documentation and ensures no carryover issues impact data reliability.

Lab Supervisor: Approves batch continuation after carryover clearance.

4. Accountability

The Head of Bioanalytical Laboratory is accountable for ensuring that carryover assessments are implemented for every analytical run as per regulatory requirements.

5. Procedure

5.1 Pre-Run Carryover Assessment

1. Prepare and inject the following sequence at the beginning of the batch:
   • Blank sample (no analyte)
   • Upper Limit of Quantification (ULOQ) standard
   • Blank sample immediately after ULOQ
2. Evaluate the blank sample following ULOQ for:
   • No peak for analyte or IS at the retention time.
   • Area ≤ 20% of LLOQ response for analyte.
   • Area ≤ 5% of average IS response for IS.

5.2 Carryover During Run

1. After injecting each calibration curve and high concentration QC sample, include a blank injection to monitor in-run carryover.
2. If any carryover is observed:
   • Stop sequence immediately.
   • Perform auto-sampler needle wash and run additional blanks until carryover clears.
   • Document actions in Annexure-1: Carryover Investigation Log.

5.3 Preventive Measures

1. Use dual or extended needle wash cycles in autosampler setup.
2. Include rinse solutions such as acetonitrile/water/formic acid mixtures in wash protocol.
3. Use partial loop or full loop with needle overfill injection modes to reduce system contact with sample residue.

5.4 Criteria for Carryover Acceptance

1. Carryover is acceptable if:
   • Post-ULOQ blank shows analyte area ≤ 20% of LLOQ.
   • Post-ULOQ blank shows IS area ≤ 5% of mean IS area.
   • No significant analyte signal visible in extracted ion chromatograms (XIC).

5.5 Documentation

1. Document every carryover evaluation in the raw data folder and record findings in Annexure-2: Carryover Evaluation Sheet.
2. Attach chromatograms of ULOQ and subsequent blank injections as evidence.

5.6 Reporting and Deviation Handling

1. If unacceptable carryover is not resolved within three rinse attempts, discard affected batch and initiate deviation report.
2. Log deviation in Annexure-3: Carryover Deviation Report and initiate CAPA.

6. Abbreviations

• BA/BE: Bioavailability/Bioequivalence
• ULOQ: Upper Limit of Quantification
• LLOQ: Lower Limit of Quantification
• XIC: Extracted Ion Chromatogram
• IS: Internal Standard

7. Documents

1. Carryover Investigation Log – Annexure-1
2. Carryover Evaluation Sheet – Annexure-2
3. Carryover Deviation Report – Annexure-3

8. References

• ICH M10: Bioanalytical Method Validation
• FDA Guidance for Industry: Bioanalytical Method Validation
• EMA Bioanalytical Guidelines

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Carryover Investigation Log

Date	Batch ID	ULOQ Response	Blank Response	Action Taken	Reviewed By
17/04/2025	BE-102	90543	1283	Needle wash repeated	Sunita Reddy

Annexure-2: Carryover Evaluation Sheet

Injection Type	Analyte Area	IS Area	Criteria Met?	Remarks
Blank after ULOQ	1356	4125	Yes	Within acceptable range

Annexure-3: Carryover Deviation Report

Date	Batch No.	Issue Description	Root Cause	CAPA Initiated
17/04/2025	BE-103	Carryover beyond 20% LLOQ	Needle clog	Scheduled needle replacement

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 CAPA handling and dual rinse strategy	Audit Compliance	QA Head