The document discusses guidelines for forced degradation studies to develop stability-indicating methods. Key points include: 1) Forced degradation studies subject drug substances, products, and placebos to stress conditions like heat, humidity, acids, bases, oxidation, and light to understand degradation pathways and develop suitable methods. 2) Common stress tests include thermal, hydrolytic, oxidative, and photolytic degradation. Conditions are specified for each test. 3) Potential degradation products are identified and the method is evaluated based on peak purity, mass balance, and other criteria. 4) Guidelines are provided for establishing relative response factors, quantitation methods, and system suitability parameters based on forced degradation results.

1. RELATED SUBSTANCES
METHOD DEVELOPMENT.
Forced degradation studies or
Stress testing studies:
What are the Objectives ?:
• To investigate the likely degradation products, which in turn
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. RS MD - Forced degradation studies or
Stress testing studies.
What we Stress?.
DRUG SUBSTANCE
DRUG PRODUCT.
PLACEBO.
All three to be stressed separately.

3. RS MD - Forced degradation studies or
Stress testing studies.
What we get to Know from this?.
Differenciation between
• Drug related and non-drug related degradation products
Discrimination between
• Synthetic process impurities, excipients,
degradation products derived from excipients alone and
drug related degradation products including the drug excipient
combinations.

4. Forced degradation studies or
Stress testing studies.
Key Point to be kept in mind :
The degradation products generated in the stressed
samples are
termed as “potential” degradation products
that may or may not be formed under relevant storage
conditions.

5. Forced degradation studies
Four major forced degradation studies are
• Thermolytic Degradation,
• Hydrolytic degradation,
• Oxidative degradation,
• Photolytic degradation.

6. Forced degradation studies
THERMOLYTIC DEGRADATION :
• Determine the Melting Point of the Analyte.
• Drugs for which melting point is <150°C,
Stress at 70°C or about 40°C below the melting point,
which ever is higher.
• Drugs for which melting point is >150°C,
Stress the samples at 105°C.

7. Forced degradation studies
THERMOLYTIC DEGRADATION :
How long to stress?.
What if the degradation is not achieved?.

8. Forced degradation studies
THERMOLYTIC DEGRADATION :
Stress for a week time,
Observe the sample.
Test the sample in between.
Achieve a degradation between 5 to 20%.
If no degradation is achieved even after
harsher stress, justification can be provided
that the molecule is stable.

9. Forced degradation studies
HYDROLYTIC DEGRADATION :
• Drug degradation that involves reaction
with water is called Hydrolysis.
Degradation which is due to hydrolysis
is called Hydrolytic Degradation.

10. Forced degradation studies
HYDROLYTIC DEGRADATION :
• Hydrolysis reactions are typically
Acid or Base catalyzed.
• Apart from water, Acidic and basic
conditions should also be employed in
order to induce potential hydrolytic
reactions.

11. Forced degradation studies
HYDROLYTIC DEGRADATION :
• As these hydrolytic stress studies are to be
conducted in aqueous solutions,
estimate the solubility of the drug molecule of
interest in water.
• If drug is hydrohobic & found to be not soluble in water,
use a co-solvent to dissolve the required quantity.

12. Forced degradation studies
HYDROLYTIC DEGRADATION :
Two most commonly used co-solvents are
Acetonitrile and Methanol.
• Methanol has the potential of participating in the
degradation chemistry, it should be used with caution
especially under acidic conditions when the compound
being tested contains a carboxylic acid, ester or amide.

13. HYDROLYTIC DEGRADATION :
Acetonitrile is typically preferable to methanol.
However,
Acetonitrile is not completely inert and can participate in
the degradation reactions.
For example,
acetonitrile is known to contribute to base catalyzed
epoxidation 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 peaks from these two products is
relatively small and use of stressed blank solutions permits
ready identification of these peaks.

14. HYDROLYTIC DEGRADATION :
In acidic acetonitrile/water solutions,
Tertiary alcohols can undergo Ritter reaction to form
amides.
In the presence of radicals acetonitrile cab be oxidized to
small amounts of formyl cyanides that will readily react
with nucleophiles (such as amines) resulting in a
formylation reaction.
Nonetheless, most of these side reactions of acetonitrile
are relatively minor and acetonitrile remains the most
frequently use co-solvent for hydrolysis studies.

15. Forced degradation studies
HYDROLYTIC DEGRADATION :
Co-solvents that are recommended for the hydrolytic stress testing
studies are shown below.
Acidic pH Neutral pH Basic pH
Acetonitrile Acetonitrile Acetonitrile
DMSO N-methyl pyrrolidine DMSO
Acetic acid Diglyme
Propionic acid p-Dioxane.

16. Forced degradation studies
HYDROLYTIC DEGRADATION :
The hydrolytic degradations are
recommended to be performed
– at a temperature of about 70°C
– with a reflux condenser installed to avoid
the loss of evaporation and
use few glass beads or porcelain pieces to
avoid bumping.
Conduct the stress reflux always in fume hood.

