Responsibilities

• Analyst: Performs extraction as per method, records all steps, and ensures sample traceability.
• QA Reviewer: Verifies documentation and investigates any deviation during extraction.
• Lab Supervisor: Ensures equipment calibration, resource availability, and environmental compliance.

4. Accountability

The Head of Bioanalytical Laboratory is accountable for ensuring validated and consistent extraction procedures to preserve analyte integrity and reproducibility.

5. Procedure

5.1 Preparation Before Extraction

1. Thaw plasma samples as per SOP (refer SOP/BA-BE/155/2025).
2. Label extraction tubes and racks appropriately (Sample ID, Batch No., Date).
3. Prepare required reagents (e.g., internal standards, precipitating agents) under validated conditions.
4. Ensure cleanliness of all glassware, pipettes, and centrifugation tubes.

5.2 General Protein Precipitation Protocol

1. Add internal standard (IS) to each thawed plasma sample using calibrated micropipettes.
2. Vortex each tube for 10 seconds to ensure complete mixing.
3. Add 3 to 4 volumes of precipitating agent (e.g., acetonitrile or methanol) to 1 volume of plasma.
4. Vortex again for 1 minute to ensure complete protein precipitation.
5. Centrifuge samples at 10,000 rpm for 10 minutes at 4°C.
6. Carefully collect the supernatant into autosampler vials or microcentrifuge tubes for evaporation.

5.3 Optional: Solid Phase Extraction (SPE)

1. Condition SPE cartridges with methanol and water.
2. Load plasma sample onto cartridge and allow it to percolate under gravity or vacuum.
3. Wash cartridges with water or weak buffer to remove impurities.
4. Elute analyte using strong organic solvent (e.g., methanol or acetonitrile).
5. Evaporate eluate under nitrogen and reconstitute with mobile phase or suitable solvent.

5.4 Optional: Liquid-Liquid Extraction (LLE)

1. To each plasma sample, add organic solvent (e.g., ethyl acetate) at a 3:1 ratio.
2. Shake vigorously on a mechanical shaker for 10–15 minutes.
3. Centrifuge at 4000 rpm for 5 minutes to separate organic and aqueous layers.
4. Transfer upper organic layer to a clean tube and evaporate under nitrogen.
5. Reconstitute with required volume of reconstitution solvent.

5.5 Evaporation and Reconstitution

1. Evaporate supernatants using nitrogen evaporator at controlled temperature (35–40°C).
2. Reconstitute dry residue with defined volume of mobile phase (as per validated method).
3. Vortex to ensure homogeneity and transfer to autosampler vials with caps.

5.6 Precautions During Extraction

1. Avoid cross-contamination by using new tips, tubes, and gloves per sample.
2. Do not leave samples unattended at room temperature beyond validated duration.
3. Use calibrated equipment and perform pipette verification periodically.

5.7 Documentation

1. Record extraction date, analyst name, reagent lot numbers, and centrifuge RPM/time in Annexure-1: Extraction Worksheet.
2. Any deviation from method to be justified and signed by QA and Supervisor.

6. Abbreviations

• BA/BE: Bioavailability/Bioequivalence
• IS: Internal Standard
• SPE: Solid Phase Extraction
• LLE: Liquid-Liquid Extraction
• LC-MS/MS: Liquid Chromatography – Mass Spectrometry

7. Documents

1. Extraction Worksheet – Annexure-1
2. Batch Preparation Checklist – Annexure-2
3. Centrifugation Log – Annexure-3

8. References

• ICH M10 Bioanalytical Method Validation Guideline
• US FDA and EMA Bioanalytical Method Validation Guidance
• Validated Bioanalytical Method SOPs

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Extraction Worksheet

Date	Sample ID	Method	IS Lot No.	Precipitant Used	Analyst
17/04/2025	PLS-1003	Protein Precipitation	IS-B123	ACN	Rajesh Kumar

Annexure-2: Batch Preparation Checklist

Step	Status	Time	Remarks
Thawing	Completed	09:30	—
IS Addition	Completed	09:45	—

Annexure-3: Centrifugation Log

Date	Sample Set	RPM	Time (min)	Temp (°C)	Done By
17/04/2025	Set-A	10,000	10	4°C	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	Incorporated SPE and LLE alternatives	Regulatory Alignment	QA Head