug left 2
Zileuton High intensity UV light — 4h Total degradation in 2
acetonitrile solutions but
less degradation in
methanolic solutions
Zileuton Fluorescent light — 2 weeks 35% and 55% degradation 2
(600 and 1500 f.c.*) respectively in acetonitrile
solution
Imipenem 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 developed
Maprotiline HCl 600 f.c. — 12 weeks Stable 2
Clioquinol 600 f.c. — 6 days 30% drug left in 2
acetonitrile solution
Chloroxazone 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 yields
cis 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 2
Loperamide 17000 lux (1580 f.c.) — 7 days No degradation 2
Nifedipine Halogen lamp (1000 W) — Conversion to nitroso 2
derivative begins after 6 h
and is completed after months
*f.c. denotes foot candles.

Pharmaceutical Technology On-Line APRIL 2000 5

Figure 1: Flow chart for performing stress studies for hydrolytic degradation under acid and alkali conditions.
● Class V: Very stable
● Class VI: Practically stable
The proposed stress conditions for the hydrolysis of each of
the six classes of drugs under acid and alkaline conditions are
given in Table VI. The term ‘sufficient decomposition’ is taken
in the broadest sense. It may mean 80–100% decomposition if
the 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 is
usually monitored up to two to three half-lives and the rise and
fall of each degradation product is checked. The monitoring of
reaction is also an essential prerequisite for the establishment
of stability-indicating assays in which it is required that even
intermediate degradation products, if any, should not interfere
in the analysis of drug at any stage of reaction.
For hydrolysis under neutral conditions (Table VII), the drugs
are classified into the same six categories. However, the time
period of exposure is suggested to be longer as the drugs are
known to hydrolyse much slower in neutral conditions than in
catalytic acid and alkaline conditions.
The classification of drugs for oxidative decomposition is
given in Table VIII. Hydrogen peroxide is recommended as the
oxidising agent at different concentrations.
6 Pharmaceutical Technology On-Line APRIL 2000
For photoreactions, unlike the above classification for hydro-
lytic and oxidative decompositions, the distribution of drugs is
limited only to two categories — photostable and photolabile
(Table IX). Here the exposure conditions are based on those
suggested 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 changes
are seen, the total exposure should be increased to 6.0  106
lux hours (10). Drugs stable to this high exposure can be safely
declared as photostable.
The decision trees
Figures 1–4 give the decision trees for investigating different
types of stress conditions for a new drug substance. The gene-
ral approach taken in the construction of these flow charts is
that 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, decision
is taken on whether to increase or decrease the strength of the
reaction conditions. The increase or decrease, if required, is
done step-wise and those stress conditions are accepted wher-
ever a sufficient decomposition is obtained.
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 Figure 1 suggests that in order to study hydrolytic degrada- formed on subjecting the drug to this harsh condition. Going
tion (under acidic and alkaline conditions) of a new drug, whose to the other side of starting condition, if a total degradation is
stability behavior is not known, one can start by refluxing the seen after refluxing in 0.1 N HCl/NaOH for 8 h, the strength of
drug in 0.1N HCl/NaOH for 8 h, considering that the drug is acid/alkali can be decreased to 0.01 N along with decrease of
labile. If a reasonable degradation is observed on subjecting the temperature to 40 C while keeping the time as same 8 h. A drug
drug to this treatment, no further studies need to be carried showing complete degradation even in these mild conditions
out. In case no degradation is seen, drug should be subjected should be treated with 0.01 N HCl/NaOH for 2 h at 25 C and
to refluxing in 1N acid/alkali for 12 h. For a drug which can if still complete degradation is taking place, drug is extremely
withstand even these conditions, more extreme conditions of labile and has to be tested under very mild conditions of tem-
acidity or alkalinity such as refluxing in 2 N HCl/NaOH for perature and pH.
24 h may be tried. The reaction should be monitored, and if Stress testing under neutral conditions can be started by re-
still satisfactory change is not obtained, the drug should be re- fluxing the drug in water for 12 h (Fig. 2). Refluxing time should
fluxed in 5 N HCl/NaOH for up to 24 h. The drug may be de- be increased to 1 day in case no degradation is seen. It should
clared to be “practically stable’’ if no hydrolytic products are be increased further to 2 days if no change is observed. In case
of negligible degradation, the drug may
Table VI: Classification system for acidic or alkaline hydrolysis. be refluxed for a period of 5 days. If still
found stable, the drug may be declared
Strength of Time of Extent of
nondegrading in neutral conditions.