17. Forced degradation studies
HYDROLYTIC DEGRADATION :
Typical conditions :
Reflux using water/ 0.1M HCl/ 0.1M NaOH for stress testing
with or without co-solvent at a temperature of about 70°C.
Reflux for 12 hours or until about 5 to 20% degradation is achieved or
which ever is earlier.
Neutralize the stressed solutions before injection.
Prepare a stressed solution at a higher concentration than that of test
concentration.
After the stress, dilute in diluent to get the test concentration, so that
peak shapes are good.

18. Forced degradation studies
HYDROLYTIC DEGRADATION :
HUMIDITY STRESS :
Stress the samples to 90% Humidity for 1 week.
Why humidity stress, when we are performing water stress?.
This is typically to know the influence of humidity on the drug.

19. Forced degradation studies
OXIDATIVE DEGRADATION :
one of the most common mechanisms of drug degradation.
Typically oxidative degradations are to be performed at room temp (about 25°C
to 30°C) in dark.
Use of higher temperatures >30°C is not recommended, because the
reaction rate in solution may actually be reduced at higher temp due to
decrease in oxygen content of the solvent.
But for some compounds, degradation may occur at higher temperature due to
free radical reaction initiation.
Typical stress conditions :
Stress with 3% hydrogen peroxide in dark at room temperature with
constant stirring.
Stress for 24 hours or until about 5 to 20% degradation is achieved or
which ever is earlier.

20. Forced degradation studies
PHOTOLYTIC DEGRADATION :
Degradation that results from exposure to UV or visible light.
Typical Exposure conditions :
Expose the samples to 3 times to
1.2 million lux-hr visible and
200 W-hr/m2 UV.

21. Forced degradation studies
MULTI COMPONENT PRODUCT
DEGRADATION :
Stress testing of placebo with each actives separately shall be
performed in order to know the information about which degradant is
from which active.
Alternatively, these can be identified by the UV spectra.
Stress testing 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.

22. EVALUATION OF FORCED DEGRADATION :
• Evaluate the peak purity of the Analyte peak and
the major degradants.
• Evaluate the peak purity for the analyte peaks for which
peak heights are less than 1000 m AU.
• If more than 1000 mAU, solutions may be diluted to
check Peak Purity.
Peak purity Criteria : Software specific.
If any of the major degradants are not passing peak purity,
modify the conditions and see that the peak purity is passing.

23. EVALUATION OF FORCED DEGRADATION :
• For GC methods and methods where RI, ELSD and FLD
detectors are used, peak homogeneity needs to be established
by doing Mass spectral study.
•Evaluate the peak purity of the Analyte peak and
the major degradants.
• Method needs be Mass compatible.

24. EVALUATION OF FORCED DEGRADATION :
In case any known impurity is observed to be increasing in stress,
Examine whether 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
A degradant 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.
Eg : 9-Hydroxy Respiridone and N-oxide of Respiridone
Dimethyl derivative of Gatifloxacin
Observe any peaks merging wrt main peak (shoulders) and or
any critical pair of peaks.

25. EVALUATION OF FORCED DEGRADATION :
• Based on the structure of the Drug, Look for possible formation of
degradants which are not UV active.
Eg : Phosphenytion degrades to give Phenytion which is UV active and the
Phoshate which is not UV active at all and elutes in viod.
Employ RI detector or ELSD in such cases to see whether those degradants
does form or not.
Examine the chromatograms at different wavelengths, especially at
Lower wavelengths such as 200 or 205 or at 210 for poorly absorbing
degradants.
• Try to establish the structure of the Major degradants by doing a mass study.

26. EVALUATION OF FORCED DEGRADATION :
• If any Major degradant is having a different absorption maxima,
Preferrably it should be identified & estimated at that wavelength.
If not identified, it is difficult to assess the exact quantification, when the
active ingradient has different absorption characteristics.
Eg : Respiridone, Only one unknown degradant is having absorption
maxima at 260 nm, where as Respirodone & other impurities & degradants
are having maximum at 280 nm.

27. EVALUATION OF FORCED DEGRADATION :
• If any of the Peaks are found to be not separating ,
Optimise the seperation using the forced degradation samples.
Mass balance study :
The process of adding together the assay value and levels of degradation
products to see how closely these add up to 100% of the initial value.
Estimate the assay of the final force degradation samples and 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.

28. EVALUATION OF FORCED DEGRADATION :
Reasons for not achieving the mass balance :
(1). Degradation products are not eluted from the HPLC column
(2). Degradation products are not detected by the detector used.
(3). Degradation products lost from the Sample matrix, due to
insolubility, volatility or adsorption losses.
(4). Parent compound lost from the sample matrix, due to insolubility,
volatility or adsorption losses.
(5). Degradation products are co-eluted with the parent compound.
(6). Degradation products are not integrated due to poor
chromatography.
(7). Inaccurate quantification due to differences in response factors.

