See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/260420032

Pharmaceutical Forced Degradation Studies with Regulatory Consideration

Article · December 2013

CITATIONS READS
6 11,131

1 author:

Namdeo Shinde
Dr. Babasaheb Ambedkar Technological University
31 PUBLICATIONS   153 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

titrations View project

All content following this page was uploaded by Namdeo Shinde on 01 March 2014.

The user has requested enhancement of the downloaded file.

Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Oct.-Dec. Pg 178-188 [AJPSci.]

ISSN- 2231–5640 (Print) www.asianpharmaonline.org

ISSN- 2231–5659 (Online)

REVIEW ARTICLE

Pharmaceutical Forced Degradation Studies with Regulatory Consideration

Namdeo G. Shinde1*, Bhaskar N. Bangar1, Sunil M. Deshmukh1, Suyog P. Sulake1,
Dipak P. Sherekar2
1
Department of Pharmaceutics, Satara College of Pharmacy, Degaon, Satara-415004, (MS) India.
2
Satara College of Pharmacy, Degaon, Satara-415004, (MS) India.
*Corresponding Author E-mail: pr.shindenamdeo@gmail.com

ABSTRACT:
Forced degradation is a powerful tool used routinely in pharmaceutical development in order to develop stability-
indicating methods that lead to quality stability data and to understand the degradation pathways of the drug substances
and drug products. These experiments generally expose the material to an external stress to assess the stability of the
constituents or formulation. External Stress mainly includes temperature, pH, light, moisture, and even exposure to
other materials within the product formulation, and their degradation products. Conventionally, degradation tests can
take very long periods of time, because standard test methods require the materials to be exposed to stress factors for
periods of weeks or longer, and then tested using standard analytical methods. Accelerated testing is of clear benefit, as
use of elevated temperature to increase the rate of interactions is the most powerful factor to shorten the length of time
required for these tests. Forced degradation studies ensure appropriate stability of final pharmaceutical products in
very early stages of pharmaceutical development.

KEYWORDS: Forced degradation study, Active Pharmaceutical Ingredient (API), and International Conference on
Harmonization (ICH).

