and documents observations.

QA Reviewer: Reviews procedure adherence, batch records, and ensures compliance with validated method.

Lab Supervisor: Ensures availability of required solvents and functioning of instruments like centrifuges and vortex mixers.

4. Accountability

The Head of the Bioanalytical Department is accountable for overall compliance, data integrity, and validated performance of the protein precipitation method.

5. Procedure

5.1 Pre-Extraction Preparation

1. Thaw plasma samples according to SOP/BA-BE/155/2025.
2. Ensure labeling of tubes with sample ID, study code, and date.
3. Prepare internal standard (IS) and precipitation solvents (methanol or acetonitrile) in advance.
4. Document lot numbers and preparation time in Annexure-1: Solvent Preparation Log.

5.2 Protein Precipitation Process

1. Add a defined volume (e.g., 50 µL) of IS to each 200 µL plasma sample.
2. Vortex the mixture for 10 seconds to ensure uniform distribution.
3. Add precipitation solvent (usually 600–800 µL) based on validated ratio (typically 3–4x plasma volume).
4. Vortex the sample vigorously for 1 minute.
5. Incubate the tubes at room temperature for 5 minutes for complete precipitation.
6. Centrifuge at 10,000 rpm for 10 minutes at 4°C to pellet proteins.
7. Carefully collect the supernatant into a clean autosampler vial or labeled Eppendorf tube.

5.3 Post-Precipitation Handling

1. If necessary, evaporate the supernatant under nitrogen and reconstitute with 200 µL of mobile phase.
2. Filter through 0.2 µm filter (optional, based on method) before LC-MS/MS injection.
3. Record processing time, centrifuge RPM, and temperature in Annexure-2: Protein Precipitation Log.

5.4 Critical Considerations

1. Avoid delay between vortexing and centrifugation to prevent re-solubilization of proteins.
2. Use freshly prepared IS and solvent each day.
3. Ensure no pellet disturbance while transferring supernatant.

5.5 Deviation and Troubleshooting

1. In case of incomplete precipitation or turbid supernatant, repeat centrifugation and report deviation in Annexure-3: Deviation Report Form.
2. Use backup aliquots only with QA approval.

6. Abbreviations

• BA/BE: Bioavailability/Bioequivalence
• IS: Internal Standard
• PPE: Personal Protective Equipment
• LC-MS/MS: Liquid Chromatography–Mass Spectrometry

7. Documents

1. Solvent Preparation Log – Annexure-1
2. Protein Precipitation Log – Annexure-2
3. Deviation Report Form – Annexure-3

8. References

• ICH M10 – Bioanalytical Method Validation
• FDA/EMA Bioanalytical Method Guidelines
• Validated Study Protocols

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Solvent Preparation Log

Solvent	Lot No.	Purity	Prepared On	Prepared By
Acetonitrile	ACN-234	HPLC Grade	17/04/2025	Vinay Pawar

Annexure-2: Protein Precipitation Log

Date	Sample ID	IS Volume	Solvent Volume	Centrifuge RPM	Analyst
17/04/2025	PLM-3012	50 µL	600 µL	10,000	Rajesh Kumar

Annexure-3: Deviation Report Form

Date	Sample ID	Description of Deviation	Corrective Action	Approved By
17/04/2025	PLM-3012	Turbid Supernatant	Re-centrifuged	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	Improved clarity in volume ratios and added deviation tracking	Annual Review	QA Head