Ingredients (APIs) to ensure reliability, accuracy, and regulatory compliance.

2. Scope

This procedure applies to all method development and validation activities performed for APIs in the Analytical Method Development (AMD) laboratory using HPLC systems. It covers reversed-phase, ion-exchange, and normal-phase chromatographic techniques used for API purity, assay, and impurity profiling.

3. Responsibilities

• Analytical Chemist: Conducts literature review, selects method conditions, prepares samples and standards, performs HPLC runs, and documents data.
• Reviewer: Reviews chromatograms, verifies peak assignments, evaluates system suitability, and ensures data integrity.
• QA Officer: Verifies compliance with GMP, GLP, and ICH Q2(R1) standards.
• Head – AMD: Authorizes finalized and validated HPLC methods for further use in QC or regulatory submission.

4. Accountability

The Head of AMD is accountable for the overall execution, validation, and approval of the HPLC method developed for API analysis.

5. Procedure

5.1 Preliminary Assessment

1. Conduct a literature review to gather information from:
   • Pharmacopoeias (USP, IP, Ph. Eur.)
   • Scientific journals
   • Reference standards and existing methods
2. Assess physicochemical properties of the API:
   • pKa, solubility, UV absorbance maxima
   • Log P, molecular weight
3. Document findings in Annexure-1: Method Feasibility Assessment Sheet.

5.2 Selection of Chromatographic Conditions

1. Column: Choose appropriate stationary phase (e.g., C18, C8, Phenyl).
2. Mobile Phase: Begin with commonly used aqueous-organic combinations (Water:Acetonitrile or Water:Methanol) and adjust pH/buffer as required.
3. Detection: Use UV at λmax of API or PDA scanning between 200–400 nm.
4. Flow Rate: Typically 1.0 mL/min (optimize as needed).
5. Injection Volume: Usually 10–20 µL.
6. Log selected conditions in Annexure-2: Chromatographic Condition Record.

5.3 Standard and Sample Preparation

1. Prepare primary standard stock solution using certified API reference substance.
2. Prepare working standards by serial dilution.
3. Prepare sample solution using appropriate solvent system; ensure clarity by filtration (0.45 µm).
4. Record preparation details in Annexure-3: Solution Preparation Log.

5.4 Method Optimization

1. Evaluate key parameters:
   • Retention time and peak shape
   • Theoretical plates (N)
   • Resolution (Rs ≥ 2.0 for adjacent peaks)
   • Tailing factor (Tf ≤ 2.0)
2. Optimize by adjusting:
   • pH of mobile phase (if ionizable compounds)
   • Gradient elution program (if peak overlap)
   • Column temperature (to improve selectivity)
3. Summarize findings in Annexure-4: Optimization Summary.

5.5 System Suitability Criteria

1. Perform 5 replicate injections of standard solution.
2. Evaluate:
   • % RSD of peak area (≤ 2.0%)
   • Retention time reproducibility
   • Plate count and resolution
3. Record results in Annexure-5: System Suitability Checklist.

5.6 Method Validation

1. Specificity: Inject blank, placebo, and known impurities to ensure no co-elution with API.
2. Linearity: Prepare at least five concentrations (e.g., 50%, 75%, 100%, 125%, 150%); R² ≥ 0.999.
3. Accuracy: Perform recovery studies at 80%, 100%, and 120% levels; acceptance 98–102%.
4. Precision: Evaluate repeatability and intermediate precision (RSD ≤ 2.0%).
5. LOD/LOQ: Determine using signal-to-noise ratio or standard deviation/slope method.
6. Robustness: Vary flow rate, temperature, pH, and column brand.
7. Compile results in Annexure-6: Validation Report.

6. Abbreviations

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

7. Documents

1. Method Feasibility Assessment Sheet – Annexure-1
2. Chromatographic Condition Record – Annexure-2
3. Solution Preparation Log – Annexure-3
4. Optimization Summary – Annexure-4
5. System Suitability Checklist – Annexure-5
6. Validation Report – Annexure-6

8. References

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

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Method Feasibility Assessment Sheet

API Name	Solubility	UV Max (nm)	Preferred Mobile Phase	Analyst
Sample API	Freely Soluble	230 nm	Water:ACN (60:40)	Sunita Reddy

Annexure-2: Chromatographic Condition Record

Column	Mobile Phase	Flow Rate	Detection	Temp
C18, 250 × 4.6 mm, 5 µm	Buffer:ACN (60:40)	1.0 mL/min	UV 230 nm	30°C

Annexure-3: Solution Preparation Log

Solution	Concentration	Solvent	Filtered	Prepared By
Standard	100 µg/mL	Mobile Phase	Yes (0.45 µm)	Rajesh Kumar

Annexure-4: Optimization Summary

Parameter	Variation	Observation	Conclusion
Mobile Phase Ratio	60:40 vs 50:50	Better resolution at 60:40	Selected

Annexure-5: System Suitability Checklist

Injection No.	Retention Time	Peak Area	Tailing Factor	Plate Count
1	6.23	145637	1.10	7200

Annexure-6: Validation Report

Parameter	Criteria	Result	Status
Accuracy	98–102%	99.5%	Pass
Precision	RSD ≤ 2.0%	0.88%	Pass
Linearity	R² ≥ 0.999	0.9994	Pass
LOD	S/N ≥ 3	Detected at 0.5 µg/mL	Pass
LOQ	S/N ≥ 10	Validated at 1.5 µg/mL	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded validation and optimization protocol for API purity and assay	Annual Review