to identify potential degradants through stress testing and ensure method suitability for stability-indicating purposes.

2. Scope

This SOP applies to the Analytical Method Development (AMD) department and covers the detection, separation, and quantification of degradation products formed under stress conditions such as acid/base hydrolysis, oxidation, photolysis, and thermal degradation.

3. Responsibilities

• Analytical Chemist: Conducts forced degradation studies, develops HPLC methods, and identifies degradation peaks.
• Reviewer: Verifies chromatographic integrity, evaluates resolution, and confirms system suitability.
• QA Officer: Reviews all method development data and ensures GMP and ICH compliance.
• Head – AMD: Approves the method for validation and inclusion in regulatory dossiers.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring the degradation profiling method is stability-indicating and scientifically validated.

5. Procedure

5.1 Stress Condition Planning

1. Design forced degradation studies to include:
   • Acid hydrolysis: 0.1N HCl, 60°C for 2 hours
   • Base hydrolysis: 0.1N NaOH, 60°C for 2 hours
   • Oxidative: 3% H₂O₂, room temperature for 1 hour
   • Thermal: 60°C for 24 hours
   • Photolytic: 1.2 million lux hours + 200 Wh/m² UV
2. Target degradation: 5–20% API degradation.
3. Document plan in Annexure-1: Degradation Study Plan.

5.2 Chromatographic Method Setup

1. Column: C18, 150 mm × 4.6 mm, 5 µm.
2. Mobile Phase:
   • A: 10 mM phosphate buffer (pH 3.5)
   • B: Acetonitrile
   • Use gradient elution (e.g., 10% to 90% B in 20 min)
3. Detection: PDA at 210–400 nm; use λ_max of API if known.
4. Flow Rate: 1.0 mL/min; Injection Volume: 20 µL.
5. Record details in Annexure-2: HPLC Conditions Log.

5.3 Sample and Standard Preparation

1. Prepare API standard solution at 100 µg/mL in diluent.
2. Prepare stressed samples using neutralization post-treatment.
3. Filter all solutions using 0.45 µm PVDF filters.
4. Document in Annexure-3: Solution Preparation Log.

5.4 System Suitability and Peak Purity

1. Inject blank, standard, and stressed samples.
2. Verify:
   • Plate count ≥ 2000
   • Tailing factor ≤ 2.0
   • Resolution ≥ 2.0 between API and nearest degradant
3. Perform peak purity using PDA (purity angle < threshold).
4. Record in Annexure-4: System Suitability and Peak Purity Log.

5.5 Identification and Quantification of Degradants

1. Identify degradation peaks by comparing retention times across stress conditions.
2. For known degradants, inject standards and match RTs.
3. For unknowns, label peaks by retention time and estimate % area.
4. Ensure degradants are well resolved and quantifiable.
5. Summarize in Annexure-5: Degradation Product Summary.

5.6 Method Validation Parameters

1. Specificity: API peak should be spectrally pure and separated from degradants.
2. LOD/LOQ: Determine for known degradants using S/N ratio method.
3. Linearity: 50–150% of reporting threshold (0.05%–0.15%).
4. Accuracy: Spike known degradants at 0.05%, 0.1%, and 0.15% w.r.t API.
5. Document validation in Annexure-6: Method Validation Summary.

6. Abbreviations

• HPLC: High-Performance Liquid Chromatography
• API: Active Pharmaceutical Ingredient
• PDA: Photodiode Array
• LOD: Limit of Detection
• LOQ: Limit of Quantification
• SOP: Standard Operating Procedure

7. Documents

1. Degradation Study Plan – Annexure-1
2. HPLC Conditions Log – Annexure-2
3. Solution Preparation Log – Annexure-3
4. System Suitability and Peak Purity Log – Annexure-4
5. Degradation Product Summary – Annexure-5
6. Method Validation Summary – Annexure-6

8. References

• ICH Q1A(R2) – Stability Testing of New Drug Substances and Products
• ICH Q3B(R2) – Impurities in New Drug Products
• ICH Q2(R1) – Validation of Analytical Procedures
• USP <621> – Chromatography

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Degradation Study Plan

Condition	Reagent	Temp	Time	Target Degradation
Acid	0.1N HCl	60°C	2 hr	10%

Annexure-2: HPLC Conditions Log

Column	Mobile Phase	Gradient	Detection	Flow Rate
C18, 150×4.6 mm	Phosphate buffer:ACN	10–90% B in 20 min	PDA 245 nm	1.0 mL/min

Annexure-3: Solution Preparation Log

ID	Type	Concentration	Diluent	Prepared By
STD-001	API Standard	100 µg/mL	Water:ACN	Sunita Reddy

Annexure-4: System Suitability and Peak Purity Log

Parameter	Value	Specification	Status
Resolution (API/Degradant)	2.8	≥ 2.0	Pass

Annexure-5: Degradation Product Summary

Peak ID	RT (min)	% Area	Condition	Comment
D1	5.42	3.4%	Oxidation	Unknown degradant

Annexure-6: Method Validation Summary

Parameter	Criteria	Result	Status
LOD	S/N ≥ 3	0.012%	Pass
LOQ	S/N ≥ 10	0.040%	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added PDA evaluation and unknown degradant annotation	Annual Update