Category of drug acid/alkali exposure Temperature decomposition
For this study, it may be advisable that
Practically stable 5N 2 days Refluxing None a sufficient volume of solution should
Very stable 2N 1 day Refluxing Sufficient be taken for the reaction initially, so that
Stable 1N 12 h Refluxing Sufficient the time period can be continually in-
Labile 0.1 N 8h Refluxing Sufficient creased, as required, and there is no
Very labile 0.01 N 8h 40 C Sufficient need to restart the reaction afresh. For
Extremely labile 0.01 N 2h 25 C Sufficient a drug undergoing complete degra-
dation on refluxing in water for 12 h,
both time and temperature of exposure
Table VII: Classification system for hydrolysis under may be decreased to 8 h and 40 C, re-
neutral conditions. spectively. More mild conditions, like
keeping the drug in water up to 2 h at
Extent of
25 C, should be tried if no intact drug
Category of drug Time of exposure Temperature decomposition
is left after exposure to above men-
Practically stable 5 days Refluxing None tioned conditions.
Very stable 2 days Refluxing Sufficient For determining the susceptibility of
Stable 1 day Refluxing Sufficient the drug to oxidative decomposition,
Labile 12 h Refluxing Sufficient testing may be started by keeping the
Very labile 8h 40 C Sufficient drug in 3% H2O2 for 6 h at room tem-
Extremely labile 2h 25 C Sufficient perature (Fig. 3). The period of reac-
tion should be increased to 24 h in case
there is no sufficient degradation. Still
Table VIII: Classification system for oxidative degradation. if there is no change, the reaction
Strength of Time of Extent of
should be conducted in 10% H2O2 for
Category of drug hydrogen peroxide exposure Temperature decomposition
24 h. For a drug which does not oxidise
even under these conditions, more ex-
Practically stable 30% 48 h R.T. None
treme conditions of 30% H 2O2 for
Very stable 10% 24 h R.T. Sufficient
24 h may be tried. The drug may be de-
Stable 3% 24 h R.T Sufficient
clared to be “practically stable’’ if no
Labile 3% 6h R.T. Sufficient
products are formed on subjecting the
Very labile 1% 3h R.T. Sufficient
drug to this condition. In an event of
Extremely labile 1% 30 min R.T. Sufficient
decomposition of whole drug under the
starting conditions, the strength of
H2O2 should be decreased from 3% to
Table IX: Classification system for photolytic degradation. 1% and the reaction may be monitored
Category of drug Total exposure Temperature Extent of decomposition for a period sufficient to yield the de-
sired percent of decomposition. The
Photolabile 1.2  106 lux h R.T. Sufficient or total
drugs undergoing complete degrada-
Photostable 6.0  106 lux h R.T. None
tion even under these conditions are
Pharmaceutical Technology On-Line APRIL 2000 7
Figure 2: Flow chart for performing stress studies for hydrolytic degradation under neutral conditions.
highly prone and should be tested in very dilute oxidising agent
with an exposure for very short duration.
In order to get an idea about photostability (Fig. 4), the drug
substance should be initially subjected to an illumination up to
1.2  106 lux hours which is the ICH recommended exposure
and 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 in
exposure to 6.0  106 lux hours has no effect on the stability of
the drug.
Postulation of the intrinsic stability behavior
from structure
What is suggested above is applicable, in general, to any drug
molecule. However, following of all the steps given in the flow
charts can be avoided, when the new drug molecule is ‘close’
in structure to the compound(s) whose inherent stability is
already known. In that event, one can go directly to the step
that 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
8 Pharmaceutical Technology On-Line APRIL 2000
the new drug can be obtained from the study of functional
groups in the structure. Drugs with specific functional groups
undergo typical type of reactions. For example, esters and -
lactams are hydrolysed when subjected to decomposition. Thi-
ols undergo oxidation. Likewise, drugs with specific functional
groups undergo photolysis. The types of functional groups re-
sponsible for decomposition of drugs by the three major routes
are listed in Tables X–XII. Examples of drugs are also included,
wherever possible.