INTRODUCTION:
Forced degradation is the process of subjecting drug
compounds to extreme chemical and environmental
conditions to determine product breakdown levels and
preliminary degradation kinetics, and to identify degradant
species. The stress testing practices that companies use can
vary significantly and can have a serious impact on the
analytical methodology used throughout the industry. Stress
testing studies can be conducted to challenge specificity of
stability indicating and impurity monitoring methods as part
of validation protocol. Forced degradation or stress testing
studies are part of the development strategy and are an
integral component of validating analytical methods that
indicate stability and detect impurities. This relates to the
specificity section of the validation studies. It is important
to recognize that forced degradation studies are not
designed to establish qualitative or quantitative limits for
change in drug substance or drug product.
Received on 30.10.2013 Accepted on 25.11.2013
© Asian Pharma Press All Right Reserved
Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Pg 178-188
178
Forced degradation studies are used for multiple purposes,
including demonstration of the specificity of separation
methods, gaining insight into degradation pathways, and
discernment of degradation products in formulations that
are related to drug substances versus those that are related
to other ingredients of a formulation. Reliable chemical
stability testing data can show how a drug product changes
over time with influence of environmental factors. The
stability of a drug product or a drug substance is a critical
parameter, which may affect purity, potency and safety.
Changes in drug stability can risk patient safety by
formation of a toxic degradation product(s) or deliver a
lower dose than expected. Therefore, it is essential to know
the purity profile and behaviour of a drug substance under
various environmental conditions.
The main purpose of forced degradation testing studies is to
evaluate the overall photosensitivity of the material for
method development purposes and/or degradation pathway
elucidation. This testing may involve the drug substance
alone and/or in simple solutions/suspensions to validate the
analytical procedures. For development and validation
purposes, it is appropriate to limit exposure and end the
studies if extensive decomposition occurs. For photostable
materials, studies may be terminated after an appropriate
Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Oct.-Dec. Pg 178-188
exposure level has been used. The initial purpose of forced
degradation studies is to investigate stability-related
properties of an API and to develop an understanding of the
degradation products and pathways. These studies should
also be used to evaluate the susceptibility of the drug
substance to hydrolysis across a wide range of pH values.
Forced degradation studies are used to provide degraded
samples for the development of stability-indicating
analytical methods for the API. The information developed
from a forced degradation study can be utilized in several
other areas of development, including analytical methods
development, formulation development and storage
conditions, manufacturing processing, safety toxicological,
identification of possible genotoxic degradants,
identification of potential metabolites and API
design/discovery.
Forced degradation is synonymous with stress testing and
purposeful degradation. Purposeful degradation can be a
useful tool to predict the stability of a drug substance or a
drug product with effects on purity, potency, and safety. It
is imperative to know the impurity profile and behaviour of
a drug substance under various stress conditions. Forced
degradation also plays an important role in the development
of analytical methods, setting specifications, and design of
formulations under the quality-by-design (QbD) paradigm.
The nature of the stress testing depends on the individual
drug substance and the type of drug product (e.g., solid oral
dosage, lyophilized powders, and liquid formulations)
involved. Forced degradation studies are most beneficial if
done early in the drug development process. The reasoning
for this is that these studies yield predictive information on
the nature of the degradants which are valuable when
assessing the appropriate synthesis routes, API salt
selection and formulation development. These early studies
can help to provide information needed for the following:
1. As a predictive tool to understand degradation
pathways and stability-related issues.
2. To predict API stability before real-time stability data
is available.
3. Development and validation of stability-indicating
methodology.
4. Determination of degradation pathways of drug
substances and drug products.
5. Discernment of degradation products in formulations
that are related to drug substances versus those that are
related to non–drug substances (e.g., excipients).
6. Structure elucidation of degradation products.
7. Determination of the intrinsic stability of drug
substances.
8. Helps to identify reactions that cause degradation of
pharmaceutical products.
9. To generate a degradation profile that mimics what
would be observed in a formal stability study under
ICH conditions.
10. To generate more stable formulation.
11. To identify impurities related to drug substances and
excipients.
12. To understand the drug molecular chemistry.
179
[AJPSci.]
13. Selection of storage conditions, packaging and better
understanding of the potential liabilities of the drug
molecule chemistry. [1]
Forced degradation studies can be done on both, the solid
state and aqueous solution or suspension forms of the API.
Furthermore, the use of analysis at multiple time points
allows for approximation of rates of degradation and such
testing at early time points can provide a distinction
between primary and secondary degradation products. This
approach allows for better degradation pathway
determination.
Forced degradation studies should be repeated as needed
throughout the drug development process. For example,
when there are changes in API impurity profile, API salt or
polymorph form. When carried out in late development,
such studies are referred as confirmatory studies.
Confirmatory studies are quantitative in nature. Full mass
accountability of the API, its impurities and degradation
products are generated from these late stage studies.
Furthermore, based upon the outcome of these studies, if
necessary, new or orthogonal methods may need to be
developed to account for all observed degradation. In
addition, confirmatory studies for API are done after
finalization of the synthetic route and form of the API. Such
studies are typically done in Phase III with one of the
registration batches of the API. For drug products,
confirmatory studies are done when final formulation(s) and
packaging are chosen. After the confirmatory studies are
completed, a report on degradation products and pathways
is generated and included in or used to support NDA filings.
DEGRADATION CONDITIONS [2, 3]
The following are general conditions that should be
considered when conducting forced degradation studies:
Solid State
• Heat
• Heat/humidity
• Light
Solution and/or Suspension
• Hydrolysis at various pHs
• Unbuffered HCl, NaOH, water
• Buffer solutions (used to determine if pH adjustment
needed to attain maximum stability)
• Oxidative stress testing
• H2O2 (to mimic possible presence of peroxides in
excipients)
• Metal ions (to mimic possible exposure during
manufacture)
• Radical initiators (to mimic autoxidation)
• Light
SOURCES OF IMPURITIES AND TYPES OF
IMPURITIES:
In a typical study, relevant stress conditions are light, heat,
humidity, hydrolysis (acid / base influence) and oxidation
or even a combination of described parameters. If it is
necessary to form degradation products, the strength of
Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Oct.-Dec. Pg 178-188
stress conditions can vary due to the chemical structure of
the drug substance, the kind of drug product, and product
specific storage requirements. An individual program has to
be set up in order to reach a target degradation of 5 to 20%.
A higher level of degradation will be out of the scope of
product stability requirements and therefore unrealistic. The
scope of the test is to generate degradation products in order
to facilitate a method development for determination of the
relevant products. Therefore, samples will be stressed in a
solid form and/or in solution. Typically, stress tests are
carried out on one batch of material. For drug products the
placebo should be stressed in a similar way in order to
exclude those impurities that are not degradation products
(e.g. impurities arising from excipients). Table 1 shows
typical stress conditions of API and drug product. Drugs
that are poorly soluble in water can be conducted either in
suspension or in solution using inert organic co-solvents
(e.g., DMSO, acetic acid or propionic acid). It is important
to avoid co-solvents that may be reactive with the drug or
complicate analysis (e.g. by LC-MS). [7, 8]
For drug substances, photostability testing should consist of
two parts: forced degradation testing and confirmatory
testing. The purpose of forced degradation testing studies is
to evaluate the overall photosensitivity of the material for
method development purposes and/or degradation pathway
elucidation. This testing may involve the drug substance
alone and/or in simple solutions/suspensions to validate the
analytical procedures. In these studies, the samples should
be in chemically inert and transparent containers. In these
forced degradation studies, a variety of exposure conditions
may be used, depending on the photosensitivity of the drug
substance involved and the intensity of the light sources
used. For development and validation purposes it is
appropriate to limit exposure and end the studies if
extensive decomposition occurs. For photostable materials,
studies may be terminated after an appropriate exposure
level has been used. The design of these experiments is left
to the applicant’s discretion although the exposure levels
used should be justified. The potential for these degradation
pathways should be assessed in both drug substance and
drug product. These mechanisms can be assessed in a
systematic way by exposure to stress conditions of heat,
humidity, photo stress, oxidative conditions, and aqueous
conditions across a broad range of pH. The ICH guidelines
Q3A (R) require identification of each impurity with
respect to both chemistry and safety perspective. The
chemistry perspective includes classification and
identification of impurities, report generation, listing of
impurities in specific guidance and a brief discussion of
analytical procedures while safety perspectives include
specific guidance for qualifying those impurities. The
impurities usually encountered in pharmaceuticals are
synthesis related, formulation related, and degradation
related. These ICH guidelines classify the impurities into
the fallowing categories.
1. Impurities associated with active pharmaceutical
ingredient (APIs).
2. Impurities that are formed during formulation, formed
with ageing and that are related to formulation forms.
180
[AJPSci.]
Impurities associated with active pharmaceutical
ingredients:
According to the ICH guidelines, impurities associated with
APIs are classified into the fallowing categories
A. Organic impurities
B. Inorganic impurities
Organic impurities:
• Starting materials
• Byproducts
• Intermediates
• Degradation products
• Residual solvents
• Reaction products with excipients
• Leachables from container closure system
Inorganic impurities
• Reagents, ligands, catalysts
• Heavy metals or other residual metals
• Inorganic salts
Other materials
• Filter aids
• Charcoal
Formulation related impurities:
During formulation, excipients added to API to render the
product elegant. In such cases, drug-excipients
incompatibility may lead to undesirable products, which
can affect the therapeutic efficiency of the product. In
addition, other solvents, which is usually present in API or
used in the synthesis of API or excipients, may also interact
with excipients resulting in impurities.
Dosage form related impurities:
1. Precipitation of main ingredient can occur due to
various factors like pH, environment, or leaching. In
general, liquid dosage forms are very much susceptible to
both degradation and microbial contamination.
Example- Precipitation of imipramine HCl with sodium
bisulfate and pH alteration of lidocaine HCl in presence of
5% dextrose solution.
2. Pharmaceutical solids: in the presence of excipients
and moisture topochemical and nucleation reactions occur.
Example- Presence of sodium CMC during granulation of
papaverine, aminopyrine and salicylic acid tablets causes
reduced coloration.
3. Microbial growth resulting from the growth of
bacteria, fungi, and yeast in a humid and warm environment
may result in oral liquid products that are unusable for
human consumption.
Method related impurities:
A known impurity 1-(2, 6 Dichloro phenyl) indoline-2one is
formed in the production of a parental dosage form
diclofenac sodium ampoules. Formation of this impurity
depends on initial pH of the preparation and the conditions
of sterilization i.e. autoclave method (1210c) that enforces
Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Oct.-Dec. Pg 178-188 [AJPSci.]

