3. Responsibilities

• Bioanalytical Scientist: Executes the method validation as per the approved plan and records data in designated templates.
• QA Personnel: Reviews validation data and ensures that all parameters meet acceptance criteria.
• Lab Manager: Oversees validation planning, resource allocation, and final review.

4. Accountability

The Head of Bioanalytical Department is accountable for ensuring that all method validations conform to ICH M10 requirements and are approved prior to study use.

5. Procedure

5.1 Validation Planning

1. Prepare a Method Validation Plan (MVP) outlining:
   • Analyte and matrix
   • Validation type (full/partial)
   • Parameters to be evaluated
   • Acceptance criteria
2. Review by QA and authorization by Department Head is mandatory.

5.2 Selectivity and Sensitivity

1. Analyze blank samples from ≥6 individual lots of matrix to assess selectivity.
2. Evaluate LLOQ signal-to-noise ratio (≥5:1) and accuracy within ±20%.

5.3 Accuracy and Precision

1. Prepare and analyze at least 5 replicates at each concentration level:
   • LLOQ, Low QC (LQC), Mid QC (MQC), High QC (HQC)
2. Acceptance Criteria:
   • Accuracy: ±15% (±20% for LLOQ)
   • Precision: CV ≤15% (≤20% for LLOQ)

5.4 Calibration Curve

1. Construct at least 6 non-zero calibration standards (excluding blank and zero).
2. Correlation coefficient (r²) must be ≥0.99; back-calculated concentrations must meet accuracy limits (±15%).

5.5 Recovery

1. Compare mean response of extracted QC samples with post-extraction spiked samples at 3 levels (LQC, MQC, HQC).
2. Document recovery percentage and variability.

5.6 Matrix Effect

1. Evaluate ion suppression/enhancement from ≥6 matrix lots by comparing post-extraction spiked responses to neat standard solutions.
2. Calculate %CV for normalized matrix factor; acceptance limit is ≤15%.

5.7 Dilution Integrity

1. Analyze samples above ULOQ after dilution to bring within range.
2. Accuracy and precision must remain within ±15% of nominal concentration.

5.8 Stability Studies

1. Assess stability under the following conditions:
   • Short-term (room temperature)
   • Long-term (storage temperature)
   • Freeze-thaw (≥3 cycles)
   • Post-preparative (auto-sampler)
2. Stability samples must meet ±15% accuracy compared to freshly prepared QCs.

5.9 Carryover

1. Inject blank samples after high concentration samples.
2. Response must be ≤20% of LLOQ and ≤5% for IS.

5.10 Reinjection Reproducibility

1. Evaluate performance of reinjected samples after an acceptable delay (≥24 hours).
2. Criteria must align with original batch acceptance.

5.11 Documentation and Reporting

1. Prepare Method Validation Report (MVR) including:
   • Raw data and chromatograms
   • Summary tables
   • Conclusion on method suitability
2. QA review and approval must precede any study sample analysis.

6. Abbreviations

• ICH: International Council for Harmonisation
• LLOQ: Lower Limit of Quantification
• ULOQ: Upper Limit of Quantification
• QC: Quality Control
• CV: Coefficient of Variation

7. Documents

1. Method Validation Plan – Annexure-1
2. Selectivity and Sensitivity Log – Annexure-2
3. Accuracy and Precision Table – Annexure-3
4. Stability Summary Sheet – Annexure-4
5. Matrix Effect Worksheet – Annexure-5
6. Method Validation Report – Annexure-6

8. References

• ICH M10 Bioanalytical Method Validation Guideline
• US FDA Bioanalytical Method Validation Guidance
• EMA Guideline on Bioanalytical Method Validation

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Method Validation Plan

Analyte	ABC-101
Matrix	Human plasma
Validation Type	Full
Planned By	Rajesh Kumar

Annexure-2: Selectivity and Sensitivity Log

Matrix Lot	Blank Interference	LLOQ S/N	Result
Lot-01	No	6.8	Pass

Annexure-3: Accuracy and Precision

QC Level	Nominal (ng/mL)	Mean	CV%	Status
LQC	10	9.8	4.5%	Pass

Annexure-4: Stability Summary

Condition	Nominal	Measured	Deviation (%)	Result
Freeze-Thaw	200	192	-4%	Pass

Annexure-5: Matrix Effect Worksheet

Lot	Matrix Factor	IS-normalized	%CV	Status
Lot-01	1.1	1.02	3.4%	Pass

Annexure-6: Method Validation Report

Validation Date	15/04/2025
Validated By	Sunita Reddy
Reviewed By	QA Officer
Conclusion	Method valid as per ICH M10

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	Aligned with ICH M10 and added matrix effect assessment	Regulatory Compliance	QA Head