Performs SPE according to validated procedures and documents all steps accurately.

Reviewer/QA: Reviews documentation, checks for deviations, and ensures compliance.

Laboratory Supervisor: Ensures availability of consumables and verifies equipment suitability.

4. Accountability

The Head of Bioanalytical Laboratory is accountable for ensuring SPE procedures are followed, validated, and meet regulatory expectations for accuracy and reproducibility.

5. Procedure

5.1 Equipment and Materials

1. Vacuum manifold
2. SPE cartridges (type specified in validated method)
3. Collection tubes, centrifuge tubes
4. Solvents: Methanol, water, buffer, elution solvent
5. Pipettes and vortex mixer
6. Nitrogen evaporator (if required)

5.2 Sample Preparation

1. Thaw and vortex plasma samples as per SOP/BA-BE/155/2025.
2. Label all SPE cartridges and collection tubes clearly with Sample ID.
3. Add internal standard (IS) to each plasma sample and vortex for 15 seconds.
4. If required, dilute samples with buffer before SPE loading step.

5.3 Conditioning of SPE Cartridge

1. Place cartridge in SPE manifold.
2. Condition the cartridge by passing:
   • 1 mL methanol
   • 1 mL water
3. Ensure complete wetting of sorbent material for efficient binding.

5.4 Sample Loading

1. Transfer plasma sample onto conditioned SPE cartridge.
2. Allow it to pass through slowly under gravity or gentle vacuum (not exceeding 5–10 psi).
3. Discard the filtrate unless method specifies collection of flow-through.

5.5 Washing Step

1. Wash the cartridge with 1 mL of wash solution (e.g., water or aqueous buffer) to remove impurities.
2. Follow with a second wash of organic solvent if specified (e.g., 5% methanol).
3. Allow wash to drain completely before elution.

5.6 Elution of Analyte

1. Elute the retained analyte by adding 1 mL of elution solvent (e.g., methanol or acetonitrile).
2. Collect the eluate into pre-labeled collection tubes.
3. If specified, repeat elution step to improve recovery.

5.7 Post-Elution Processing

1. Evaporate eluates to dryness under nitrogen at 35–40°C.
2. Reconstitute the residue with appropriate mobile phase (e.g., 200 µL).
3. Transfer to autosampler vials and cap securely.

5.8 Precautions

1. Do not allow SPE cartridges to run dry between steps.
2. Use fresh pipette tips and tubes for each sample to avoid cross-contamination.
3. Document each step and ensure traceability using Annexure-1: SPE Extraction Log.

6. Abbreviations

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

7. Documents

1. SPE Extraction Log – Annexure-1
2. SPE Cartridge Batch Record – Annexure-2
3. Solvent Preparation Log – Annexure-3

8. References

• ICH M10: Bioanalytical Method Validation
• US FDA and EMA Bioanalytical Method Validation Guidelines
• Internal validated method documentation

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: SPE Extraction Log

Date	Sample ID	IS Added	Wash Volume	Elution Volume	Analyst
17/04/2025	PLS-1005	Yes	2 mL	1 mL	Rajesh Kumar

Annexure-2: SPE Cartridge Batch Record

Cartridge Type	Lot No.	Expiry	Conditioning Verified	Checked By
Oasis HLB	HLB1023	12/2025	Yes	Sunita Reddy

Annexure-3: Solvent Preparation Log

Solvent	Composition	Preparation Date	Prepared By
Elution Solvent	100% Methanol	16/04/2025	Vinay Pawar

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	Added cartridge log and elution volume checks	Regulatory Alignment	QA Head