A major percentage of new drugs are just modifications of
existing drugs. In general, a new drug which is a variant of the
already known homologous series would show a close inherent
stability behavior to the existing drug(s), provided there is no
influence of modifying groups or major change at the site of
reaction. 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 study
was 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 most
of them clustered around one point and that of the remaining
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were also not very different. Similar is the case expected of
cephalosporins or other homologous series of compounds where
the 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 a
chart drawn in a manner in which drugs are listed functional
group 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 search
of the literature for stability information, including deep study
of reports on decomposition kinetics of a large number of com-
pounds. It may be considered as a subject matter of a future
report.
Other general considerations
Alternate stress conditions for each class. Figure 1 suggests testing
to be initiated by refluxing the drug in 0.1 N acid or alkali for
8 h. There exists a possibility of using alternate conditions in
which the acid strength is increased and the same is compen-
sated by reducing the period of reaction. In sum, there may be
two or more possibilities for each reaction condition where the
same extent of decomposition can be obtained. In such situa-

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, Spironolactone
open chain acid and alcohol
Imides Lysis of imide ring to Glutethimide, Ethosuximide,
open chain products Barbiturates (di-imides)
Alkyl chlorides Conversion to Chloramphenicol
corresponding alcohols
Azomethines Bond breakage Nitrofurantoin
*The list is only representative and not comprehensive. Based on information in reference 32.

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, Ranitidine
Bicyclic or tricyclic Formation of dimers Naphthol, Morphine
phenolic compounds
-ketols Formation of glyoxals Steroids
Polyhydroxybenzenes Formation of quinones Epinephrine, Hydroxyquinone
Thiazines Formation of S-oxides Phenothiazines
Unsaturated compounds Formation of hydroperoxides Amphotericin B, Fatty acids
e.g. oleic acid, linoleic acid, etc.
N-isopropyl Oxidation of N-isopropyl Practolol, Propranolol, Sotalol
ethanolamine derivatives ethanolamine groupl
Indole derivatives Oxidation of N–H group Sumatriptan
in the indole moiety
*The list is only representative and not comprehensive.

Pharmaceutical Technology On-Line APRIL 2000 9

 Table XII: Functional group based classification of drugs (with examples) that undergo different types of photolytic
reactions.*
Functional group Degradation reaction
Olefins Isomeric conversion
Isomeric conversion
and/or photocyclization
Formation of epoxide
Formation of hydroperoxide
2–2 cycloaddition of singlet
oxygen
Dimerisation mediated by
2–2 cycloaddition
Aryl acetic acids Decarboxylation
Aromatic nitro Reduction to nitroso group and
compounds oxidation of the ring
Aryl halo derivatives Dehalogenation
N-alkyl derivatives N-dealkylation
Benzophenones Formation of radical derivatives
N-Oxides Formation of oxaziridines
*The list is only representative and not comprehensive. Based on information in reference 33.
tion, should one prefer using a harsher condition spending lesser
time or use the normal conditions, as suggested in either tables
or flow charts? The answer should always be in favor of the lat-
ter. This is for the following reasons: i) there may be a change
in mechanism of reaction when a harsh condition is used, and
ii) there is a practical problem in neutralising or diluting every
sample, 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 as
normal as possible conditions should be used for causing the
decomposition 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 at
the step where sufficient degradation is obtained. If this hap-
pens at the first one or two steps, then the drug generally is not
to 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 the
degradation 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 the
HPLC was done, a new peak was shown to appear. The height
of the new peak was very small in 0.01 N NaOH which increased
almost equal to the drug peak in 1 N NaOH. For further con-
version of the drug to product, the concentration of alkali was
increased to 2 N and the solution was refluxed for 12 hours.
Good crystals of the products were obtained after overnight
storage. In a subsequent experiment, the same product was
found to be formed immediately when the drug was brought
in contact with 5 N NaOH at room temperature. It was, there-
10 Pharmaceutical Technology On-Line APRIL 2000
Drug examples
Retinoic acid, Cinnamic acid, Nitrofurazone
Stilboestrol, Dienoesterol, Clomiphene
Menadione, Norethisterone
Cyclobarbitone
Menaquinone-I, Phylloquinone
Thymine
Flurbiprofen, Naproxen, Ketoprofen, Butibufen
Nifedipine, Furnidipine, Nirendipine,
Nicardipine, Nimodipine, Nitrazepam,
Chloramphenicol
Chlorpromazine (and other chlorosubstituted
phenothiazines), Amiodarone, Frusemide,
Diclofenac, Perphenazine
Diphenhydramine, Methotrexate, Folic acid,
Chloroquine
Fenofibrate, Ketoprofen, Dithranol
Chlordiazepoxide, Methaqualone-N-oxide
fore, very easy to isolate this unknown degradation product in
pure form.
