mobile phase according to method-specific requirements and performs verification steps.

QA Officer: Reviews mobile phase logs and verifies adherence to procedures.

Lab Manager: Maintains oversight of mobile phase records and ensures compliance with this SOP.

4. Accountability

The Head of Bioanalytical Department is accountable for ensuring proper preparation, verification, and documentation of mobile phases used in regulated bioanalytical methods.

5. Procedure

5.1 Reagent and Solvent Quality

1. Use HPLC-grade solvents (e.g., acetonitrile, methanol, water) and reagents with CoA from approved vendors.
2. Ensure chemical labels clearly indicate:
   • Name and grade
   • Lot number
   • Expiry date
3. Do not use expired solvents or those with signs of contamination or precipitation.

5.2 Mobile Phase Preparation

1. Refer to validated method SOP for exact composition, order of mixing, and pH adjustment requirements.
2. Use volumetric glassware or calibrated dispensers for accurate measurement.
3. Prepare mobile phase in a clean, designated preparation area.
4. For buffered mobile phases:
   • Ensure pH is measured using a calibrated pH meter.
   • Adjust pH using approved acid/base agents only.
5. Record preparation details in Annexure-1: Mobile Phase Preparation Log.

5.3 Filtration and Degassing

1. Filter the mobile phase using 0.22 µm or 0.45 µm nylon or PVDF membrane filters (as per method).
2. Degas by sonication for at least 10 minutes or by helium sparging, if required.
3. Label final container with:
   • Mobile phase name
   • Date of preparation
   • Prepared by
   • Storage conditions
   • Expiry date (max 7 days or method-specific)

5.4 Verification of Composition

1. Verify mobile phase composition via:
   • Gravimetric check for solvent ratios (optional)
   • pH confirmation for aqueous phases
   • Retention time stability of system suitability standard
2. Record verification results in Annexure-2: Mobile Phase Verification Checklist.
3. Do not proceed with analysis if:
   • pH deviates ±0.2 units from specified range
   • Appearance is turbid or phase separation is observed

5.5 Storage and Disposal

1. Store mobile phase in clean, airtight glass bottles, preferably amber-colored for light-sensitive preparations.
2. Label containers with expiry and discard after designated time.
3. Dispose unused or expired mobile phases according to SOP for Hazardous Waste Disposal.

6. Abbreviations

• BA/BE: Bioavailability/Bioequivalence
• LC-MS/MS: Liquid Chromatography Tandem Mass Spectrometry
• pH: Hydrogen Ion Concentration
• SOP: Standard Operating Procedure
• CoA: Certificate of Analysis

7. Documents

1. Mobile Phase Preparation Log – Annexure-1
2. Mobile Phase Verification Checklist – Annexure-2

8. References

• ICH M10: Bioanalytical Method Validation
• US FDA Bioanalytical Method Validation Guidance (2022)
• 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 Log

Date	Mobile Phase Composition	Volume Prepared	Prepared By	Expiry Date
16/04/2025	Water:Acetonitrile (60:40)	2 L	Rajesh Kumar	23/04/2025

Annexure-2: Mobile Phase Verification Checklist

Date	Parameter	Expected Value	Observed Value	Verified By
16/04/2025	pH	4.5 ± 0.2	4.6	Sunita Reddy

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
01/01/2022	1.0	Initial Release	New SOP	QA Head
17/04/2025	2.0	Expanded verification and documentation requirements	Compliance Enhancement	QA Head