precision, accuracy, and specificity as per ICH Q2(R1) guidelines.

2. Scope

This SOP applies to the Analytical Method Development (AMD) department and covers assay method development for solid (tablets, capsules), liquid (syrups, suspensions), and injectable dosage forms using HPLC with UV or PDA detection systems.

3. Responsibilities

• Analytical Chemist: Conducts experimental trials for assay development, prepares standards and samples, records chromatograms, and evaluates results.
• Reviewer: Ensures compliance of developed method with specificity, linearity, accuracy, and robustness requirements.
• QA Officer: Verifies documentation, method protocol, and ensures regulatory alignment.
• Head – AMD: Approves assay methods for validation and subsequent QC or regulatory use.

4. Accountability

The Head of Analytical Method Development is accountable for the scientific justification, reproducibility, and regulatory compliance of all assay methods developed using HPLC.

5. Procedure

5.1 Pre-Development Considerations

1. Review existing pharmacopoeial monographs, literature data, and historical method performance.
2. Collect information on API solubility, pKa, UV absorbance (λmax), and stability under assay conditions.
3. Document all findings in Annexure-1: Assay Method Planning Sheet.

5.2 Chromatographic Condition Selection

1. Begin with commonly used C18 reversed-phase column (150 × 4.6 mm, 5 µm) unless specificity issues arise.
2. Use UV absorbance max (λmax) as the detection wavelength for sensitivity.
3. Select mobile phase:
   • Aqueous buffer (pH 3.0–5.0) + acetonitrile/methanol in isocratic or gradient mode
   • Buffer: phosphate, acetate, or formate (10–20 mM)
4. Initial mobile phase composition: 60:40 or 70:30 (A:B) for good retention and symmetry.
5. Record initial parameters in Annexure-2: Chromatographic Setup Log.

5.3 Sample and Standard Preparation

1. Standard:
   • Weigh suitable quantity of working standard and dilute to target concentration using diluent.
   • Filter through 0.45 µm nylon/PVDF membrane filter.
2. Sample:
   • Crush tablets or reconstitute dosage form and dilute to equivalent assay concentration.
   • Ensure uniformity and clarity of solution.
3. Document in Annexure-3: Sample and Standard Preparation Log.

5.4 Assay Method Optimization

1. Inject blank, standard, and sample to assess:
   • Peak shape (Tailing factor ≤ 2.0)
   • Retention time (2–10 min target)
   • No interference from excipients or impurities
2. Fine-tune mobile phase pH, organic solvent ratio, or flow rate (0.8–1.2 mL/min).
3. Overlay sample and standard chromatograms for specificity and reproducibility.
4. Document each change and result in Annexure-4: Optimization Log.

5.5 System Suitability Criteria

1. Inject 5 replicate standard injections and check:
   • RSD of peak area ≤ 2.0%
   • Retention time consistency (≤ 2% variation)
   • Theoretical plates ≥ 2000
2. Confirm signal stability and baseline integrity.
3. Record in Annexure-5: System Suitability Report.

5.6 Assay Method Validation Parameters

1. Specificity: Confirm that no interference is observed at API RT in placebo or excipient matrix.
2. Linearity: Validate across 50–150% of test concentration; R² ≥ 0.999.
3. Accuracy: Perform recovery at 80%, 100%, and 120% levels (98–102% recovery).
4. Precision: Repeatability (intra-day) and intermediate precision (inter-day); RSD ≤ 2.0%.
5. Robustness: Assess impact of small variations in pH, flow rate, and temperature.
6. Summarize in Annexure-6: Assay Validation Summary.

6. Abbreviations

• HPLC: High-Performance Liquid Chromatography
• API: Active Pharmaceutical Ingredient
• PDA: Photodiode Array
• RSD: Relative Standard Deviation
• SOP: Standard Operating Procedure

7. Documents

1. Assay Method Planning Sheet – Annexure-1
2. Chromatographic Setup Log – Annexure-2
3. Sample and Standard Preparation Log – Annexure-3
4. Optimization Log – Annexure-4
5. System Suitability Report – Annexure-5
6. Assay Validation Summary – Annexure-6

8. References

• ICH Q2(R1) – Validation of Analytical Procedures
• USP General Chapter <621> – Chromatography
• FDA Guidance on Analytical Procedures and Methods Validation
• Pharmacopoeia Monographs (IP/BP/USP)

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Assay Method Planning Sheet

API	λmax	Solubility	Reference Method	Analyst
XYZ	245 nm	Freely soluble	USP	Sunita Reddy

Annexure-2: Chromatographic Setup Log

Column	Mobile Phase	Flow Rate	Wavelength	Temperature
C18, 150×4.6mm	60:40 Phosphate buffer:ACN	1.0 mL/min	245 nm	30°C

Annexure-3: Sample and Standard Preparation Log

Type	Concentration	Diluent	Filtered	Prepared By
Standard	100 µg/mL	Mobile Phase	Yes	Rajesh Kumar

Annexure-4: Optimization Log

Trial	Modification	Observation	Conclusion
1	Increased ACN from 40% to 45%	Improved symmetry	Accepted

Annexure-5: System Suitability Report

Injection	Retention Time	Area	Tailing	Plate Count
1	3.42	132485	1.15	7200

Annexure-6: Assay Validation Summary

Parameter	Criteria	Result	Status
Accuracy	98–102%	99.4%	Pass
Precision	RSD ≤ 2.0%	0.82%	Pass
Linearity	R² ≥ 0.999	0.9992	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Updated flow rate range and added validation summary structure	Annual SOP Review