the intramolecular cyclic reaction of diclofenac sodium Example- Aspirin combined with water and hydrolyzed to
forming indolineone derivative and sodium hydroxide.[5] form salicylic acid and acetic acid. The hydrolytic
degradation of new drug in acidic and alkaline condition
Environmental related impurities: can be studied by refluxing the drug in 0.1N HCl or 0.1N
The environmental factors that can reduce stability are NaOH. If degradation is seen testing can be stopped at that
point. In case of no degradation under these conditions, the
i. Temperature: drug should be refluxed in acid and alkali of higher strength
There is many APIs that are liable to heat or topical and for a long duration time. Hydrolysis of most of drugs is
temperature. Ex: Vitamins as drug substances are very heat depending upon the relative concentration of hydronium
sensitive and degradation frequently leads to loss of and hydroxyl ions.
potency in vitamin products, especially in liquid Example- Anatrazole – degraded in basic pH
formulations. Degradation caused by exposure to Doxophylline – shows degradation in acidic pH
temperatures include bond breakage i.e. pyrolysis. Any
degradation mechanism that is enhanced at elevated A reaction in which water is an reactant causing
temperatures are thermolytic pathways include Hydrolysis, precipitation. Hydrolysis is a common phenomenon for
Dehydration, Isomerization, Decorboxylation, ester type of drugs. Esters, amides, lactones, lactams,
Rearrangement, Polymerization reactions, pyrolysis. In imides, and carbamates, are susceptible for acid base
general, rate of reaction increases with increase in hydrolysis.
temperature. Many APIs are sensitive to heat or topical Examples- Aspirin, Chloramphenicol, Barbiturates,
temperature such as vitamins and peptides. Effect of Chlordiazepoxide, Oxazepam, Examples includes Aspirin,
temperature on thermal degradation of a substance is Benzocaine, Cefolaxime, Cocaine, and Cefpodoxime.
studied through Arrhenius equation.
2) Oxidation:
K = Ae -Ea/RT Many drug substances undergo autoxidation i.e. oxidation
Where K = specific reaction rate under normal conditions and involving ground state
A = frequency factor elemental oxidation. Autoxidation is a free radical reaction
Ea = energy of activation that requires free radical initiator to begin the chain
reaction. Hydrogen peroxide, metal ions, traces level of
Thermal degradation study is carried out at 400C. The impurities in a drug substance act as initiator for oxidation.
mostly accepted temperature is 700C at low and high The oxidative stress testing is initially carried out in 3%
humidity for 1-2 months. The use of high-temperatures in H2O at room temperature for 6 h and it can be increased or
predictive degradation studies assumes that the drug decreased to achieve sufficient degradation. The time can
molecule will follow the same pathway of decomposition at also be increased up to 24 hr 3% or decreased up to 30 min
all temperatures.[9] with 1% of H2O The mechanism of oxidative degradation
of drug substance involves an electron transfer mechanism
ii. Light (UV light): Light is one of the means by which the to form reactive anions and cations. The mechanism of
formulation degrades because of photolytic reaction. Ex: oxidative degradation of drug substance involves an
sunlight having about 8000 foot-candles can destruct nearly electron transfer mechanism to form reactive anions and
34% of vitamin B in 24 hrs. cations. Drugs In pharmaceuticals most common form of
oxidative decomposition is oxidation through a free radical
iii. Humidity: Humidity is one of the important key factors chain process.
in case of hygroscopic compounds. Ex: Ranitidine,
Aspirin[9] Example- Amines, sulphide, and phenols are susceptible to
electron transfer oxidation. Drugs which prone to oxidation
Functional group related: are hydrocortisone, methotrexate, adinazolam,
1) Hydrolysis: catecholamine conjugated dienes, heterocyclic aromatic
Drug degradation that involves reaction with water is called rings, nitraso and nitrate derivatives.
hydrolysis. PH, buffer salts, ionic strength, solvents,
complexing agents, surfactants and excipients affect a) Rapamycin, an immunosuppressant drug reacts with
hydrolysis. Hydrolysis reactions are typically acid or base oxygen to form a complex mixture of monomeric and
catalyzed. Hydrolysis is the most common degradation oligomeric products. Formation of the majority of the
chemical reaction over wide range of pH. Water either as rapamycin degradation products could be rationalized with
solvent or as moisture in the air comes in contact with free radical-mediated autoxidation reactions involving
pharmaceutical dosage form is responsible for degradation alkenes and alcohol sites.
of most drugs. Hydrolytic study under acidic and basic
condition involves catalyzation of ionisable functional b) Auto-oxidation of ascorbic acid studies reveals that
groups present in the molecule. cupric ion known to oxidize ascorbic acid rapidly in to
dehydro ascorbic acid and potassium cyanide.

181
Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Oct.-Dec. Pg 178-188 [AJPSci.]

3) Photolysis: Stress conditions

Exposure of drug molecule to light may produce photolytic
degradation products. The rate of photo degradation
depends upon the intensity of incident light and quantity of
light absorbed by drug molecule. Photolytic degradation is
carried out by exposing the drug substance to or drug
product to a combination of visible and UV light. The
photolytic degradation can occur through non-oxidative and
oxidative photolytic reaction.
a) The non-oxidative photolytic reaction includes
isomerization, diamerization, cyclization, rearrangement,
decorboxylation and haemolytic cleavage.
b)
Oxidative photolytic reaction occurs through either
single oxidation or triple oxidation mechanism. Ex:
Barnidipin.[1,10]
Photolytic cleavage on aging includes examples of
pharmaceutical drugs or products that are prone to
degradation on exposure to UV-light. During manufacturing
process or packing or on storage drugs like ergometrine,
nifedipine, nitroprucide, riboflavin, are liable to photo
oxidation. Most of the compounds will degrade as solutions
when exposed to high-energy UV exposure. This photo
oxidation involves generation of free-radical intermediates,
which will degrade the product.
Example- The formulation of ciprofloxacin eye drops 0.3%
on exposure to UV-light induces photolysis, which results
in formation of ethylene di-amine analogue of
ciprofloxacin.
Typical stress tests include four main degradation
mechanisms: heat, hydrolytic, oxidative, and photolytic
degradation. Selecting suitable reagents such as the
concentration of acid, base, or oxidizing agent and varying
the conditions (e.g., temperature) and length of exposure
can achieve the preferred level of degradation. Over-
stressing a sample may lead to the formation of secondary
degradants that would not be seen in formal shelf-life
stability studies and under-stressing may not serve the
purpose of stress testing. Therefore, it is necessary to
control the degradation to a desired level. A generic
approach for stress testing has been proposed to achieve
purposeful degradation that is predictive of long-term and
accelerated storage conditions. The generally recommended
degradation varies between 5-20% degradation. This range
covers the generally permissible 10% degradation for small
molecule pharmaceutical drug products, for which the
stability limit is 90%-110% of the label claim. Although
there are references in the literature that mention a wider
recommended range (e.g., 10-30%), the more extreme stress
conditions often provide data that are confounded with
secondary degradation products.
Table 1. The four climate zones (ICH Stability guidelines)
Zone I Temperate 21°C ± 2°C/ 45% RH ± 5% RH
Zone II Subtropical 25°C ± 2°C/ 60% RH ± 5% RH
and Mediterranean
Zone III Hot and Dry 30°C ± 2°C/ 35% RH ± 5% RH
Zone IV Hot and Humid 30°C ± 2°C/ 65% RH ± 5% RH