In another case, a research group had reported in literature
the degradation chemistry of a drug at pH 10 and it was indi-
cated that an unknown product was formed along with the ones
that were identified. In our laboratories, it was of our interest to
isolate 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. The
drug was heated under the stressful condition of 2 N NaOH at
80 C up to 10 minutes and the solution was left overnight. In
the 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 those
for the unknown. A separate reaction in concentrated ammo-
nia solution resulted in formation of another product in pure
form. This was a previously reported degradation product but
procedure 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 conditions
of acid and alkali for the complete conversion of the drugs to
products.
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 reaction
up to several thousand times, for example, copper is considered
to be an extremely effective catalyst with the rate of monovalent
ascorbic acid oxidation increasing by ten thousand times. Should
then degradation studies in presence of metal ions be a part of
standard stress testing protocol?
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Figure 3: Flow chart for performing stress studies for degradation under oxidative conditions.
In the author’s personal viewpoint, the answer to this ques-
tion should be ‘yes’. Logically, high acid and alkaline conditions
are also used in stress studies due to their catalytic activity. No
doubt 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 a
small amount of hydrochloric acid to a formulation to bring
down the pH to maintain the color of the drug which was
whitish in neutral form. This formulation was being packed in
a metallic container. A browning of the formulation was ob-
served in samples kept on stability. It was later found that the
drug had a nitro group and in the presence of hydrochloric acid
and the metal species (contributed by packaging and/or excipi-
ents), the nitro group was getting reduced to amino group. This
amino compound was further unstable resulting in brown col-
ored products. In the absence of metal ions, the drug did not
follow 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. To
what 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 in
stress testing protocols.
Important practical aspects in conduct of stress testing
Drug concentration. Till this point, no discussion has been done
on the drug concentration of the reaction solution during stress
testing. 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 a
concentration of 1 mg/ml. If solubility is a limitation, varying
amounts of methanol may be used to get a clear solution. By
using drug concentration of 1 mg/ml, it is usually possible to get
even minor decomposition products in the range of detection.
It is suggested that some degradation studies should also be
done at a concentration at which the drug is expected to be pres-
ent in the final formulations. The reason for proposing this are
the examples of aminopenicillins and aminocephalosporins
where a range of polymeric products have been found to be
formed in commercial preparations of containing drug in high
concentrations (14). Incidentally, little or no polymerization is
shown at lower drug concentrations.
Pharmaceutical Technology On-Line APRIL 2000 11
Figure 4: Flow chart for performing stress studies for photolytic degradation.
Handling the reaction samples for chromatographic studies. There
is another practical aspect of stress testing that generates in-
quiries from practitioners. The question is generally put in the
form — What is the best way to handle samples containing high
concentrations 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 that
the concentration of reagent falls within the tolerable range.
For HPLC, the dilution can be done in the mobile phase, while
it can be a suitable solvent like methanol, ethanol, etc., for TLC
studies. The second approach involves neutralization of acid
and alkali solutions to a tolerable pH. In the experience of the
authors, the dilution is a more easy method than neutraliza-
tion. The former applies to acid/alkali solutions and even to
samples containing oxidizing agents. The problems with
neutralization are that it becomes difficult to carry it out in a
quantitative manner and, moreover, it generally leads to pre-
cipitation of the dissolved ingredients of the sample. Both these
problems 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 of
100 times or even more. The starting drug concentration of
1 mg/ml suggested in this paper is considered optimum in this
respect also. However, if in any situation the response in diluted
samples 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 of
the buffer used in the mobile phase.
Design of the studies. For every stress study, it is advised to
12 Pharmaceutical Technology On-Line APRIL 2000
generate four samples and report the results of each. First is the
blank solution stored under normal conditions, second is the
blank subjected to stress in the same manner as the drug solu-
tion, third is the zero time sample containing the drug which
is stored under normal conditions and fourth is the drug solu-
tion subjected to stress treatment. A real assessment of changes
is only made through the comparison of the results of all these.
