Standard Operating Procedure for Preparation of HPLC Mobile Phase in API Manufacturing

Department	API Manufacturing
SOP No.	SOP/API/122/2025
Supersedes	SOP/API/122/2022
Page No.	Page 1 of 8
Issue Date	13/04/2025
Effective Date	15/04/2025
Review Date	13/04/2026

1. Purpose

To lay down the procedure for the preparation of mobile phases used in HPLC analysis to ensure consistency, accuracy, and compliance with cGMP in the QC laboratory for API manufacturing.

2. Scope

This SOP is applicable to all QC chemists involved in the preparation of aqueous, non-aqueous, and buffer-based mobile phases for HPLC systems in the analysis of raw materials, intermediates, and final APIs.

3. Responsibilities

• QC Chemist: Prepare mobile phases as per analytical method, record preparation details, and ensure proper labeling and storage.
• QC Executive: Review preparation and filtration records and approve mobile phase usage.
• QA Officer: Verify and audit compliance during inspections and reviews.

4. Accountability

The Head – QC is accountable for ensuring that all mobile phases used for HPLC are prepared and handled according to validated procedures and that records are maintained.

5. Procedure

5.1 General Instructions

1. Use only approved and AR/HPLC-grade solvents and reagents.
2. All glassware must be rinsed with the same solvents being used for mobile phase preparation.
3. Record batch number, solvent grade, and expiry date of chemicals used.

5.2 Preparation Steps

1. Refer to the approved analytical method or STP for mobile phase composition and mixing ratio.
2. Measure components using calibrated measuring cylinders or balances.
3. Prepare buffer solutions freshly and adjust pH using calibrated pH meter.
4. Mix components in a clean, dry glass bottle using magnetic stirrer (if applicable).

5.3 Filtration and Degassing

1. Filter the prepared mobile phase through 0.45 μm or 0.22 μm membrane filter using vacuum filtration assembly.
2. Degas using:
   • Ultrasonic bath for 15–20 minutes
   • Or helium sparging (if required by method)
3. Do not reuse previously filtered mobile phase without proper justification and QA approval.

5.4 Labeling Requirements

1. Each mobile phase container shall be labeled with:
   • Name: HPLC Mobile Phase
   • Composition
   • Prepared By
   • Date of Preparation
   • Use Before Date (max 7 days unless specified)
   • Storage Condition

5.5 Documentation

1. Enter details in Mobile Phase Preparation Logbook (Annexure-1).
2. Attach buffer pH adjustment records (if applicable).
3. QC Executive shall review and sign-off before first use.

5.6 Disposal of Expired Mobile Phase

1. Discard unused mobile phase after expiry or as per defined usage limit.
2. Document the disposal activity in the Mobile Phase Disposal Record (Annexure-2).

6. Abbreviations

• HPLC: High-Performance Liquid Chromatography
• SOP: Standard Operating Procedure
• STP: Standard Test Procedure
• QA: Quality Assurance
• QC: Quality Control

7. Documents

1. Mobile Phase Preparation Logbook (Annexure-1)
2. Mobile Phase Disposal Record (Annexure-2)
3. Analytical Method or STP

8. References

• ICH Q2 (R1) – Validation of Analytical Procedures
• 21 CFR Part 211 – cGMP for Finished Pharmaceuticals
• USP <621> – Chromatography

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Mobile Phase Preparation Logbook

Date	Prepared By	Composition	pH	Volume	Use Before	Reviewed By
13/04/2025	Ravi Kumar	Buffer:Acetonitrile (60:40)	3.2	1000 mL	20/04/2025	Sunita Reddy

Annexure-2: Mobile Phase Disposal Record

Date	Composition	Volume Disposed	Disposed By	Remarks
21/04/2025	Buffer:ACN (60:40)	100 mL	Rajesh Kumar	Expired

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
01/01/2022	1.0	Initial SOP Release	New Procedure	QA Head
13/04/2025	2.0	Added filtration and disposal documentation	Regulatory Observation	QA Head