method can measure the target analyte accurately in the presence of other potential components such as impurities, degradation products, matrix excipients, and placebo formulations.

2. Scope

This SOP applies to the Analytical Method Development (AMD) laboratory for specificity testing of raw materials, APIs, excipients, finished pharmaceutical products, and stability-indicating assays using HPLC systems.

3. Responsibilities

• Analytical Chemist: Prepares samples and standards, performs HPLC runs, evaluates chromatographic separation, and documents the specificity study.
• Method Reviewer: Confirms spectral integrity, resolution, and peak identification.
• QA Officer: Ensures compliance with ICH Q2(R1) and applicable regulatory standards.
• Head – AMD: Reviews and approves the specificity study results and conclusion.

4. Accountability

The Head of Analytical Method Development is accountable for confirming that the developed method is specific and suitable for further validation and regulatory submission.

5. Procedure

5.1 Preparation of Samples for Specificity Study

1. Prepare the following sets of solutions:
   • Blank (diluent only)
   • Placebo (formulation matrix without API)
   • API standard solution
   • Impurity/Degradation products (known degradants or spiked samples)
   • Sample from stress testing (acid/base, peroxide, heat, photolysis)
2. Filter all solutions through 0.45 µm syringe filters.
3. Document preparations in Annexure-1: Sample Preparation Log.

5.2 HPLC Instrument Setup

1. Ensure the HPLC system is calibrated and qualification status is current.
2. Use validated chromatographic conditions or method under development.
3. Set detection wavelength (preferably λmax) and enable PDA acquisition if applicable.
4. Inject the prepared solutions sequentially and save data for overlay analysis.

5.3 Data Acquisition and Evaluation

1. Overlay the chromatograms of:
   • Blank vs Sample
   • Placebo vs API
   • API vs Known Impurities
   • Sample vs Stress Degradation Profiles
2. Confirm absence of peaks at retention time (RT) of API in blank and placebo chromatograms.
3. Check resolution between API and impurity peaks (Rs ≥ 2.0).
4. Use PDA spectral analysis or peak purity tool if co-elution is suspected.
5. Document findings in Annexure-2: Chromatographic Overlay Log.

5.4 Forced Degradation Studies

1. Perform stress studies on API or formulation under the following conditions:
   • Acid degradation (e.g., 0.1N HCl, 2 hr at 60°C)
   • Base degradation (e.g., 0.1N NaOH, 2 hr at 60°C)
   • Oxidation (e.g., 3% H₂O₂, 1 hr at room temp)
   • Thermal degradation (60°C for 24 hours)
   • Photostability (1.2 million lux hours + 200 Wh/m² UV)
2. Ensure significant degradation (5–20%) occurs to confirm method’s ability to detect changes.
3. Record stress conditions and observations in Annexure-3: Degradation Study Summary.

5.5 Specificity Conclusion

1. If no interference is observed from blank, placebo, and degradation products at API RT, and resolution and peak purity criteria are met, the method is considered specific.
2. Document final interpretation in Annexure-4: Specificity Assessment Certificate.

6. Abbreviations

• HPLC: High Performance Liquid Chromatography
• API: Active Pharmaceutical Ingredient
• PDA: Photodiode Array
• Rs: Resolution
• SOP: Standard Operating Procedure

7. Documents

1. Sample Preparation Log – Annexure-1
2. Chromatographic Overlay Log – Annexure-2
3. Degradation Study Summary – Annexure-3
4. Specificity Assessment Certificate – Annexure-4

8. References

• ICH Q2(R1) – Validation of Analytical Procedures
• USP <621> – Chromatography
• FDA Guidance for Analytical Procedure Development
• ICH Q1A(R2) – Stability Testing of New Drug Substances

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Sample Preparation Log

Sample Code	Type	Concentration	Diluent	Filtered	Prepared By
STD-API	Standard	100 µg/mL	Mobile Phase	Yes	Sunita Reddy

Annexure-2: Chromatographic Overlay Log

Overlay Type	Observation	Interference	Resolution	Status
Placebo vs API	No overlapping peaks	No	Rs = 2.8	Pass

Annexure-3: Degradation Study Summary

Stress Condition	Duration	Degradation (%)	Peak Shape	Resolution
0.1N HCl, 60°C	2 hr	12%	Symmetrical	3.1

Annexure-4: Specificity Assessment Certificate

Method ID	Analyte	Matrix	Interference	Conclusion	Reviewed By
MTH-074	Compound X	Tablet Placebo	None	Specific	Rajesh Kumar

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded degradation condition details and overlay documentation	Annual Review