Equipment for stress studies. Although there is nothing big to
discuss as far as equipment for stress studies is concerned, as it
is 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 reaction
being studied is hydrolytic, oxidative or photolytic.
For hydrolytic studies in dilute acid and alkali conditions at
temperatures between 5 C above room temperature up to
70 C, the reactions can simply be carried out in containers like
volumetric flasks or stoppered culture tubes and stored in a water
bath set at the desired temperature. If precision water bath is not
available, a simple student water bath can be converted to give
precise control of temperature by tying the thermostat sensor
directly to the heater (15). A single odd reaction at a time can
also be carried out using the rotary film evaporator employing
a round bottom (RB) or pear shaped flask of the optimum size.
If it is found that there is a change in reaction mechanism at
high temperatures and the reaction needs to be carried out at
lower temperatures for longer period running into days, it is ad-
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visable to use dry block thermostatic device for maintaining the
temperature. Capped containers made of glass of suitable size
to 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 use
a boiling water bath equipped with a voltage regulator. Alterna-
tively, an oil bath with a voltage regulator may be employed. For
refluxing, a RB flask-condenser combination can be used, but
ampoules may be a better choice for use in oil baths. Another
possibility of carrying out reactions at varied temperatures is by
making use of constant boiling systems. In this set-up, a suitable
quantity of a single solvent or an azeotropic mixture of solvents
is added to a RB flask and sealed ampoules containing different
reaction mixtures are dipped into the solvent in the flask. The
refluxing is done after attaching the flask to a condenser and
making use of a heating mantle. Here the necessary precaution
is that water supply must be continuously available otherwise
the solvent in the flask would evaporate and may lead to dan-
gerous situation due to overheating of sealed ampoules. The use
of recirculating chiller is suggested here. The major advantage
of using constant boiling solvents is that one can achieve a pre-
cise control of temperature even in absence of precision water
baths. In addition, in the experience of authors, a large number
of reactions under different conditions can be done at one time
as simply the flask size and the mantle need to be optimised to
accommodate all the ampoules containing the intended reaction
solutions.
In general, while carrying out the stress reactions by the above
said approaches, one must use containers made of thick boro-
silicate glass or laminated containers. When studies are being
carried out in multiple ampoules at one time, care should be
taken on labeling, so that the ampoules for different reaction
conditions are identifiable till the end of the study. Labeling
with marker pens sometimes does not work if the reactions are
being carried out in oil baths or boiling in solvent or water. In
our laboratories, we use with success ringing of ampoules with
different color wires. Different samples are differentiated by the
number 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 that
stress studies should be discouraged from being conducted in
mechanical convection ovens or heating chambers. This is im-
portant, particularly, if the sealed containers like ampoules are
being used as the containers. There have been incidents where
the thermostat relay failure during the study resulted in shoot-
ing up of the temperature and consequent explosion of the
sealed ampoules, and the whole chamber was thrown away from
its 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 than
the 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 chambers
are 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 be
done in a leak proof stoppered containers. A caution is that the
headspace left above the solution during the study should be
small, for which, either the solution volume can be increased
or the size of the container can be selected so that it is sufficient
just 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 sources
defined in the guideline might be used. The output of the lamp
should meet D65/ID65 emission standards defined by ISO. The
guideline suggests use of artificial daylight fluorescent lamp
combining visible and UV outputs, xenon or metal halide lamp.
Photostability test equipment using these types of lamps and
based on the international standards are sold commercially. In
a typical xenon lamp equipment, the defined 1.2  106 lux hours
exposure is obtained in around 4 hours. In comparison, fluo-
rescent and near UV lamps based equipment give an output of
5000–10,000 lux and almost 5–10 days are required to get the
total exposure. Hence, the more intense xenon or even metal
halide lamps are more useful for stress studies as results are
rapidly obtained.
The author has at times been asked how an exposure of
1.2 million lux hours is calculated. The answer is that if a lamp
gives a total light of 6250 lux and if the exposure is done for
one hour, then it means an exposure of 6250 lux hours. One
full day exposure means 150 kilo lux hours (6250  24). An
8 days exposure accordingly is equivalent to 1.2 million lux
hours (6250  24  8).
