Stress testing involves subjecting drug substances and products to conditions beyond typical accelerated testing to induce degradation. This helps identify degradation products and establish degradation pathways. Stress testing is performed during development and manufacturing to validate stability-indicating analytical methods. Methods may involve exposure to heat, humidity, acids, bases, oxidation or light. Identification and characterization of degradation products is important for regulatory approval and safety evaluation.

1. DETECTION, IDENTIFICATION AND
CHARACTERIZATION OF DEGRADATION
PRODUCTS
PRESENTED BY
V. KAMESWARA RAO
SEMII
PHARMACEUTICAL ANALYSIS

2. Stress Testing
Stress testing is defined as the stability testing of drug substances and drug products under conditions exceeding
those used for accelerated testing.
Pharmaceutical companies perform stress testing (also called forced-degradation studies) during preformulation to
help select compounds and excipients for further development
Stress testing often is repeated when manufacturing processes, product composition, and analytical procedures are
refined and reach a more final state
Stress testing is part of stability studies.
Quality of drug substance and drug product can vary with time under the influence of different stress conditions,
such as acid/base hydrolysis, thermal, oxidation and light exposure.
These studies provide information about intrinsic stability of a drug molecule, establishment of the degradation
pathway and degradation kinetics of the drug substances

3. The presence of degradation products (DPs) in drug substance beyond certain limits raise quality and safety related
issue for the regulatory agencies.
Degradation chemistry of drug, identification and characterization of DPs, establishment of degradation pathway by
mechanistic explanation and in silico toxicity study of DPs are required to be carried out.
Predict changes that could happen when the drug substance and drug products are exposed to extremes of environment

4. Regulatory status of stress testing
As per ICH Q1A(R2), stress testing of the drug substances can help in
 Identification of all the likely DPs
 Establishment of degradation pathways and intrinsic stability of the drug molecule
 Validation of stability indicating analytical procedures
 Effect of temperatures above that for accelerated stability testing by 10 oC increments (e.g., 50 oC,
60 oC, etc.), ≥75% RH, oxidation and photolysis on the drug substance. Solution or suspension state
stress studies evaluate liability of the drug substance to hydrolysis over a wide range of pH values
 Photostability should be an integral part of stress testing study design
 DPs that do not form in accelerated or long term stability may not be necessary to be examined, but
may be required as a part of QbD knowledge space.
 Result from stress testing form an integral part of the information provided to regulatory
authorities. Stress testing studies are carried out using single batch of the drug substance

5. USFDA
• .
• 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.
• Requirements of stress testing data at the IND and NDA stage

7. Start
labile
Accept
Declare drug to
be practically
stable
Practically stable
Very stable
Stable
Carry out studies
under milder
conditions
Extremely labile
Very labile
No degradation
No degradation
No degradation
No degradation
Total degradation
Total
degradation
Total
degradation
Sufficient
Sufficient
Sufficient
Sufficient
Sufficient
degradation
degradation
degradation
degradation
degradation
degradation
The stress conditions are accepted where a sufficient degradation (10-30%) of the drug is obtained.
Decision is taken based on step wise increase or decrease in the strength of the stressor and reaction conditions

8. Drug category Oxidative stress at
R.T., with H2O2
Hydrolytic stress Photolytic stress at R.T.
Acid (HCl)/ base
(NaOH)
Neutral
Practically stable 30%, 48 h 5 N, 2 days, refluxing 5 days, refluxing Photostable
6.0×106 lux·h,
1000 W·h/m2
Very stable 10%, 24 h 2 N,1 day, refluxing 2 days, refluxing
Stable 3%, 24 h 1 N, 12 h, refluxing 1 day, refluxing No intermediates are
classifiedLabile 3%, 6 h 0.1 N, 8 h, refluxing 12 h, refluxing
Very labile 1%, 3 h 0.01 N, 8 h, 40 °C 8 h, 40 °C Photolabile
1.2×106 lux·h,
200 W·h/m2
Extremely labile 1%, 30 min 0.01 N, 2 h, 25 °C 2 h, 40 °C

9. Stress
study
Drug +
(5 mg)
Stressor State Conditions Exposure time
Acid Stress
Boric acid/ Oxalic acid (10 mg)
Dry open 40 °C/75% RH 1 month
Alkaline Stress
Na2CO3 (10 mg)
Moist closed
(20 µl water)
40 °C
( or 40 °C/75%RH as may be
available)Neutral Stress Wet closed
(200 µl water)
Oxidative Stress
O2 (g)
Closed 25 °C 15 days
Photolytic Stress Open in petridish UV and Visible light Upto 6 × 106 lux·h and 1000
W·h/m2
Thermal Stress Dry Open 50 °C 21 days
Controls Acid/Alkali/Neutral Closed
(Drug alone
Stressor alone
Drug+Stressor)
40 °C
( or 40°C/75%RH as may be
available)
1 month
Oxidative Closed 25 °C 15 days
Photolytic Open Dark Same as that of stress samples
Thermal Closed 25 °C 21 days