Selection of Stress Conditions Table 2. Type of study with Storage conditions (ICH Stability
Forced degradation is normally carried out under more guidelines)
severe conditions than those used for accelerated studies. Study Storage condition Min. time
The choice of stress conditions should be consistent with period
the products decomposition under normal manufacturing, Long term 25°C ± 2°C/ 60% RH ± 5% RH 12 months
or
storage, and use conditions, which are specific in each case.
30°C ± 2°C/ 65% RH ± 5% RH
The ICH guidance recognize that it is impossible to provide
Intermediate 30°C ± 2°C/ 65% RH ± 5% RH 6 months
strict degradation guidelines and allows certain freedom in
Accelerated 40°C ± 2°C/ 75% RH ± 5% RH 6 months
selecting stress conditions for biologics. The choice of
forced degradation conditions should be based on data from Table 3. Drug products intended for storage in refrigerator (ICH
accelerated pharmaceutical studies and sound scientific Stability guidelines)
understanding of the product’s decomposition mechanism Study Storage condition Min. time
under typical use conditions. A minimal list of stress factors period
suggested for forced degradation studies must include acid Long term 5°C ± 3°C 12 months
and base hydrolysis, thermal degradation, photolysis, Accelerated 25°C ± 2°C/ 60% RH ± 5% RH 6 months
oxidation, and may include freeze-thaw cycles and shear.
Table 4. Drug products intended for storage in freezer
Study Storage condition Min. time period
Regulatory guidance does not specify pH, temperature
Long term -20°C ± 5°C 12 months
ranges, specific oxidizing agents, or conditions to use, the
number of freeze-thaw cycles, or specific wavelengths and
Photostability
light intensities. The design of photolysis studies is left to
Photostability testing should be an integral part of stress
the applicant's discretion although Q1B recommends that
testing, especially for photo-labile compounds. Some
the light source should produce combined visible and
recommended conditions for photostability testing are
ultraviolet (UV, 320-400 nm) outputs, and that exposure
described in ICH Q1B Photostability Testing of New Drug
levels should be justified. Consult the appropriate
Substances and Products. Samples of drug substance, and
regulatory authorities on a case-by-case basis to determine
solid/liquid drug product, should be exposed to a minimum
guidance for light-induced stress.
of 1.2 million lux hours and 200-watt hours per square
meter light. The same samples should be exposed to both
white and UV light. To minimize the effect of temperature

182
Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Oct.-Dec. Pg 178-188 [AJPSci.]