Although not suggested in Fig. 4, advantage of direct sunlight
exposure can also be taken for forcing decomposition of drug.
In tropical climates, an exposure for 4 hours on a hot sunny day
would almost give same degradation as can be obtained with a
xenon lamp in the same period or in a fluorescent and UV lamps
fitted photostability chamber in 8 days. The advantage of using
sun test equipment, however, should not be undermined as one
gets an equal standard exposure round the year while sunlight
testing is highly dependent upon vagaries of nature.
Stress testing of biological or biotechnological products
Stress studies are also required to be conducted on biotechno-
logical and biological products. This is a requirement under the
ICH guideline Q5C (17). The stress studies are said to be useful
i) in determining if accidental exposures to conditions other than
those proposed are deleterious to the product, ii) for evaluating
which specific test parameters may be the best indicators of prod-
uct stability and iii) in revealing patterns of degradation. The
guideline emphasizes that the conditions of the stress study as
enshrined in the tripartite stability guideline Q1A may not be
appropriate for biotechnological/biological products. It is stated
that ‘conditions should be carefully selected on a case-by-case
basis’.
Accordingly, it is not claimed that the classification system
and decision trees, as discussed in this paper, would apply to
biotechnological/biological drugs and their products. Unlike,
chemical drugs, the bio products are particularly sensitive to
factors such as temperature changes, oxidation, light, ionic
strength and shear. Their main routes of decomposition also
Pharmaceutical Technology On-Line APRIL 2000 13
vary from chemical drugs. The bio products undergo reactions
such as deamidation, oxidation, aggregation, proteolysis, etc.
(17). The exact classification system for these products need to
be developed, by making a survey of the reports on intrinsic
stability of these drugs. This exercise remains to be done.
Conclusions
The determination of inherent stability of compounds using
stress testing, in exact terms, is not something new. It has been
practiced since decades as evident from the reports in literature.
The only difference is that it is now spelt out as a requirement
in 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 of
the chemistry part of the dossier and remains closed till the in-
novator itself decides to make it open. Sometimes studies are
done and reported by independent groups and they appear in
literature. Except that there is some laxity given on existing and
pharmacopoeial drugs, like done by CPMP guideline QWP/
556/96 (18), stress studies otherwise need to be done on all new
drugs.
As it mostly applies to new drugs, the activity related to stress
testing has been practiced mainly by those who were involved
in the process of new drug discovery, meaning that the activity
was restricted to countries in the developed world from where
most 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 hence
the activity of stress testing also is going to be wide spread.
From this aspect, it is hoped that the guidance provided in
this paper would be of general and wide interest. However, it
may be pertinent to add here that the opinions expressed are
purely personal to the authors and do not represent thinking
of the regulatory agencies.
References
1. 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 Examination
of 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 of
Isoxazolyl Penicillins using Dual Wavelength High-Performance Liq-
uid Chromatography,” J. Chromatogr. 708, 153–159 (1998).
9. ICH, “Stability Testing: Photostability Testing of New Drug Substances
and Products” (International Conference on Harmonization, Geneva,
November 1996).
10. N.H. Anderson, “Photostability Testing: Design and Interpretation of
Tests on Drug Substances and Dosage Forms,” in The Photostability of
14 Pharmaceutical Technology On-Line APRIL 2000
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 of
Temperature, 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. Part
1. Spectroscopic and Electrochemical Investigation of Interactions
under 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 Antigenic
Polymerization 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 for
the 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 Liquid
Chromatographic Determination of Trifluoperazine hydrochloride in
Bulk Form and Tablets,” Eur. J. Pharm. Sci. 5, 1–5 (1997).
22. R.L. Garnick et al., “High-Performance Liquid Chromatographic Assay
for Sodium levothyroxine in Tablet Formulations: Content Uniformity
Applications,” 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 Liquid
Chromatographic 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 and
Aqueous 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-Performance
Liquid 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 Dosage
Forms 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 of
Pharmaceuticals (J. Wiley & Sons, New York, 1986).
33. H.H. Tonnerson, Photostability Testing: Design and Interpretation of
Tests on Drug Substances and Dosage Forms (Taylor and Francis, Lon-
don, 1996).
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