records.

Reviewer: Verifies LLE performance and documents compliance with method requirements.

Laboratory Supervisor: Ensures availability of required solvents, consumables, and equipment.

4. Accountability

The Head of the Bioanalytical Laboratory is accountable for ensuring validated LLE methods are followed, equipment is calibrated, and safety precautions are maintained during volatile solvent handling.

5. Procedure

5.1 Preparation Before Extraction

1. Thaw plasma samples as per SOP/BA-BE/155/2025.
2. Prepare required organic solvents as per validated method (e.g., ethyl acetate, methyl tert-butyl ether).
3. Label tubes clearly with Sample IDs, batch number, and date.
4. Ensure fume hood is operational and PPE is worn during volatile solvent handling.

5.2 Sample Spiking

1. Add internal standard (IS) to thawed plasma samples using calibrated pipette.
2. Vortex for 10–15 seconds to ensure mixing.

5.3 Addition of Organic Solvent

1. Add 3 to 5 times the volume of organic solvent to each sample (e.g., 3 mL solvent to 1 mL plasma).
2. Seal or cap the tubes properly.

5.4 Mixing and Extraction

1. Shake tubes on a mechanical shaker for 10–15 minutes at defined speed.
2. Ensure complete mixing to promote phase separation and analyte extraction.

5.5 Phase Separation

1. Centrifuge samples at 4000–5000 rpm for 5–10 minutes to separate aqueous and organic layers.
2. Carefully collect the upper (organic) layer into new pre-labeled tubes using glass Pasteur pipettes or auto-pipettors.
3. Ensure no carryover of aqueous phase or precipitates.

5.6 Drying and Reconstitution

1. Evaporate the collected organic phase under nitrogen stream at 35–40°C until completely dry.
2. Reconstitute residue in a defined volume of mobile phase or reconstitution buffer (e.g., 200 µL).
3. Vortex to ensure complete solubilization and transfer to autosampler vials.

5.7 Safety and Waste Disposal

1. Handle all solvents inside fume hoods and dispose of waste in labeled organic solvent containers.
2. Follow institutional EHS procedures for hazardous waste management.

5.8 Documentation

1. Record extraction details in Annexure-1: LLE Batch Record.
2. Include date, sample ID, volumes used, analyst name, and any deviations observed.

6. Abbreviations

• BA/BE: Bioavailability/Bioequivalence
• LLE: Liquid-Liquid Extraction
• IS: Internal Standard
• PPE: Personal Protective Equipment

7. Documents

1. LLE Batch Record – Annexure-1
2. Solvent Preparation Log – Annexure-2
3. Evaporation and Reconstitution Log – Annexure-3

8. References

• ICH M10 – Bioanalytical Method Validation
• FDA/EMA Bioanalytical Method Guidelines
• Internal Method Validation Reports

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: LLE Batch Record

Date	Sample ID	Solvent Used	Volume (mL)	Reconstitution Volume	Analyst
17/04/2025	PLM-2023	Ethyl Acetate	3.0	200 µL	Rajesh Kumar

Annexure-2: Solvent Preparation Log

Solvent	Purity	Lot No.	Prepared By	Date
Ethyl Acetate	HPLC Grade	EA0425	Sunita Reddy	16/04/2025

Annexure-3: Evaporation and Reconstitution Log

Sample ID	Evap. Temp	Reconst. Solvent	Volume	Remarks
PLM-2023	37°C	Mobile Phase	200 µL	Clear

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	Integrated new annexures and solvent safety guidelines	Compliance Update	QA Head