ointments, and gels using High Performance Liquid Chromatography (HPLC).

2. Scope

This SOP is applicable to Analytical Method Development (AMD) and Quality Control (QC) departments for the assay of APIs in semi-solid dosage forms during formulation development, stability studies, and routine analysis.

3. Responsibilities

• Analytical Scientist: Conducts method development, optimization, and validation.
• QC Analyst: Prepares samples, performs HPLC analysis, and maintains logbooks.
• QA Executive: Reviews validation reports and approves assay methodology for implementation.

4. Accountability

The Head of Analytical Method Development is accountable for the scientific validity, regulatory compliance, and fitness-for-purpose of the developed assay method.

5. Procedure

5.1 Sample Preparation

1. Weigh accurately 1 g of the topical formulation into a 50 mL volumetric flask.
2. Add 30 mL of diluent (e.g., Methanol:Water 70:30), sonicate for 15 minutes, and vortex.
3. Cool, make up the volume, and filter through 0.45 µm PVDF membrane.

5.2 Standard Preparation

1. Weigh 25 mg of API reference standard into a 50 mL volumetric flask.
2. Dissolve in diluent, sonicate, and dilute to volume.
3. Filter before HPLC injection.

5.3 Chromatographic Conditions (Example)

1. Column: C18, 250 mm × 4.6 mm, 5 µm
2. Mobile Phase: 60:40 (v/v) Acetonitrile:Water with 0.1% phosphoric acid
3. Flow Rate: 1.0 mL/min
4. Detection: UV at 254 nm
5. Injection Volume: 20 µL

5.4 System Suitability

1. Inject standard solution five times.
2. % RSD of peak area ≤ 2.0%, theoretical plates ≥ 2000, tailing factor ≤ 2.0.

5.5 Assay Calculation

Assay (% w/w) = (Sample Area / Standard Area) × (Standard Concentration / Sample Weight) × Dilution Factor × 100

5.6 Acceptance Criteria

• Assay value: 90–110% of label claim (unless otherwise specified)
• Method precision: % RSD ≤ 2.0%

5.7 Method Validation Parameters

1. Specificity: No interference at retention time of API
2. Linearity: Across 50–150% of label claim with r ≥ 0.999
3. Accuracy: Recovery between 98.0–102.0%
4. Precision: Repeatability and intermediate precision within 2% RSD
5. Robustness: Assessed by altering flow rate, temperature, wavelength

5.8 Documentation

1. Record chromatograms, peak areas, retention times, and calculations in assay log.
2. Compile validation report with raw data and summary.

6. Abbreviations

• HPLC: High Performance Liquid Chromatography
• API: Active Pharmaceutical Ingredient
• RSD: Relative Standard Deviation
• QA: Quality Assurance

7. Documents

1. Assay Data Log – Annexure-1
2. System Suitability Log – Annexure-2
3. Method Validation Summary – Annexure-3

8. References

• ICH Q2(R1): Validation of Analytical Procedures
• USP General Chapter <1225>: Validation of Compendial Procedures
• EP 2.2.29: Liquid Chromatography

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Meenakshi Rao	Prashant Menon	Sunita Reddy
Designation	Analytical Scientist	QA Reviewer	Head – AMD
Department	Analytical Method Development	QA	Analytical Method Development

11. Annexures

Annexure-1: Assay Data Log

Sample ID	Retention Time (min)	Peak Area	Assay (% w/w)	Status
TOP-251-01	5.32	14567	99.8%	Pass

Annexure-2: System Suitability Log

Injection	Peak Area	Retention Time	Tailing	Plate Count
1	14560	5.31	1.2	3050
2	14565	5.32	1.2	3025
3	14567	5.32	1.1	3100

Annexure-3: Method Validation Summary

The HPLC method was validated for assay of topical gel formulation. The method showed good specificity, linearity (r = 0.9993), precision (% RSD = 1.2%), and accuracy (99.2–101.3%). The method was found suitable for routine and stability testing.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
21/05/2025	2.0	Updated chromatographic parameters and acceptance limits	Annual Review	Sunita Reddy
15/03/2022	1.0	Initial Release	New SOP	QA Head