impurity determination in tablets, capsules, liquids, and injectables.

2. Scope

This SOP applies to the Analytical Method Development (AMD) laboratory and encompasses chromatographic method design, optimization, and validation for finished product formulations of single or multi-API drugs. It covers immediate-release, sustained-release, and combination drug products.

3. Responsibilities

• Analytical Chemist: Executes experimental work for method design, prepares test samples and standards, operates the HPLC system, and documents observations.
• Reviewer: Reviews chromatograms, confirms accuracy and robustness, and validates peak assignments and system suitability.
• QA Officer: Ensures adherence to GMP, ICH Q2(R1), and internal compliance systems.
• Head – AMD: Reviews and approves the final method and validation documentation for regulatory and routine use.

4. Accountability

The Head of Analytical Method Development is accountable for the integrity and approval of the HPLC method developed for finished pharmaceutical products.

5. Procedure

5.1 Pre-Method Development Review

1. Review:
   • Formulation composition (API and excipients)
   • Dose strength and expected degradation pathways
   • Existing pharmacopoeial monographs or validated methods
2. Perform UV/PDA spectral scanning to determine optimal detection wavelength.
3. Record findings in Annexure-1: Pre-Development Assessment Sheet.

5.2 Chromatographic Condition Selection

1. Select column:
   • Reversed-phase C18 or C8 for general applications
   • Specialty columns (phenyl, CN, ion-pair) if required by polarity or retention
2. Choose mobile phase:
   • Water-methanol or water-acetonitrile with/without buffer (e.g., phosphate, acetate)
   • Adjust pH depending on API ionization
3. Optimize flow rate (typically 0.8–1.2 mL/min), column oven temperature (25–40°C), and injection volume (10–50 µL).
4. Document settings in Annexure-2: HPLC Method Setup Log.

5.3 Standard and Sample Preparation

1. Prepare API standard solution using certified working standard.
2. For finished product:
   • Crush solid dosage units uniformly and dissolve in suitable diluent
   • For liquids/injectables, use volumetric dilution and filtration through 0.45 µm filter
3. Ensure clarity, reproducibility, and compatibility of diluent with mobile phase.
4. Document in Annexure-3: Sample & Standard Preparation Record.

5.4 Method Optimization

1. Inject placebo to check for interference with API and impurity peaks.
2. Optimize:
   • Resolution between APIs and excipient interference
   • Tailing factor, plate count, and peak symmetry
   • Gradient elution if multiple APIs or impurities co-elute
3. Apply PDA detection or dual wavelength monitoring if necessary.
4. Summarize results in Annexure-4: Optimization Log.

5.5 System Suitability

1. Inject 5 replicates of standard solution and assess:
   • % RSD of peak area (≤ 2.0%)
   • Plate count (≥ 2000)
   • Tailing factor (≤ 2.0)
   • Resolution (≥ 2.0 for adjacent peaks)
2. Record in Annexure-5: System Suitability Evaluation.

5.6 Validation Protocol

1. Specificity: Demonstrate that API and impurities are distinguishable from excipients.
2. Linearity: Establish over 5 concentration levels; R² ≥ 0.999.
3. Accuracy: Spike placebo at 80%, 100%, and 120% recovery levels; acceptance: 98–102%.
4. Precision: Repeatability and inter-analyst precision RSD ≤ 2.0%.
5. LOD/LOQ: Determined by S/N ratio or calibration slope method.
6. Robustness: Assess effect of pH, column temperature, flow rate, and mobile phase ratio variations.
7. Compile in Annexure-6: Validation Summary.

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. Pre-Development Assessment Sheet – Annexure-1
2. HPLC Method Setup Log – Annexure-2
3. Sample & Standard Preparation Record – Annexure-3
4. Optimization Log – Annexure-4
5. System Suitability Evaluation – Annexure-5
6. Validation Summary – Annexure-6

8. References

• USP <621> – Chromatography
• ICH Q2(R1) – Validation of Analytical Procedures
• Pharmacopoeia of India (IP) – Finished Product Standards
• FDA Guidance for Industry – 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: Pre-Development Assessment Sheet

Formulation	Dosage Form	Matrix Complexity	Existing Method	Analyst
Drug XYZ	Tablet	Moderate	USP Monograph	Sunita Reddy

Annexure-2: HPLC Method Setup Log

Column	Mobile Phase	Flow Rate	Detection	Temperature
C18, 250×4.6 mm, 5 µm	Buffer:ACN (65:35)	1.0 mL/min	UV 225 nm	30°C

Annexure-3: Sample & Standard Preparation Record

Sample	Weight	Diluent	Filtered	Prepared By
Crushed Tablet	500 mg	Mobile Phase	0.45 µm	Rajesh Kumar

Annexure-4: Optimization Log

Parameter	Trial Conditions	Observation	Conclusion
Gradient vs Isocratic	Isocratic: 65:35 vs Gradient 50–80%	Better resolution in gradient	Gradient selected

Annexure-5: System Suitability Evaluation

Injection No.	Retention Time	Area	Tailing Factor	Plate Count
1	6.42	132458	1.12	7800

Annexure-6: Validation Summary

Parameter	Criteria	Result	Status
Accuracy	98–102%	99.2%	Pass
Precision	RSD ≤ 2.0%	0.92%	Pass
Linearity	R² ≥ 0.999	0.9995	Pass
LOD	S/N ≥ 3	Detected at 0.4 µg/mL	Pass
LOQ	S/N ≥ 10	1.2 µg/mL	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Included gradient method logic and robustness verification	Annual Review