changes during exposure, temperature control may be such as metal ions, oxygen, and radical initiators (e.g.,
necessary. The light-exposed samples should be analyzed azobisisobutyronitrile) can also be used. Selection of an
for any changes in physical properties such as appearance, oxidizing agent, its concentration, and conditions depends
clarity, color of solution, and for assay and degradants. The on the drug substance. Solutions of drug substances and
decision tree outlined in the ICH Q1B can be used to solid/liquid drug products can be subjected to oxidative
determine the photo stability testing conditions for drug degradation. It is reported that subjecting the solutions to
products. The product labelling should reflect the 0.1%-3% hydrogen peroxide at neutral pH and room
appropriate storage conditions. It is also important to note temperature for seven days or up to a maximum 20%
that the labelling for generic drug products should be degradation could potentially generate relevant degradation
concordant with that of the Reference Listed Drug (RLD) products. Samples can be analyzed at different time
and with United States Pharmacopeia (USP) monograph intervals to determine the desired level of degradation.
recommendations, as applicable.
Analysis method: The preferred method of analysis for a
Heat: stability indicating assay is reverse-phase high-performance
Thermal stress testing (e.g., dry heat and wet heat) should liquid chromatography. Reverse-phase HPLC is preferred
be more strenuous than recommended ICH Q1A accelerated for several reasons, such as its compatibility with aqueous
testing conditions. Samples of solid-state drug substances and organic solutions, high precision, sensitivity, and ability
and drug products should be exposed to dry and wet heat, to detect polar compounds. Separation of peaks can be
whereas liquid drug products can be exposed to dry heat. It carried out by selecting appropriate column type, column
is recommended that the effect of temperature be studied in temperature, and making adjustment to mobile phase pH.
10°C increments above that for routine accelerated testing, Poorly retained, highly polar impurities should be resolved
and humidity at 75% relative humidity or greater. Studies from the solvent front. As part of method development, a
may be conducted at higher temperatures for a shorter gradient elution method with varying mobile phase
period. Testing at multiple time points could provide composition (very low organic composition to high organic
information on the rate of degradation and primary and composition) may be carried out to capture early eluting
secondary degradation products. In the event that the stress highly polar compounds and highly retained nonpolar
conditions produce little or no degradation due to the compounds. Stressed samples can also be screened with the
stability of a drug molecule, one should ensure that the gradient method to assess potential elution pattern. Sample
stress applied is in excess of the energy applied by solvent and mobile phase should be selected to afford
accelerated conditions (40°C for 6 months) before compatibility with the drug substance, potential impurities,
terminating the stress study. and degradants. Stress sample preparation should mimic the
sample preparation outlined in the analytical procedure as
Acid and base hydrolysis: closely as possible. Neutralization or dilution of samples
Acid and base hydrolytic stress testing can be carried out may be necessary for acid and base hydrolyzed samples.
for drug substances and drug products in solution at Chromatographic profiles of stressed samples should be
ambient temperature or at elevated temperatures. The compared to those of relevant blanks (containing no active)
selection of the type and concentrations of an acid or a base and unstressed samples to determine the origin of peaks.
depends on the stability of the drug substance. A strategy The blank peaks should be excluded from calculations. The
for generating relevant stressed samples for hydrolysis is amount of impurities (known and unknown) obtained under
stated as subjecting the drug substance solution to various each stress condition should be provided along with the
pH (e.g., 2, 7, 10–12) at room temperature for two weeks or chromatograms (full scale and expanded scale showing all
up to a maximum of 15% degradation. Hydrochloric acid or the peaks) of blanks, unstressed, and stressed samples.
sulphuric acid (0.1M to 1M) for acid hydrolysis and sodium Additionally, chiral drugs should be analyzed with chiral
hydroxide or potassium hydroxide (0.1M to 1M) for base methods to establish stereo-chemical purity and stability.
hydrolysis are suggested as suitable reagents for hydrolysis.
For lipophilic drugs, inert co-solvents can be used to The analytical method of choice should be sensitive enough
solubilize the drug substance. Attention should be given to to detect impurities at low levels (i.e., 0.05% of the analyte
the functional groups present in the drug molecule when of interest or lower), and the peak responses should fall
selecting a co-solvent. Prior knowledge of a compound can within the range of detector's linearity. The analytical
method should be capable of capturing all the impurities
be useful in selecting the stress conditions. For instance, if a
compound contains ester functionality and is very labile to formed during a formal stability study at or below ICH
base hydrolysis, low concentrations of a base can be used. threshold limits. Degradation product identification and
Analysis of samples at various intervals can provide characterization are to be performed based on formal
information on the progress of degradation and help to stability results in accordance with ICH requirements.
distinguish primary degradants from secondary degradants. Conventional methods (e.g., column chromatography) or
hyphenated techniques (e.g., LC–MS, LC–NMR) can be
Oxidation: used in the identification and characterization of the
Oxidative degradation can be complex. Although hydrogen degradation products. Use of these techniques can provide
peroxide is used predominantly because it mimics possible better insight into the structure of the impurities that could
presence of peroxides in excipients, other oxidizing agents add to the knowledge space of potential structural alerts for
183
Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Oct.-Dec. Pg 178-188
genotoxicity and the control of such impurities with tighter
limits. It should be noted that structural characterization of
degradation products is necessary for those impurities that
are formed during formal shelf-life stability studies and are
above the qualification threshold limit. Various detection
types can be used to analyze stressed samples such as UV
and mass spectroscopy. The detector should contain 3D
data capabilities such as diode array detectors or mass
spectrometers to be able to detect spectral non-
homogeneity. Diode array detection also offers the
possibility of checking peak profile for multiple
wavelengths. The limitation of diode array arises when the
UV profiles are similar for analyte peak and impurity or
degradant peak and the noise level of the system is high to
mask the co-eluting impurities or degradants. Compounds
of similar molecular weights and functional groups such as
diastereoisomers may exhibit similar UV profiles. In such
cases, attempts must be made to modify the
chromatographic parameters to achieve necessary
separation. An optimal wavelength should be selected to
detect and quantitate all the potential impurities and
degradants. Use of more than one wavelength may be
necessary, if there is no overlap in the UV profile of an
analyte and impurity or degradant peaks. A valuable tool in
method development is the overlay of separation signals at
different wavelengths to discover dissimilarities in peak
profiles.
Table 5. Stress conditions for drug substance
Type of study Conditions Time
Acid hydrolysis 0.1 N- 1.0 N HCl, RT or higher 1-7 days
Base hydrolysis 0.1 N- 1.0 N NaOH, RT or higher 1-7 days
Thermal Aqueous solution, 70°C 1-7 days
hydrolysis
Oxidative/ O2 + Initiator (AIBN) in CAN/ 1-7 days
solution H2O; 80/20,40°C
0.3-3.0 % H2O/ Ambient in the Few hours
dark to 7 days
Thermal solid, 70°C Upto 3
weeks
Thermal/ solid, 70°C/ 75% RH Upto 3
Humidity weeks
Photo Solid, fluorescent and UV light >2× ICH
degradation
Peak purity analysis:
Peak purity is used as an aid in stability indicating method
development. The spectral uniqueness of a compound is
used to establish peak purity when co-eluting compounds
are present. Peak purity or peak homogeneity of the peaks
of interest of unstressed and stressed samples should be
established using spectral information from a diode array
detector. When instrument software is used for the
determination of spectral purity of a peak, relevant
parameters should be set up in accordance with the
manufacturer's guidance. Attention should be given to the
peak height requirement for establishing spectral purity. UV
184
[AJPSci.]
detection becomes non linear at higher absorbance values.
thresholds should be set such that co-eluting peaks can be
detected. Optimum location of reference spectra should also
be selected. The ability of the software to automatically
correct spectra for continuously changing solvent
background in gradient separations should be ascertained.
Termination of study:
Stress testing studies are terminated after ensuring adequate
exposure to stress conditions. Typical activation energy of
drug substance molecules varies from 12–24 kcal/mol. A
compound may not necessarily degrade under every single
stress condition. In circumstances, where some stable drugs
do not show any degradation under any of the stress
conditions, specificity of an analytical method can be
established by spiking the drug substance or placebo with
known impurities and establishing adequate separation.
