Analytical method development finalization for Drug substance or Drug product is dependent on finalization of Forced degradation studies or Stress studies as it indicates a clear scenario of potential degradant impurities which may arise during the course of study and we can be prepared for further preparation, isolation and characterization of impurities for Regulatory requirement. 1. Objective: To investigate possible degradation products, arising due to degradation mechanism carried out on either drug substance or drug product which further helps to establish the degradation pathways and the intrinsic stability of the drug molecule. To provide foundation for developing a suitable stability indicating method

1. Analytical method development – Forced degradation study
Analytical method development finalization for Drug substance or Drug product is
dependent on finalization of Forced degradation studies or Stress studies as it indicates a
clearscenarioof potential degradantimpurities which may arise during the course of study
and we can be preparedforfurther preparation, isolation andcharacterization of impurities
for Regulatory requirement.
1. Objective:
To investigate possible degradationproducts, arisingdue todegradationmechanism carried
out on either drug substance or drug product which further helps to establish the
degradation pathways and the intrinsic stability of the drug molecule.
To provide foundation for developing a suitable stability indicating method
2. Samples used for study:
Forced degradation studies should be carried out on drug substance, drug products and
placebo.
3. Information explored from forced degradation studies:
Stress study shows difference in degradation products arising either from drug substance,
drug product or placebo.
Completionof studyshowsdiscriminationbetween;Syntheticprocessimpurities,excipients,
degradation products derived from excipients, product and due to drug excipient
interaction.
The degradation products generated during stress study are termed as “potential”
degradation products that may or may not be formed under relevant storage conditions.
4. Major forced degradationstudies:Thermal Degradation,Hydrolytic degradation, Oxidative
degradation, Photolytic degradation.
Study should be carried out using Photo-diode array detector or suitable mass compatible
method to gain information on potentialdegradant arising from stress study and for further
preparation and qualification of relevant impurities.
a. Thermal degradation:

2. Analyte having melting point less than 150°C, stress study should be carried out at 70°C or
about 40°C below the melting point.
Analyte havingmeltingpointgreaterthan 150°C, stressstudyshouldbe carried out at 105°C.
Stress study should be done at relevant temperature conditions and exposure time can be
varied to achieve degradation between 5 to 20%.
If no degradation isachieved even after harsher stress, justification can be provided that the
molecule is stable.
b. Hydrolytic degradation:
This involves treatment of analyte using water.
Hydrolytic forced degradation studies involve treatment with dilute acid and alkali at
different exposure time to achieve target degradation
Estimate solubility of analyte in water as hydrolytic stress studies are to be conducted in
aqueoussolutions.If drugis hydrophobic&foundto be insoluble in water, use a co-solvent
to dissolve the required quantity.
Commonly used co-solvents are Acetonitrile and Methanol.
# Methanol has the potential of participating in the degradation mechanism; it should be
used with caution especially under acidic conditions when the compound being tested
contains a carboxylic acid, ester or amide.
# Acetonitrile is not completely inert and can participate in the degradation reactions. For
such as itAcetonitrilecan contribute tobase to catalyse oxidation reactions in the presence
of peroxides. Acetonitrile will also degrade in presence of base (at pH 13)and /or acid (at
pH1) under elevated temperatures to detectable levels of Acetamide or Acetic acid which
can show up as early eluting peaks in RP-HPLC when monitored at lower wavelengths. The
size of the HPLC peaksfromthese twoproducts is relatively small and use of stressed blank
solutions permits ready identification of these peaks.
# Hydrolyticrefluxdegradationstudy should be performed infume hood ata temperature of
about 70°C using a reflux condenser with few glass beads or porcelain pieces to avoid
bumping and further loss due to evaporation.
# Typical conditions are, Reflux using water/ 0.1MHCl/ 0.1MNaOH for stress testing with or
withoutco-solventata temperature of about70°C.Reflux for about12 hours or until about 5
to 20% degradation is achieved or whichever is earlier. Neutralize the stressed solutions
before injection. Prepare a stressed solution at a higher concentration than that of test
concentration. Afterthe stress,dilute with diluenttoachieve required testconcentration, so
that peak shapes are good.
c. Humidity treated samples: Stress the samples to 90% Humidity for 1 week to gain
information on influence of humidity on the drug.
d. Oxidative degradation:Stress with 3% hydrogen peroxide in dark at room temperature for
24 hours or until about 5 to 20% degradation is achieved
e. Photolyticdegradation: Degradation resultingfromexposure toUV or visible light is termed
as Photolyticdegradation.Samplesshouldbe exposed to1.2 millionlux-hrvisible and200 W-
hr/m2 UV.
5. # Multi-componentStressStudy:Stresstestingof placebo with each active separately shall
be performedinordertoknowthe informationaboutwhichdegradantis fromwhichactive.

3. Alternatively,thesecanbe identifiedbythe UV spectra. Stresstesting of placebo with other
actives excluding the one at a time, shall be performed, in order to know the non-
interference from each other. Alternatively, these can be identified by the Peak purity. If
methods are different, placebo shall include the other actives.
6. Evaluation of results:
# Evaluate the peak purity of the Analyte peak and the major degradant peaks having peak
height less than 1000 m AU.
If more than 1000 mAU, solutions should be diluted to check for Peak Purity as per the
Software being used.
If any of the majordegradant is not passing peak purity, modify the stress study conditions
and check that the peak purity is passing.
# For GC methods and methods where RI, ELSD and FLD detectors are used, peak
homogeneity needs to be established by doing Mass spectral study and evaluate the peak
purity of the Analyte peak and the major degradant. Ensure that the method is Mass
compatible.
# If any known impurity is observed to be increasing in stress study, check if it is process
impurity or a possible degradant. In case a known compound is a process impurity and the
peak is found to be increasing in forced degradation, it indicates that degradant peak is
eluting at the same RT as that of the process impurity or there could be a secondary
pathway of formation of process impurity via some other degradation route
# Based on the structure of Analyte, check for possible formation of degradant
# If any of the Peaks are found to be not separating, Optimise the separation using the
forced degradation samples
7. Mass balance study: The process of adding together the assay value and levels of
degradationproductstosee how closelythese add up to 100% of the initial value. Estimate
the assay of the final force degradationsamplesand assess the mass balance. Mass balance
is to be achieved at least up to 95% level. If the mass balance is less than the required
criteria investigation to be done and justified.
8. Reasons for not achieving the mass balance:
# Degradation products are not eluted from the HPLC column or are not detected by
detector used for study
# Loss in degradation products or analyte due to extraction from sample placebo, due to
poor solubility, volatility or adsorption losses
# Co-elution of degradation products or impurity with parent Analyte peak
# Issues in integration of peaks due to poor chromatography
# In-accurate quantification due to differences in response factors
9. Finalization of detector wavelengths:
Afterseparationof all impurities from Analyte peak check overlaid spectra and finalize the
wavelength
10. Finalization of Chromatographic conditions & System suitability:
Perform the robustness of the method for the following parameters;
Mobile phase composition (organic component) change by (±10%),
pH of mobile phase change by (± 0.2) units
Gradient composition change (± 0.2 % per min)
Verify in different brand of HPLC’s columns
Flow rate (± 0.2 ml/min)

4. Temperature (± 5°C)
Fine-tune the method in the range where it is most robust.
In case any parameter is sensitive, specify the same in the test method so that it will be
monitored.