SOP for Determination of Absorptivity Values in UV Spectrophotometry

Department	Analytical Method Development
SOP No.	SOP/AMD/092/2025
Supersedes	SOP/AMD/092/2022
Page No.	Page 1 of 14
Issue Date	19/05/2025
Effective Date	20/05/2025
Review Date	19/05/2026

1. Purpose

This SOP outlines the procedure for determining the absorptivity value (A 1%, 1cm) of Active Pharmaceutical Ingredients (APIs) using UV spectrophotometry, ensuring method standardization, reproducibility, and regulatory compliance with USP and ICH guidelines.

2. Scope

This procedure is applicable to the Analytical Method Development (AMD) department for the determination of absorptivity values of APIs and reference standards used in pharmaceutical formulation and quality control.

3. Responsibilities

• Analytical Chemist: Prepares dilutions, performs UV scans, calculates absorptivity values.
• Reviewer: Validates accuracy of calculations and spectrophotometric readings.
• QA Officer: Ensures adherence to documentation practices and compliance with regulations.
• Head – AMD: Approves determined absorptivity values and ensures SOP compliance.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring that absorptivity values determined using this procedure are scientifically justified, reproducible, and suitable for regulatory submissions and specification building.

5. Procedure

5.1 Principle

1. Absorptivity (A 1%, 1cm) is the absorbance of a 1% w/v solution in a 1 cm path length cuvette.
2. Calculated using:

   A (1%, 1cm) = (A × 100) / C
   

   Where:

   • A = absorbance of test solution
   • C = concentration in mg/mL

5.2 Selection of Wavelength (λmax)

1. Scan test solution from 200–400 nm using UV spectrophotometer.
2. Determine wavelength with highest and stable absorbance (λmax).
3. Confirm absence of interference from solvent or excipients.
4. Document in Annexure-1: Wavelength Scanning Report.

5.3 Standard Solution Preparation

1. Accurately weigh 100 mg of API or reference standard.
2. Dissolve in solvent (e.g., water, ethanol, methanol) and make up to 100 mL volume to achieve 1 mg/mL (0.1% w/v).
3. Further dilute to 0.01% w/v (100 µg/mL) if absorbance exceeds 1.0.
4. Use matched quartz cuvettes and record absorbance at λmax.
5. Record in Annexure-2: Standard Preparation Log.

5.4 Blank and Instrument Setup

1. Use blank solvent (same as used for sample) in reference cell.
2. Use spectrophotometer set to 1 nm bandwidth and 1.000 path length (cm).
3. Zero instrument with blank before taking absorbance of test solution.
4. Confirm baseline stability and document in Annexure-3: Blank Verification Log.

5.5 Calculation of Absorptivity Value

1. Use following formula:

   A (1%, 1cm) = (Absorbance × 100) / Concentration in mg/mL
   

2. Example: Absorbance = 0.875 at 260 nm; Concentration = 0.01 mg/mL (0.001% w/v)
   A (1%, 1cm) = (0.875 × 100) / 0.01 = 8750
3. Repeat with at least three concentrations to ensure linearity.
4. Record in Annexure-4: Absorptivity Calculation Sheet.

5.6 Verification of Linearity and Precision

1. Prepare three dilutions (e.g., 80, 100, 120 µg/mL).
2. Measure absorbance and calculate A (1%, 1cm) for each.
3. Acceptance Criteria:
   • %RSD of A (1%, 1cm) ≤ 2.0%
   • Linearity: R² ≥ 0.995
4. Document in Annexure-5: Linearity and Precision Report.

5.7 Application in Assay and Dissolution

1. Use A (1%, 1cm) value to convert absorbance to concentration for:
   • Single-point assay
   • Routine dissolution testing
   • Analytical method equivalency checks
2. Ensure that same solvent, cuvette, and path length are used in all subsequent applications.

6. Abbreviations

• UV: Ultraviolet
• API: Active Pharmaceutical Ingredient
• λmax: Wavelength of Maximum Absorbance
• A (1%, 1cm): Absorptivity of 1% solution in 1 cm cell
• RSD: Relative Standard Deviation

7. Documents

1. Wavelength Scanning Report – Annexure-1
2. Standard Preparation Log – Annexure-2
3. Blank Verification Log – Annexure-3
4. Absorptivity Calculation Sheet – Annexure-4
5. Linearity and Precision Report – Annexure-5

8. References

• USP <857> – Ultraviolet-Visible Spectroscopy
• ICH Q2(R1) – Validation of Analytical Procedures
• Ph. Eur. and IP Monographs on UV Absorptivity
• FDA Guidance on Analytical Methods

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 Scanning Report

Sample ID	Solvent	Scan Range	λmax (nm)
API-UV-212	Methanol	200–400 nm	262

Annexure-2: Standard Preparation Log

Substance	Weight (mg)	Solvent	Volume (mL)	Prepared By
API-212	100.0	Methanol	100	Sunita Reddy

Annexure-3: Blank Verification Log

Blank Solvent	Absorbance at λmax	Conclusion
Methanol	0.001	No Interference

Annexure-4: Absorptivity Calculation Sheet

Sample	Concentration (mg/mL)	Absorbance	A (1%, 1cm)
API-212	0.01	0.853	8530

Annexure-5: Linearity and Precision Report

Concentration (µg/mL)	Absorbance	A (1%, 1cm)
80	0.682	8525
100	0.853	8530
120	1.021	8517

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Annexures expanded to include linearity and blank interference	Annual SOP Update