29. EVALUATION OF FORCED DEGRADATION :
Reasons for not achieving the mass balance :
(1). Degradation products are not eluted from the HPLC column
(2). Degradation products are not detected by the detector used.
(3). Degradation products lost from the Sample matrix, due to
insolubility, volatility or adsorption losses.
(4). Parent compound lost from the sample matrix, due to insolubility,
volatility or adsorption losses.
(5). Degradation products are co-eluted with the parent compound.
(6). Degradation products are not integrated due to poor
chromatography.
(7). Inaccurate quantification due to differences in response factors.

30. EVALUATION OF FORCED DEGRADATION :
Finalization of Detector wavelengths:
After finalization of the separation of all impurities and degradants,
take the overlaid spectra of all impurities for each of the stress
conditions, along with the spectra of the analyte.
Select a wavelength where all impurities are detected and have
maximum absorbance.
In case if this is not feasible, select different wavelengths to estimate
all impurities.
Exclusion criteria is to be specified at the other wavelengths.

31. EVALUATION OF FORCED DEGRADATION :
Finalization of Chromatograhic conditions & System
suitability :
Can be done only after the study of the Robustness of the method.
Perform the robustness of the method with regard to
– mobile phase composition (±10%),
– pH (± 0.2),
– gradient (± 0.2 % per min) : Verify in different brand of HPLC’s,
– flow rate ± 0.2 ml/min),
– 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.

32. EVALUATION OF FORCED DEGRADATION :
Finalization of System suitability :
based on the criticality of separation.
In general, resolution factor for the closely eluting compounds is
selected as a system suitability requirement.
Try to avoid injecting the impurities solutions by improving the
separations
If the separation is found to be satisfactory, there is no need to keep a
resolution factor as a system suitability parameter.
In such a case, only a diluted standard reproducibility can be adopted as a
system suitability requirement.
Reproducibility can be measured with two injections also, by monitoring the
ratio of peak areas of two injections.
Ratio should be between 0.9 to 1.1

33. RS METHOD DEVELOPMENT
RRF (Relative Response Factor) :
Establish the stability of solutions & Filter suitability
studies before conducting RRF study.
Response Factor = Area / amount (Concentration).
RRF = Ratio of Response factors of impurity to API.
Correction is given in denominator or Numerator depending on the way
the RRF is calculated.
IF RRF =(RF of Impurity/ RF of API), Then RRF is used in Denominator.
IF RRF =(RF of API/ RF of Impurity), Then RRF is used in Numerator.
Typically called as ‘correction factor ‘also.

34. RS METHOD DEVELOPMENT
RRF (Relative Response Factor) :
Response Factor = Area / amount (Concentration).
While Calculating the Concentration, use potencies of Complete molecule
for both API and for Impurities.
For Example,
If API / impurity is a salt or a complex, calculate the concentration as salt or as
drug complex.
Concen. In mg/ml of fluoxetine HCL ( Not as fluoxetine base)
Concen. In mg/ml of Omeprazole Magnesium ( Not as Omerazole base)
Consideration is same for Impurities also.

35. RS METHOD DEVELOPMENT
RRF (Relative Response Factor) :
Best way to establish the RRF is by “Slope method”(By linearity curve).
Where Slope = RF.
Typically using 5 Concentrations ranging between 0.1% to 1.0%.
"Do not include Zero" in linearity plot.
NOTE: If impurity quantity is a constraint, draw the curve with 2 or 3
points instead of five points.
RRF =(SLOPE of Impurity/ SLOPE of API), Then RRF is used in
Denominator.
RRF =(SLOPE of API/ SLOPE of Impurity), Then RRF is used in
Numerator.
Conduct recovery studies to prove that the established RRF's are
correct. Acceptance criteria : 90% to 110%.

36. QUANTITATION METHODS :
Area normalization:
If the Impurities RRF close to the Analyte (0.9 to 1.1)
Diluted Standard Method:
If the impurities RRF are different from the analyte.
External standard Method :
If the impurities are estimating with out Standard peak in same
chromatogram.
Internal standard method:
If the sample preparation procedure involves different extraction steps
(Normally when Derivitisation techniques or Liquid/Liquid Extraction
techniques are used)

37. QUANTITATION METHODS :
For Multi Component products,
If method is common ,
Use the diluted standard area for which the response
is low
If the methods are different,
Ensure that impurities are not estimated twice.

38. QUANTITATION METHODS :
DISREGARD CRITERIA :
Disregard any peak which is from Blank, Placebo.
Disregard any impurity which is less than 0.01%.
Placebo degradants can be disregarded if found not toxic.

39. QUANTITATION EQUIVALENCY :
Between API method and Formulation methods :
Analyse API in both methods.
Compare the % of known & number & % of
unknown impurities.
Assess the difference & its impact.
Typically, it should be ± 0.04% for all impurities
individually.

40. RS METHOD DEVELOMENT
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