SOP for UV Spectrophotometric Method Development in AMD Laboratory

Department	Analytical Method Development
SOP No.	SOP/AMD/054/2025
Supersedes	SOP/AMD/054/2022
Page No.	Page 1 of 14
Issue Date	19/05/2025
Effective Date	20/05/2025
Review Date	19/05/2026

1. Purpose

The purpose of this SOP is to describe the standard procedure for developing, optimizing, and validating UV spectrophotometric methods for the quantitative analysis of active pharmaceutical ingredients (APIs), intermediates, and finished dosage forms using a UV-visible spectrophotometer.

2. Scope

This procedure applies to the Analytical Method Development (AMD) laboratory and includes all steps from wavelength scanning, method optimization, calibration curve generation, and full validation according to ICH Q2(R1) and pharmacopeial guidelines.

3. Responsibilities

• Analytical Scientist: Responsible for scanning, method development, standardization, and performing validation parameters.
• Reviewer: Verifies raw data, calculations, and validation outcomes for compliance.
• QA Personnel: Reviews final method reports and ensures SOP compliance with regulatory standards.
• Head – AMD: Oversees the method development process and approves final documentation for use in QC or regulatory filings.

4. Accountability

The Head of AMD is accountable for ensuring UV spectrophotometric methods are scientifically sound, validated, and regulatory-compliant for intended pharmaceutical use.

5. Procedure

5.1 Selection of Analytical Wavelength

1. Perform a UV spectrum scan of a known concentration of analyte between 200 nm and 400 nm.
2. Identify the wavelength of maximum absorbance (λ_max).
3. Ensure the λ_max is not overlapped by known excipients or impurities.
4. Record the λ_max and corresponding absorbance in Annexure-1: Wavelength Selection Log.

5.2 Preparation of Standard Stock and Working Solutions

1. Prepare a standard stock solution of the analyte using a certified reference standard in a suitable solvent (typically water, methanol, or buffer).
2. Dilute the stock to prepare 5–7 concentrations over the intended working range (e.g., 5 ppm to 50 ppm).
3. Use the same diluent as the sample matrix to avoid matrix mismatch.
4. Record details in Annexure-2: Standard Preparation Sheet.

5.3 Calibration Curve Construction

1. Measure absorbance of each standard solution at the selected λ_max using a blank as reference.
2. Plot absorbance vs. concentration using appropriate software or manually.
3. Calculate the regression line equation and determine correlation coefficient (R²).
4. Acceptable linearity: R² ≥ 0.999.
5. Record in Annexure-3: Calibration Curve Log.

5.4 Sample Preparation

1. Prepare sample solutions in the same matrix/diluent as standards.
2. Filter or centrifuge if required to remove insoluble matter.
3. Ensure concentration of test solution falls within standard curve range.
4. Document results in Annexure-4: Sample Analysis Report.

5.5 Method Validation

1. Validate the method as per ICH Q2(R1) and pharmacopeial expectations.
2. Specificity: Ensure blank and excipients do not absorb at λ_max.
3. Linearity: Prepare at least 5 concentrations (80–120%) and confirm R² ≥ 0.999.
4. Accuracy (Recovery): Spike placebo at 80%, 100%, and 120% levels. Recovery should be 98–102%.
5. Precision:
   • Repeatability (intra-day): 6 replicates at 100% level, RSD ≤ 2.0%
   • Intermediate precision (inter-day/different analyst): RSD ≤ 2.0%
6. LOD/LOQ: Use calibration curve method or signal-to-noise (S/N) method (LOD S/N = 3.3, LOQ S/N = 10).
7. Robustness: Vary wavelength (±1 nm), temperature (±2°C), and diluent ratio (±5%).
8. Solution Stability: Measure absorbance over 6 hours at room temperature. Acceptable deviation ≤ 2%.
9. All validation data must be documented in Annexure-5: Method Validation Summary.

5.6 Final Method Documentation

1. Compile complete method including:
   • Title and scope
   • Reagents and solutions
   • Instrument settings
   • Calculation formulae
   • Acceptance criteria
   • System suitability tests
2. Ensure peer-review and QA approval before use in stability or routine analysis.

6. Abbreviations

• UV: Ultraviolet
• SOP: Standard Operating Procedure
• RSD: Relative Standard Deviation
• ICH: International Council for Harmonisation
• QA: Quality Assurance
• λ_max: Wavelength of maximum absorbance

7. Documents

1. Wavelength Selection Log – Annexure-1
2. Standard Preparation Sheet – Annexure-2
3. Calibration Curve Log – Annexure-3
4. Sample Analysis Report – Annexure-4
5. Method Validation Summary – Annexure-5

8. References

• ICH Q2(R1): Validation of Analytical Procedures
• USP <857>: Ultraviolet-Visible Spectroscopy
• IP 2022: Appendix – UV Methods
• Ph. Eur. 2.2.25: UV-Vis Spectrophotometry

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Wavelength Selection Log

Sample	λmax (nm)	Absorbance	Selected By
API-A	272	1.215	Ajay Mehra

Annexure-2: Standard Preparation Sheet

Stock Conc. (ppm)	Dilution Volume	Final Conc. (ppm)	Prepared By
100	10 mL in 100 mL	10	Sunita Reddy

Annexure-3: Calibration Curve Log

Conc. (ppm)	Absorbance
5	0.255
10	0.512
20	1.021
30	1.527
40	2.038

Annexure-4: Sample Analysis Report

Sample	Absorbance	Calculated Conc. (ppm)	Analyst
Formulation X	0.984	19.3	Rajesh Kumar

Annexure-5: Method Validation Summary

Parameter	Criteria	Result	Status
Linearity	R² ≥ 0.999	0.9996	Pass
Accuracy	98–102%	99.2%	Pass
Precision	RSD ≤ 2.0%	1.1%	Pass
LOD	Calculated	1.5 ppm	Pass
LOQ	Calculated	4.8 ppm	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded validation parameters and added solution stability study	Audit Preparedness