is precise, accurate, and robust for quality control and validation.

2. Scope

This SOP applies to the Analytical Method Development (AMD) department and covers all UV-based assay method development activities for APIs in powder or solution form, either as raw materials or within finished dosage forms.

3. Responsibilities

• Analytical Chemist: Performs method development, standard/sample preparation, wavelength selection, and result documentation.
• Reviewer: Verifies method linearity, precision, and assay results.
• QA Officer: Reviews all records for compliance with GMP, GLP, and ICH standards.
• Head – AMD: Approves the developed method and authorizes it for validation and routine use.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring the integrity, reliability, and regulatory compliance of all UV-based assay methods developed and implemented in the department.

5. Procedure

5.1 Wavelength Selection

1. Scan the UV spectrum of the API (200–400 nm) using a UV-visible spectrophotometer.
2. Identify the wavelength of maximum absorbance (λmax) and verify absence of interferences.
3. Ensure molar absorptivity is sufficient (A > 0.1 for 1% solution at 1 cm path length).
4. Record spectrum and selected λmax in Annexure-1: UV Scanning Report.

5.2 Standard and Sample Preparation

1. Weigh accurately ~10 mg of API (standard) and transfer to a 100 mL volumetric flask.
2. Dissolve in suitable solvent (water, methanol, ethanol, etc.).
3. Sonicate if required. Dilute to mark to obtain working concentration (e.g., 10–25 µg/mL).
4. Prepare sample using identical solvent and conditions as standard.
5. Log preparation details in Annexure-2: Preparation Log.

5.3 Calibration Curve and Linearity

1. Prepare standard solutions at five concentrations: 50%, 75%, 100%, 125%, and 150% of target assay level.
2. Measure absorbance at λmax using matched quartz cuvettes (1 cm path length).
3. Plot absorbance vs concentration and determine linearity (R² ≥ 0.995).
4. Calculate slope, intercept, and %RSD of replicate readings.
5. Document results in Annexure-3: Linearity and Calibration Record.

5.4 Assay Calculation

1. Use Beer–Lambert Law for single-point assay:

   Assay (%) = (Abs_sample / Abs_standard) × (Conc_standard / Conc_sample) × 100
   

2. Apply appropriate dilution factors and record final assay %.
3. Document in Annexure-4: Assay Result Sheet.

5.5 Method Precision and Accuracy

1. Perform six replicate determinations at 100% concentration level.
2. Calculate %RSD for precision (acceptable: ≤2.0%).
3. Conduct recovery studies by spiking placebo with known quantities of standard (80%, 100%, 120%).
4. Calculate % recovery and log in Annexure-5: Precision and Recovery Report.

5.6 Specificity and Placebo Interference

1. Scan placebo solution to ensure no absorbance at selected λmax.
2. Overlay placebo and standard spectra to verify absence of overlap or interfering peaks.
3. Document results in Annexure-6: Specificity Evaluation Sheet.

5.7 System Suitability

1. Measure absorbance of six replicates of standard solution at assay level.
2. Acceptance Criteria:
   • %RSD ≤ 2.0%
   • Absorbance range: ±0.010 from mean
3. Record results in Annexure-7: System Suitability Log.

6. Abbreviations

• UV: Ultraviolet
• API: Active Pharmaceutical Ingredient
• λmax: Wavelength of Maximum Absorbance
• RSD: Relative Standard Deviation
• SOP: Standard Operating Procedure

7. Documents

1. UV Scanning Report – Annexure-1
2. Preparation Log – Annexure-2
3. Linearity and Calibration Record – Annexure-3
4. Assay Result Sheet – Annexure-4
5. Precision and Recovery Report – Annexure-5
6. Specificity Evaluation Sheet – Annexure-6
7. System Suitability Log – Annexure-7

8. References

• ICH Q2(R1) – Validation of Analytical Procedures
• USP <857> – Ultraviolet-Visible Spectroscopy
• USP <1225> – Validation of Compendial Procedures
• FDA Guidance for Industry – Analytical Procedures and Methods 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: UV Scanning Report

Sample ID	Solvent	Scan Range	λmax (nm)
API-UV-101	Water	200–400 nm	274

Annexure-2: Preparation Log

Type	Amount Weighed	Diluent	Final Volume	Prepared By
Standard	10.2 mg	Water	100 mL	Rajesh Kumar

Annexure-3: Linearity and Calibration Record

Concentration (µg/mL)	Absorbance
10	0.246
15	0.370
20	0.491
25	0.612
30	0.735

Annexure-4: Assay Result Sheet

Sample ID	Absorbance	Assay (%)
SMP-001	0.485	98.7%

Annexure-5: Precision and Recovery Report

Spike Level	% Recovery	% RSD
100%	99.2%	1.2%

Annexure-6: Specificity Evaluation Sheet

Solution	Absorbance at λmax	Inference
Placebo	0.002	No interference

Annexure-7: System Suitability Log

Replicate	Absorbance
1	0.491
2	0.490
3	0.489
4	0.492
5	0.491
6	0.490

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded linearity range and updated specificity protocol	Annual SOP Review