Forced degradation in QbD paradigm
A well-designed, forced degradation study is indispensable
for analytical method development in a QbD paradigm. A
systematic process of manufacturing quality drug products
that meet the predefined targets for the critical quality
attributes (CQA) necessitates the use of knowledge
obtained in forced degradation studies. It helps to establish
the specificity of a stability indicating method and to predict
potential degradation products that could form during
formal stability studies. Incorporating all potential
impurities in the analytical method and establishing the
peak purity of the peaks of interest helps to avoid
unnecessary method re-development and revalidation.
Knowledge of chemical behaviour of drug substances under
various stress conditions can also provide useful
information regarding the selection of excipients for
formulation development. Excipients compatibility is an
integral part of understanding potential formulation
interactions during product development and is a key part of
product understanding. Degradation products due to drug-
excipient interaction or drug-drug interaction in
combination products can be examined by stressing samples
of drug substance, drug product, and placebo separately and
comparing the impurity profiles. Information obtained
regarding drug-related peaks and non-drug-related peaks
can be used in the selection and development of more stable
formulations. For instance, if a drug substance is labile to
oxidation, addition of an antioxidant may be considered for
the formulation. For drug substances that are labile to acid
or undergo stereochemical conversion in acidic medium,
delayed-release formulations may be necessary. Acid/base
hydrolysis testing can also provide useful insight in the
formulation of drug products that are liquids or suspensions.
Knowledge gained in forced degradation studies can
facilitate improvements in the manufacturing process.
Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Oct.-Dec. Pg 178-188
Table 6. Recommended labeling statements for finished pharmaceutical products (FPP)
Test conditions of FPP
25°C/ 60 % RH (long term)40°C/ 75 % RH (accelerated)
25°C/ 60 % RH (long term)30°C/ 65 % RH (intermediate, failure of accelerated)
30° C/ 65 % RH (long term)40°C/ 75 % RH (accelerated)
30° C/ 75 % RH ( long term)40°C/ 75 % RH (accelerated)
5° C ± 3° C
-20° C ± 5° C
If a photostability study shows a drug substance to be
photolabile, caution should be taken during the
manufacturing process of the drug product. Useful
information regarding process development (e.g., wet
versus dry processing, temperature selection) can be
obtained from thermal stress testing of drug substance and
drug product. Additionally, increased scientific
understanding of degradation products and mechanisms
may help to determine the factors that could contribute to
stability failures such as ambient temperature, humidity,
and light. Appropriate selection of packaging materials can
be made to protect against such factors.
GUIDELINES FOR FORCED DEGRADATON
STUDY
Impurities in new drug substances (CPMP/ICH/2737/99)
This document are intended to provide guidance for
registration applications on the content and qualification of
impurities in new drug substances produced by chemical
syntheses and not previously registered in a region or
member state. It is not intended to apply to new drug
substances used during the clinical research stage of
development. The following types of drug substances are
not covered in this guideline:
• Biological/biotechnological products
• Peptides
• Oligonucleotide
• Radiopharmaceuticals
• Fermentation products
• Semi-synthetic products
• Herbal products, crude products of animal or plant
origin.
Impurities in new drug products (CPMP/ICH/2738/99)
This guideline addresses only those impurities in new drug
products classified as degradation products of the drug
substance or reaction products of the drug substance with an
excipients and/or immediate container closure system
(referred to as "degradation products"). Generally,
impurities present in the new drug substance need not be
monitored or specified in the new drug product, unless they
are also degradation products. Impurities arising from
excipients present in the new drug product, extracted, or
leached from the container closure system are not covered
by this guideline. [5]
Stability Testing of New Drug Substances and Products
(ICH Q1A (R2))
Drug substance:
Stress testing a drug substance can help identify the likely
degradation products, which can in turn help establish the
degradation pathways and the intrinsic stability of the
185
[AJPSci.]
Recommended labeling statement
“Do not store above 25° C”
“Do not store above 25° C”
“Do not store above 30° C”
“Do not store above 30° C”
“Store in refrigerator” (2° C to 8° C)
“Store in freezer”
molecule and validate the stability-indicating power of the
analytical procedures used. The nature of the stress testing
will depend on the individual drug substance and the type
of drug product. Stress testing is likely to be carried out on
a single batch of the drug substance. It should include the
effect of temperatures (in 100C increments [e.g., 500C,
600C] above that for accelerated testing), humidity (e.g.,
75% RH or greater) where appropriate, oxidation, and
photolysis on the drug substance. Testing should evaluate
the susceptibility of the drug substance to hydrolysis across
a wide range of pH values when in solution or suspension.
Photostability testing should be an integral part of stress
testing. Examining degradation products under stress
conditions is useful for establishing degradation pathways,
developing and validating suitable analytical procedures. It
may not be necessary, however, to examine for specific
degradation products if previous studies have demonstrated
that these products are not formed under accelerated or
long-term storage conditions.
Drug product:
The design of formal stability studies for a drug product
should be based on the behavior and properties of the drug
substance, the results from stability studies on the drug
substance, and the experience gained from clinical
formulation studies. The likely changes to storage
conditions and the rationale for the selection of attributes to
be tested in the formal stability studies should be stated.
Photostability testing should be conducted on at least one
primary batch of the drug product if appropriate.
Stability Testing: Photostability Testing of New Drug
Substances and Products (ICH Q1B)
Drug substance:
Photostability testing should consist of two parts: forced-
degradation testing and confirmatory testing. The purpose
of forced-degradation testing is to evaluate the overall
photosensitivity of the material for method-development
purposes and/or degradation pathway elucidation. This
testing may involve the drug substance alone and/or the
substance in simple solutions and suspensions to validate
the analytical procedures. In these studies, the samples
should be in chemically inert and transparent containers.
For forced-degradation studies, various exposure conditions
may be used, depending on the photosensitivity of the drug
substance and the intensity of the light sources. For
development and validation purposes, it is appropriate to
limit exposure and end the studies if extensive
decomposition occurs. For photostable materials, studies
may be terminated after an appropriate exposure level has
been used. The design of these experiments is left to the
applicant’s discretion although the exposure levels used
Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Oct.-Dec. Pg 178-188
should be justified. Under forcing conditions,
decomposition products may be observed that are unlikely
to be formed under the conditions used for confirmatory
studies. This information may be useful in developing and
validating suitable analytical methods. If in practice it has
been demonstrated that they are not formed in the
confirmatory studies, these degradation products need not
be further examined.
Validation of Analytical Procedures: Methodology (ICH
Q2B)
Drug substance/drug product:
If impurity or degradation product standards are
unavailable, specificity can be demonstrated by comparing
the test results of samples containing impurities or
degradation products with a second well-characterized
procedure; e.g., pharmacopeial method or other validated
analytical procedure (independent procedure). As
appropriate, this should include samples stored under
relevant stress conditions (light, heat, humidity, acid–base
hydrolysis, and oxidation).
Impurities in New Drug Substances (ICH Q3A(R))
Drug substance:
The applicant should summarize the actual and potential
impurities most likely to arise during the synthesis,
purification, and storage of the new drug substance. This
summary should be based on sound scientific appraisal of
the chemical reactions involved in the synthesis, impurities
associated with raw materials that could contribute to the
impurity profile of the new drug substance, and possible
degradation products. This discussion can be limited to
those impurities that might reasonably be expected based on
knowledge of the chemical reactions and conditions
involved. In addition, the applicant should summarize the
laboratory studies conducted to detect impurities in the new
drug substance. This summary should include test results of
batches manufactured during the development process and
batches from the proposed commercial processes well as
the results of stress testing (see ICH Guideline Q1A on
Stability) used to identify potential impurities arising during
storage. The impurity profile of the drug substance batches
intended for marketing should be compared with those used
in development and any differences discussed.
Impurities in New Drug Products (ICH Q3B(R))
Drug product:
Analytical procedures should be validated to demonstrate
