pharmaceutical ingredients (APIs) and formulations. It supports the development of stability-indicating methods under ICH Q1A(R2) guidelines.

2. Scope

This procedure is applicable to APIs, raw materials, and finished pharmaceutical products analyzed in the Analytical Method Development (AMD) department. It includes conditions for mild and strong acid/base degradation.

3. Responsibilities

• Analytical Scientist: Designs hydrolysis study protocol and selects analytical method.
• Lab Analyst: Prepares and analyzes hydrolyzed samples and documents degradation profiles.
• QA Officer: Reviews data and verifies that test conditions meet compliance standards.
• Head – AMD: Authorizes the study and validates its use in regulatory submissions.

4. Accountability

The Head of AMD is accountable for the scientific integrity, documentation, and regulatory alignment of all hydrolysis stress testing studies conducted in the laboratory.

5. Procedure

5.1 Study Planning

1. Develop a stress testing protocol outlining:
   • API/formulation details
   • Acid/base concentration
   • Temperature and time conditions
   • Neutralization strategy
   • Dilution and filtration instructions
2. Obtain approvals prior to initiating the study.

5.2 Acid Hydrolysis Conditions

1. Prepare a 0.1N hydrochloric acid solution.
2. Weigh required quantity of API/formulation and dissolve in suitable solvent.
3. Add acid solution and incubate at 60°C for 1 to 3 hours.
4. At the end of incubation, neutralize with 0.1N sodium hydroxide (molar equivalence).
5. Filter through 0.45 µm PVDF filter and dilute to required volume with mobile phase.

5.3 Base Hydrolysis Conditions

1. Prepare a 0.1N sodium hydroxide solution.
2. Repeat the same process as acid hydrolysis, incubating at 60°C for 1 to 3 hours.
3. Neutralize with 0.1N hydrochloric acid and proceed with filtration and dilution.

5.4 Sample Analysis

1. Analyze neutralized and diluted samples using validated HPLC/UPLC/UV method.
2. Compare assay values, peak area, retention time, and degradation profile with unstressed control sample.
3. Record all data in Annexure-1: Hydrolysis Test Report.

5.5 Interpretation Criteria

1. Degradation level should ideally be 5–20% to demonstrate method’s ability to detect changes.
2. No interference of degradants with the main peak (assess peak purity).
3. Calculate % degradation and assess formation of new peaks.
4. Draw conclusion on specificity of the method and behavior of API.

6. Abbreviations

• API: Active Pharmaceutical Ingredient
• HPLC: High-Performance Liquid Chromatography
• SOP: Standard Operating Procedure
• ICH: International Council for Harmonisation

7. Documents

1. Hydrolysis Test Report – Annexure-1

8. References

• ICH Q1A(R2) – Stability Testing of New Drug Substances and Products
• ICH Q2(R2) – Validation of Analytical Procedures
• FDA Guidance on Stability Testing

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Hydrolysis Test Report

Sample ID	Condition	Time (hrs)	% Degradation	New Peaks	Conclusion
API-HYD-001	0.1N HCl, 60°C	2	11.4%	Yes	Method Specific
API-HYD-001	0.1N NaOH, 60°C	3	9.7%	No	Stable in Alkali

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded neutralization steps and clarified dilution instructions	Regulatory clarification and audit compliance