applies to the Analytical Method Development (AMD) laboratory and includes selection of wavelength, preparation of standards, method linearity, system suitability, and validation activities in accordance with ICH Q2(R1) and pharmacopeial requirements.

3. Responsibilities

• Analytical Scientist: Develops method, prepares standards/samples, records and interprets data.
• Method Reviewer: Reviews calculations, spectrums, and validation reports.
• QA Team: Ensures the method meets GMP documentation and compliance requirements.
• Head – AMD: Provides final method approval for transfer to QC or for regulatory submission.

4. Accountability

The Head of the AMD department is accountable for ensuring all UV spectrophotometric methods developed are scientifically sound, validated, and regulatory compliant.

5. Procedure

5.1 Wavelength Selection

1. Prepare a 10 ppm standard solution of the analyte using a suitable solvent (e.g., methanol, water).
2. Scan from 200 to 400 nm using a UV-visible spectrophotometer.
3. Record the UV spectrum and identify the λ_max (wavelength of maximum absorbance).
4. Select λ_max with maximum absorbance and minimum baseline noise for quantification.
5. Record spectrum in Annexure-1: UV Spectrum Report.

5.2 Standard and Sample Preparation

1. Use a certified reference material to prepare a 1000 ppm stock solution.
2. Prepare working standard solutions in the range of 50–150% of the expected analyte concentration (typically 5–25 ppm).
3. Use the same diluent as used in final product preparation.
4. Filter all solutions using a 0.45 µm filter before analysis.
5. Prepare test sample solution following product-specific procedures.

5.3 Calibration Curve and Linearity

1. Measure absorbance of each standard solution at λ_max.
2. Plot absorbance vs. concentration to generate calibration curve.
3. Calculate correlation coefficient (R²); accept R² ≥ 0.999.
4. Document calibration data in Annexure-2: Calibration Curve Worksheet.

5.4 Method Optimization Parameters

1. Study the effect of:
   • Solvent selection (water, methanol, ethanol)
   • pH (using phosphate or acetate buffer if needed)
   • Reaction time (if applicable)
   • Path length of cuvette (1 cm standard)
2. Identify and justify the most stable and reproducible conditions.
3. Record all optimization trials in Annexure-3: Method Optimization Log.

5.5 Method Validation

1. Validate the method as per ICH Q2(R1) guidelines:
2. Specificity: Confirm no interference at λ_max from excipients or diluents.
3. Linearity: R² ≥ 0.999 across working range.
4. Accuracy: Recovery between 98% and 102% at 80%, 100%, and 120% levels.
5. Precision:
   • Repeatability: RSD ≤ 2.0% for 6 replicates at 100% level.
   • Intermediate Precision: Repeat on different days or analysts.
6. LOD and LOQ: Based on standard deviation of response and slope (3.3×σ/S for LOD, 10×σ/S for LOQ).
7. Robustness: Vary parameters such as wavelength (±2 nm), temperature, and solvent.
8. Solution Stability: Evaluate sample/standard stability at room temperature for up to 24 hours.
9. Document validation results in Annexure-4: Validation Summary Report.

5.6 System Suitability

1. Inject standard solution six times.
2. Calculate % RSD of absorbance values.
3. Acceptable RSD ≤ 2.0% for absorbance measurements.
4. Document results in Annexure-5: System Suitability Record.

6. Abbreviations

• SOP: Standard Operating Procedure
• UV: Ultraviolet
• ICH: International Council for Harmonisation
• RSD: Relative Standard Deviation
• LOD: Limit of Detection
• LOQ: Limit of Quantification
• λ_max: Wavelength of Maximum Absorbance

7. Documents

1. UV Spectrum Report – Annexure-1
2. Calibration Curve Worksheet – Annexure-2
3. Method Optimization Log – Annexure-3
4. Validation Summary Report – Annexure-4
5. System Suitability Record – Annexure-5

8. References

• ICH Q2(R1) – Validation of Analytical Procedures
• USP <857> – Ultraviolet-Visible Spectroscopy
• IP Appendix – UV Spectrophotometric Assays
• Ph. Eur. 2.2.25 – UV-visible absorption 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: UV Spectrum Report

Sample Name	Concentration (ppm)	λmax (nm)	Observed Peak	Analyst
API-A	10	272	0.864	Sunita Reddy

Annexure-2: Calibration Curve Worksheet

Concentration (ppm)	Absorbance
5	0.425
10	0.851
15	1.275
20	1.694
25	2.115

Annexure-3: Method Optimization Log

Trial No.	Solvent	λmax (nm)	Observation	Conclusion
1	Methanol	272	Stable peak	Accepted
2	Water	271	Peak broadening	Rejected

Annexure-4: Validation Summary Report

Parameter	Acceptance Criteria	Result	Status
Precision	RSD ≤ 2%	1.3%	Pass
Accuracy	98–102%	99.4%	Pass
Linearity	R² ≥ 0.999	0.9998	Pass

Annexure-5: System Suitability Record

Injection	Absorbance
1	0.852
2	0.856
3	0.851
4	0.854
5	0.853
6	0.855

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded method validation and updated annexures	Regulatory alignment