specificity for the specified and unspecified degradation
products. As appropriate, this validation should include
samples stored under relevant stress conditions: light, heat,
humidity, acid/base hydrolysis, and oxidation.
FDA Guidance for Industry: Stability Testing of Drug
Substances and Drug Products
Drug substance/drug product:
Stress testing helps determine the intrinsic stability
characteristics of a molecule by establishing degradation
pathways to identify the likely degradation products, and to
validate the stability-indicating power of the analytical
186
[AJPSci.]
procedures used. Stress testing provides data about the
forced-decomposition products and decomposition
mechanisms of the drug substance. The severe conditions
that may be encountered during distribution can be covered
by stress testing definitive batches of the drug substance.
These studies should establish the inherent stability
characteristics of the molecule such as the degradation
pathways, lead to identification of degradation products,
and hence support the suitability of the proposed analytical
procedures. The detailed nature of the studies will depend
on the particular drug substance and type of drug product.
This testing is likely to be carried out on a single batch of a
drug substance. Testing should include the effects of
temperatures in 100C increments above the accelerated
temperature test condition (e.g., 500C, 600C) and humidity,
where appropriate (e.g., 75% or greater). In addition,
oxidation and photolysis on the drug substance plus its
susceptibility to hydrolysis across a wide range of pH
values when in solution or suspension should be evaluated.
Results from these studies will form an integral part of the
information provided to regulatory authorities. Light testing
should be an integral part of stress testing. Some
degradation pathways can be complex and under forced
conditions, decomposition products may be observed that
are unlikely to be formed under accelerated or long-term
testing. This information may be useful in developing and
validating suitable analytical methods, but it may no always
be necessary to examine specifically for all degradation
products if, in practice, it has been demonstrated that these
are not formed.
FDA Guidance for Industry: Analytical Procedures and
Methods Validation
Drug substance/drug product:
Degradation information obtained from stress studies (e.g.,
products of acid and base hydrolysis, thermal degradation,
photolysis, and oxidation) for the drug substance and for the
active ingredient in the drug product should be provided to
demonstrate the specificity of the assay and analytical
procedures for impurities. The stress studies should
demonstrate that impurities and degradants from the active
ingredient and drug product excipients do not interfere with
the quantization of the active ingredient.
FDA Guidance for Industry: Submitting Documentation
for the Stability of Human Drugs and Biologics
Drug substance:
A program for the stability assessment might include
storage at ambient temperature and under stress conditions.
Stress testing conditions ordinarily include temperature
(e.g., 5, 50, and 750C), humidity, where appropriate (e.g.,
75% or greater) and exposure to various wavelengths of
electromagnetic radiation (e.g., 190–780 nm UV and visible
ranges), preferably in open containers, where applicable. It
is also suggested that the following conditions be evaluated
in stability studies on solutions or suspensions of the bulk-
drug substance: acidic and alkaline pH, high oxygen
atmosphere, and the presence of added substances under
consideration for product formulation.
Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Oct.-Dec. Pg 178-188
Drug product: REFERENCES:
Stress testing the drug product helps identify potential 1. Kallam.Vinusha, Srinath Nissankararao, Ramadevi Bhimavarapu,
problems during storage and transportation and provides an K.Renuka, J. Naga Silpa, S. Lakshmi Sravanthi. Retrospect of
estimate of the expiration-dating period. impurity profiling – an industry perspective. int.j.inv.pharm.sci, 1
FDA Guidance for Industry: INDs for Phase II and
Phase III Studies, Chemistry Manufacturing, and
Controls Information
Drug substance:
Performance of stability-stress studies with the drug
substance early in drug development is encouraged because
these studies provide information crucial to selecting
stability indicating analytical procedures for real-time
studies. If not performed earlier, stress studies should be
conducted during Phase 3 to demonstrate the inherent
stability of the drug substance, potential degradation
pathways, and the capability and suitability of proposed
analytical procedures. Stress studies should assess the
stability of the drug substance in different pH solutions, in
the presence of oxygen and light, and at elevated levels of
temperatures and humidity. These one-time stress studies
on a single batch are not considered part of the formal
stability program.
Drug product:
Drug products, one-time stress testing can be warranted to
11.
assess the potential for changes in the physical (e.g., phase
separation, precipitation, aggregation, changes in particular-
size distribution) and/or chemical (e.g., degradation and/or
interaction of components) characteristics of the drug
product. The studies could include testing to assess the 12.
effect of high temperature, humidity, oxidation, photolysis
and/or thermal cycling. [6] 13.
CONCLUSION:
Forced degradation studies, plays important role in
formulation development process. A well-designed stress
study can provide insight in choosing the appropriate
formulation for a proposed product prior to intensive
formulation development studies. A thorough knowledge of
degradation, including mechanistic understanding of
potential degradation pathways, stress conditions etc. are
essential for success of these studies. Stress testing can
provide useful insight into the selection of physical form,
stereo-chemical stability of a drug substance, packaging,
and storage conditions. It is important to perform stress
testing for generic drugs due to allowable qualitative and
quantitative differences in formulation with respect to the
RLD, selection of manufacturing process, processing
parameters, and packaging materials.
ACKNOWLEDGEMENT:
The authors express their sense of gratitude towards 22.
management of Satara College of Pharmacy, Degaon,
Satara for providing all obligatory facilities necessary to 23.
carry out present work.
187
[AJPSci.]
(2) 2013; 89-98.
2. J Menoutis, A Paris. Technical Guide Series Forced Degradation
Studies. Quantex Laboratory, 2012.
3. Kishor K Hotha, sattho phani kumar reddy, V Kishor Raju.
Forced degradation studies: practical approach: overview of
regulatory guidance and literature for the drug products and drug
substances. Int. Res. J. Pharm. 4 (5): 2013.
4. Gabriel K Kaddu. Stability studies. National Drug Authority,
WHO, Uganda, 2009: 1-38.
5. Stefanie Seitz. Purity testing and analytical method requirements.
Pharma solutions, 2012: 1-46.
6. Kathy Waddle, Wai Pan. Stability studies in pharmaceutical
development. Catalant Pharma Solutions, 2009: 1-56.
7. Hildegard Brummer. How to approach a forced degradation
study. Technical bulletin, (31) 2011: 1-4.
8. Michael Kats. Forced Degradation Studies: Regulatory
Considerations and Implementation. BioPharm International,
2005: 1-08.
9. Yande Huang, Wei Ding. Forced degradation study of
BMS582664 a prodrug of Brivanib for the treatment of cancer.
Bristol Mayers Squib, USA, 2013: 1-23.
10. Doredla Narasimha rao, M. Prasada rao, J. Naga Hussain, S.
Lakshmi Sumanoja and V. Rajeswara rao. Method development
and validation of forced degradation studies of metformin
hydrochloride by using UV spectroscopy. IJPCBS, 3(3) 2013:
546-553.
Michael Kats. Forced Degradation Studies: Regulatory
Considerations and Implementation Stress testing studies are
conducted to challenge specificity of stability- indicating and
impurity- monitoring methods as part of validation protocol.
BioPharm Int, (1) 2005: 1-7.
FDA. Guidance for Industry. Analytical Procedures and Methods
Validation: Chemistry, Manufacturing, and Controls
Documentation, Draft Guidance, 2000.
ICH Guidance for Industry, Quality of Biotechnological
Products: Stability Testing of Biotechnological/Biological
Products. ICH-Q5C. 1996 July 10.
14. Kovaleski J, Kraut B, Matiuz A, Giangiulio M, Brosbst G, Cango
W. Impurities in generic pharmaceutical development. Adverse
Drug Rev (59) 2007: 56-63.
15. Rao RN, Nagaraju V. A overview of the recent trends in
development of HPLC methods for determination of impurities in
drugs. J Phramaceut Biomed, 33, 2003: 335-337.
16. Zhou L, Mao B, Novak T, Ge Z. Impurity profile tracking for act
pharmaceutical ingredients: Case reports. J Pharmaceut Biomed,
44, 2007: 421-429.
17. ICH Q3A (R2) (October 2006). Impurities in New Drug
Substances and Products (Step 4), International Conference on
Harmonization.
18. ICH Q3B (R2) (June 2006). Impurities in New Drug.
19. Substances and Products (Step 4), International Conference on
Harmonization.
20. Ragine Maheswaran Ragine. Scientific Considerations of Forced
Degradation Studies in ANDA. Pharmaceutical Technology 36
(5), 2012: 73-80.
21. ICH Q3C (R3) (September 2002). Impurities: Guidelines for
Residual solvents (Step 5), International Conference on
Harmonization.
Ahuja S. Impurities Evaluation of Pharmaceuticals. Marcel
Dekker, New York, 1998.
Shirazi D, Ware E, Harper T, Pflaumer A. The Benefits of Using
AAI Pharma Services for Elemental Impurity Analysis Using
ICP-MS Technology. Am Pharmaceut Rev 2012: 1-10.
24. Qiu F Norwood DL, Identification of pharmaceutical impurities.
J Liq Chromatogr R T 2007; 30: 877-935.
25. Matthews BR. Regulatory aspects of stability testing in Europe.
Drug Dev Ind Pharm 1999; 25: 831-856.
 Asian J. Res. Pharm. Sci. 2013; Vol. 3: Issue 4, Oct.-Dec. Pg 178-188 [AJPSci.]

