ensure methods meet sensitivity and quantification requirements as per ICH Q2(R1).

2. Scope

This SOP applies to all newly developed and validated methods in the Analytical Method Development laboratory for APIs and formulated products, where LOD and LOQ assessments are required.

3. Responsibilities

• Analytical Scientist: Prepares calibration curve data and calculates LOD/LOQ.
• Group Leader: Reviews the calculation and assessment report.
• QA Executive: Verifies data integrity and compliance to ICH requirements.
• Department Head: Approves the LOD/LOQ report and method validation package.

4. Accountability

The Department Head of Analytical Method Development is accountable for the implementation and compliance of this SOP across all applicable methods.

5. Procedure

5.1 Definitions

• LOD: The lowest amount of analyte in a sample which can be detected but not necessarily quantified.
• LOQ: The lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy.

5.2 Methods for Determination

1. LOD and LOQ can be established using:
   • Signal-to-Noise Ratio (S/N)
   • Calibration Curve Approach (Standard Deviation of response and slope)
   • Visual Evaluation
2. The selection of method shall be documented and justified based on the type of method and sensitivity required.

5.3 Signal-to-Noise Method

1. Prepare a series of dilute solutions of known concentration near the expected LOD/LOQ.
2. Inject solutions into HPLC/GC system and measure signal-to-noise ratio.
3. Acceptance Criteria:
   • LOD: S/N ≥ 3
   • LOQ: S/N ≥ 10
4. Document results in Annexure-1: S/N LOD-LOQ Log.

5.4 Calibration Curve Method

1. Prepare at least 6 concentrations of the analyte covering the low concentration range.
2. Plot the linearity curve and determine:
   • Standard deviation of y-intercepts (σ)
   • Slope of the curve (S)
3. Calculate:
   • LOD = 3.3 × (σ/S)
   • LOQ = 10 × (σ/S)
4. Document results in Annexure-2: Calibration-Based LOD/LOQ Log.

5.5 Documentation

1. All raw data, chromatograms, and calculations must be retained and attached to the method validation report.
2. Results must be reviewed by a second analyst and authorized by the QA reviewer.

6. Abbreviations

• SOP: Standard Operating Procedure
• LOD: Limit of Detection
• LOQ: Limit of Quantification
• QA: Quality Assurance
• S/N: Signal-to-Noise Ratio

7. Documents

1. S/N LOD-LOQ Log – Annexure-1
2. Calibration-Based LOD/LOQ Log – Annexure-2
3. Method Validation Summary Sheet – Annexure-3

8. References

• ICH Q2(R1): Validation of Analytical Procedures
• FDA Guidance for Industry: Analytical Procedures and Methods Validation
• WHO Technical Report Series 996

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name	Deepika Joshi	Ashwin Rao	Sunita Reddy
Designation	Analytical Chemist	QA Executive	Dept. Head
Department	AMD	QA	Analytical Method Development

11. Annexures

Annexure-1: S/N LOD-LOQ Log

Sample ID	Concentration (µg/mL)	S/N Ratio	LOD/LOQ	Tested By
LOD-01	0.05	3.2	LOD	Deepika Joshi
LOQ-01	0.15	10.4	LOQ	Deepika Joshi

Annexure-2: Calibration-Based LOD/LOQ Log

σ (SD of y-intercepts)	Slope (S)	LOD (µg/mL)	LOQ (µg/mL)
0.005	0.085	0.194	0.588

Annexure-3: Method Validation Summary Sheet

Method Name	Assay of Paracetamol
LOD Method Used	S/N Ratio
LOQ Method Used	Calibration Curve
Final LOD	0.05 µg/mL
Final LOQ	0.15 µg/mL
Validated By	QA Department

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
21/05/2025	2.0	Updated to include calibration curve method	Regulatory Update	Sunita Reddy
05/03/2022	1.0	Initial Issue	New SOP	QA Head