detection wavelength, and flow rate to achieve reproducible retention times and efficient separation with minimal baseline noise.

2. Scope

This SOP applies to all activities within the Analytical Method Development (AMD) laboratory related to optimizing isocratic HPLC methods for raw materials, APIs, excipients, and finished pharmaceutical products when gradient methods are not required or preferred.

3. Responsibilities

• Analytical Chemist: Executes method trials, records data, interprets chromatograms, and suggests optimization changes.
• Reviewer: Reviews method optimization sheets and approves final isocratic method conditions.
• QA Officer: Ensures regulatory compliance and documentation completeness.
• Head – AMD: Approves the optimized method and authorizes its use in method validation or transfer.

4. Accountability

The Head of AMD is accountable for ensuring the optimized isocratic methods meet analytical performance and regulatory standards.

5. Procedure

5.1 Initial Method Setup

1. Begin with default isocratic conditions using 60:40 (v/v) water:acetonitrile or water:methanol as mobile phase.
2. Select a reversed-phase C18 column (150 mm × 4.6 mm, 5 µm particle size) for general testing unless the API is hydrophilic or ionic.
3. Choose detection wavelength based on UV absorbance maxima from PDA scan or literature.
4. Set flow rate to 1.0 mL/min and column temperature at 30°C initially.
5. Document initial method parameters in Annexure-1: Method Initialization Log.

5.2 Retention Time and Peak Shape Evaluation

1. Inject standard API solution and observe:
   • Retention time (target 2–10 min)
   • Peak shape and tailing factor (≤ 2.0)
   • Theoretical plates (≥ 2000)
2. If API elutes too early (<1.5 min), increase aqueous content; if too late (>12 min), increase organic content.
3. Adjust flow rate if needed for retention tuning.
4. Document observations in Annexure-2: Retention Optimization Log.

5.3 Mobile Phase Selection and Adjustment

1. Select the optimal organic solvent (ACN vs MeOH) based on peak sharpness and resolution.
2. Use buffer in aqueous phase if the API is ionizable:
   • Buffer range: pH 3.0–7.0 (e.g., phosphate, acetate)
   • Buffer strength: 10–20 mM
3. Maintain buffer solubility and compatibility with UV detection.
4. Record buffer details and mobile phase adjustments in Annexure-3: Mobile Phase Optimization Sheet.

5.4 Method Robustness Trials

1. Assess the effect of small deliberate variations:
   • ±0.2 mL/min flow rate
   • ±2°C column oven temperature
   • ±2% change in mobile phase ratio
2. Evaluate peak area, resolution, and retention time drift.
3. Document results in Annexure-4: Robustness Evaluation Log.

5.5 Final Method Parameters

1. Summarize optimized parameters:
   • Mobile phase ratio (A:B)
   • pH and buffer strength
   • Flow rate
   • Injection volume
   • Run time
2. Confirm system suitability with 5 replicate injections.
3. Transfer method to validation phase after approval.
4. Enter finalized conditions in Annexure-5: Final Optimized Method Record.

6. Abbreviations

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

7. Documents

1. Method Initialization Log – Annexure-1
2. Retention Optimization Log – Annexure-2
3. Mobile Phase Optimization Sheet – Annexure-3
4. Robustness Evaluation Log – Annexure-4
5. Final Optimized Method Record – Annexure-5

8. References

• USP General Chapter <621> – Chromatography
• ICH Q2(R1) – Validation of Analytical Procedures
• 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 Initialization Log

Column	Mobile Phase	Flow Rate	Detection	Temperature
C18, 150 × 4.6 mm	Water:ACN (60:40)	1.0 mL/min	UV 254 nm	30°C

Annexure-2: Retention Optimization Log

Trial	Retention Time	Tailing	Resolution	Remarks
1	2.3	1.2	N/A	Too early elution

Annexure-3: Mobile Phase Optimization Sheet

Trial	A:B Ratio	pH	Peak Shape	Conclusion
2	70:30	4.5	Sharp	Accepted

Annexure-4: Robustness Evaluation Log

Parameter	Variation	Impact	Status
Flow Rate	0.8 mL/min	Slight increase in retention time	Acceptable

Annexure-5: Final Optimized Method Record

Column	Mobile Phase	Flow Rate	Detection	Injection Volume	Run Time
C18	70:30 Water:ACN	1.0 mL/min	254 nm	20 µL	10 min

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded optimization trials, added robustness testing	Annual SOP Review