flags abnormalities.

Reviewer (QA/Scientific): Reviews integration accuracy and compliance with established rules.

Lab Supervisor: Approves manual integrations and verifies method adherence.

4. Accountability

The Bioanalytical Lab Head is accountable for ensuring consistent application of integration rules and documentation of any deviations.

5. Procedure

5.1 Integration Software Settings

1. Use validated integration methods (e.g., centroid, height-based, area-based) pre-set in software (Analyst™, Empower™, MassLynx™, etc.).
2. Ensure peak width, threshold, and smoothing settings match parameters established in bioanalytical method validation.
3. Do not alter default integration parameters unless justified and documented.

5.2 Peak Selection Rules

1. Accept peaks that:
   • Match expected retention time ±2%
   • Have S/N ratio ≥ 10 at LLOQ
   • Do not show peak fronting or tailing beyond symmetry factor limits (0.8–1.5)
2. Flag peaks with co-elution, doublets, or inconsistent baseline for scientific review.

5.3 Manual Integration Policy

1. Manual integration may be applied when:
   • Baseline noise causes incorrect automatic peak delineation
   • Split peaks need unification into a single quantifiable peak
   • Software fails to detect known peak above threshold
2. Document every manual integration in Annexure-1: Manual Integration Log and include justification.

5.4 Peak Area and Retention Time Validation

1. Cross-check peak area and retention time (RT) against reference injections of standards and QCs.
2. Evaluate any unexpected shift in RT (greater than ±2%) or change in area (RSD > 5% for replicates).

5.5 Peak Shape and Chromatogram Quality

1. Acceptable peak shapes:
   • Gaussian or symmetrical
   • Symmetry factor between 0.8 and 1.5
2. Reject results with:
   • Baseline drift or instability
   • Negative peaks, spike noise, or ghost peaks

5.6 Data Integrity and Audit Trail

1. Ensure that software audit trails are enabled and capture:
   • Date/time of integration
   • Username and reason for manual edits
   • Chromatogram overlay before and after edits
2. QA must review audit trail for all manual integrations prior to batch approval.

5.7 Reviewer Checks

1. Scientific reviewer must:
   • Verify peak integration accuracy for at least 10% of chromatograms
   • Cross-validate LLOQ, ULOQ, and IS peaks across calibration curves and unknown samples
   • Approve batch only after resolving discrepancies

6. Abbreviations

• BA/BE: Bioavailability/Bioequivalence
• LC-MS/MS: Liquid Chromatography-Tandem Mass Spectrometry
• S/N: Signal-to-Noise Ratio
• IS: Internal Standard
• RT: Retention Time

7. Documents

1. Manual Integration Log – Annexure-1

8. References

• ICH M10: Bioanalytical Method Validation
• FDA and EMA Bioanalytical Guidelines
• Vendor User Manuals for Chromatographic Software

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Manual Integration Log

Date	Analyst	Sample ID	Integration Type	Reason for Manual Integration	Reviewer Remarks
16/04/2025	Rajesh Kumar	BE012-TP5	Area-based	Split peak due to baseline noise	Accepted

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	Updated with audit trail checks and reviewer roles	Data Integrity Compliance	QA Head