26. Kunzle IS, Swartz ME. Validated viewpoint: Laboratory

Notebook Documuentation. Lc Gc N. Am 1997; 15: 1122-1129.
27. Reynolds DW, Fachine KL, Mullaney JF, Alsante KM, Hatajik
TD, Motto MG. Availiable guidance and best practices for
conducting forced degradation studies. Pharm Tech, 26, 2002:
48-56.
28. ICH Q1A (R2) (2003). Stability Testing of New Drug Substances
and Products. International Conference on Harmonization ,
IFPMA, Geneva.
29. Kovarikova P, Jiri K, Jiri D, Lucie T. HPLC study of glimepiride
under hydrolytic stress conditions. J. Pharmaceut Biomed 36,
2004: 205-209.
30. Singh S, Bakshi M. Guidance on conduct of stress tests to
determine inherent stability of drugs. Phrama Tech 24, 2000: 1-
14.
31. Skoog DA, West DM, Holler FJ. Analytical Chemistry, An;
Introduction, Saunders College Publishing, Philadelphia. 2002: 3-
11
32. Cheekatla S, Ravichandrababu R, Reddy BN. Determination and
characterization of degradation products of Anastrozole by LC–
MS/MS and NMR spectroscopy. J. Pharmaceut Biomed, 56,
2011: 962-968.
33. Gupta A, Yadav JS, Rawat S, Gandhi M. Method Development
and Hydrolytic Degradation Study of Doxofylline by RPHPLC
and LC-MS/MS. Asian J Pharm Ana , 11, 2011:14-18.
34. Skoog, DA, West DM. Principles of Instrumental Analysis.
Saunderg Golden, Japan. 1980: 2-3.
35. Boccardi G. Oxidative susceptibility testing. In; pharmaceutical
Stress Testing-Predicting Drug Degradation; Baertschi SW,
editors, Taylor and Francis, New York. 2005: 220.
36. Alsante KM, Hatajik TD, Lohr LL, Santafianos D Sharp TR.
Solving impurity/degradation problems: case studies, In;
handbook of Isolation and Characterization of impurities in
Pharmaceutical, Ahuja S, Alsante K, editors, Academics Press,
New York. 2003: 380.
37. Alan RO, Brigitte ES, Yanqiu SA, Polshyna NM, Dunphy R,
Barbara LM, et al. Forced degradation studies of rapamycin:
Identification of autoxidation products. J Pharmaceut Biomed
2012; 59: 194-200
38. Bojana P, Markus D, Kappe CO. Microwave-assisted forced
degradation using high-throughput microtiter platforms. J
Pharmaceut Biomed 56, 2011: 867-873
39. Susanne CM, Lisa AM, Amina SW, Victor VL, Vladimir MD,
Robert JC. Atmospheric pressure matrix-assisted laser
desorption/ionization (AP MALDI) on a quadrupole ion trap
mass spectrometer. J Mass Spectrom, 226, 2003: 133-150.
40. Takats Z, Wiseman JM, Gologan B, Cooks RG. Mass
spectroscopy sampling under ambient conditions with desorption
electrospray ionization. Science, 306, 2004: 471- 473.

188

View publication stats