10. Klick’s generic approach

11. • According to this approach the drug substance degrades by ~10% from its initial amount is supposed to be sufficient
degradation.
• Drugs need only be stressed to 5-10% or under conditions kinetically equivalent to long-term storage conditions
whichever comes first.
• 0.1N HCl, phosphate buffer (pH 7) and 0.1N NaOH can be employed for generating acidic, neutral and basic
conditions, respectively.
• If the drug degrades into acidic or basic compounds, the value of pH one unit above and below the nominal value may
be optimum for unbuffered conditions.
• Stress conditions for solid state: studies conducted include 80/75% RH upto 2 weeks and light exposure in excess of
ICH requirements and at 80C upto 2 weeks or at 60C up to 6 weeks in solutions.
Reynolds approach

12. • Acid-base solution, oxidative, thermal-humidity and photostability methods are used by the companies on drug substance
whereas thermal-humidity and photostability studies are performed on drug products.
• Companies attempts to induce at least 5-20% degradation of the drug substances.
• Stress testing on drug substance in solutions at different pH (1-13) and the temperatures used was ambient to 70°C.
• During stress testing drug concentration were used between 0.1 and 1 mg/mL, whenever there is solubility problem co-
solvents are used to solubilise the drug substance.
• Study of the effect of thermal-humidity both in open and closed conditions at 50- 70°C was carried out. If sufficient
degradation was not achieved then testing should be done at temperatures > 70°C and the humidity employed is 50-75%
for both drug substance and drug product for a duration of >3-6 weeks.
• For photostability studies majority of companies use the ICH visible-light dose range (i.e. overall illumination ≥1.2
million lux h) and UV light dose range (near-UV energy of ≥200 W.h/m2).
• For oxidative stress studies most companies used peroxides (1-3%) as well as a radical initiator AIBN 2-20 mol, pressured
oxygen, transition metals Cu (II) 0.05 mM, Fe (III) 1 mM and bubbled oxygen.
Alsante-benchmarking study

13. Stability-indicating HPLC method (SIM)
• Critical study of drug structure to assess the likely degradation pathway(s),
• Collection of information on physicochemical properties of drug,
• Stress testing of drug,
• Preliminary separation studies on stressed samples,
• Method development and optimization,
• Identification and characterization of dps and validation
Two types specific and selective SIM.
• Specific SIM is defined as “a method that is able to measure unequivocally the drug(s) in the presence of all degradation
products, excipients and additives, expected to be present in the formulation.”
• Selective SIM is defined as “a method that is able to measure unequivocally the drug(s) and all degradation products in
the presence of excipients and additives, expected to be present in the formulation .”

14. Regulatory and compendia status for SIM
1.2.1.1. ICH
The ICH Q1A(R2) guideline emphasizes that the testing of those features which are susceptible
change during storage and are likely to influence quality, safety and/or efficacy must be done by
validated stability-indicating method.
1.2.1.2. USFDA
As per draft guideline, “Validated quantitative analytical methods that can detect the changes
time in the chemical, physical, or microbiological properties of the drug substance and drug
product, and that are specific so that the contents of the active ingredient, DPs, and other
components of interest can be accurately measured without interference”.
1.2.1.3. United States Pharmacopeia (USP)
A requirement listed under ‘Pharmaceutical Stability’, which says that the stability-indicating
method should be able to accurately differentiate between the intact drug molecules and their

15. Identification and characterization of degradation products
• All volatile and non-volatile DPs are identified and separated by GC and HPLC chromatography techniques, respectively.
• Hyphenated GC-MS technique is used for the confirmation of organic volatile DPs and residual solvents present in a
sample.
• Hyphenated techniques like GC-MS, LC-MS, LC-MS/TOF and LC-NMR are used in characterization and structure
elucidation of DPs present in small amount.
• LC-MS/TOF is used to find accurate mass of molecular ions and fragments and helps in generation of molecular formula
for fragments.
• Multiple stage mass spectrometry (MSn) gives information about origin of fragments.

16. Reported analytical methods of selected drug(s)
Physicochemical properties of selected drug(s)
Structural formula
Empirical formula
IUPAC name
Exact mass (g/mol)
λmax (nm)
Melting point
pKa
LogP value
Solubility
Solid state