absorbance, essential for quantitative analysis of Active Pharmaceutical Ingredients (APIs).

2. Scope

This SOP applies to the Analytical Method Development (AMD) department and is relevant for all methods requiring quantification of APIs or finished formulations through UV spectrophotometric techniques using a standard calibration curve.

3. Responsibilities

• Analytical Chemist: Prepares standard dilutions, measures absorbance, and generates the calibration curve.
• Reviewer: Verifies dilution accuracy, regression analysis, and correlation coefficient.
• QA Officer: Reviews calibration data, curve accuracy, and documentation integrity.
• Head – AMD: Approves the final calibration curve and data for method validation and routine analysis.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring that all calibration curves are scientifically valid, statistically evaluated, and compliant with ICH Q2(R1) and pharmacopeial requirements.

5. Procedure

5.1 Standard Stock Solution Preparation

1. Weigh accurately about 100 mg of API (or reference standard).
2. Dissolve in a suitable solvent and make up to 100 mL to achieve 1 mg/mL (1000 µg/mL) solution.
3. Sonicate if required for complete dissolution.
4. Label as “Standard Stock Solution” and record in Annexure-1: Standard Preparation Log.

5.2 Working Standard Dilutions

1. Prepare a minimum of five standard dilutions in the range of 50% to 150% of the target concentration (e.g., 10, 15, 20, 25, 30 µg/mL).
2. Use Class A volumetric flasks and pipettes for accurate volume transfer.
3. Document dilution details in Annexure-2: Dilution Worksheet.

5.3 Measurement of Absorbance

1. Turn on UV-visible spectrophotometer and allow warm-up for 30 minutes.
2. Set wavelength to previously determined λ_max (e.g., 274 nm).
3. Use the solvent as blank and measure absorbance of each concentration in triplicate using 1 cm path length quartz cuvettes.
4. Log readings in Annexure-3: Absorbance Record Sheet.

5.4 Plotting Calibration Curve

1. Plot mean absorbance (Y-axis) against concentration (X-axis).
2. Perform linear regression using Excel or validated software.
3. Record slope, intercept, and correlation coefficient (r or R²).
4. Sample output: y = 0.032x + 0.005; R² = 0.9998
5. Include plot and equation in Annexure-4: Calibration Curve Summary.

5.5 Acceptance Criteria

1. Correlation coefficient (R²): ≥ 0.995
2. %RSD of absorbance: ≤ 2.0% at each level
3. Linearity: Slope should be consistent across batches
4. Intercept: Close to zero (acceptable if within ±0.010)
5. If criteria are not met, repeat dilution preparation and measurement.

5.6 Use of Calibration Curve

1. Use the equation y = mx + c to calculate unknown sample concentrations.
2. Example: If absorbance = 0.678, and curve equation is y = 0.032x + 0.005

   x = (0.678 – 0.005) / 0.032 = 21 µg/mL
   

3. Report final result after applying dilution factors.

6. Abbreviations

• UV: Ultraviolet
• API: Active Pharmaceutical Ingredient
• RSD: Relative Standard Deviation
• λ_max: Wavelength of Maximum Absorbance
• SOP: Standard Operating Procedure
• R²: Coefficient of Determination

7. Documents

1. Standard Preparation Log – Annexure-1
2. Dilution Worksheet – Annexure-2
3. Absorbance Record Sheet – Annexure-3
4. Calibration Curve Summary – Annexure-4

8. References

• ICH Q2(R1) – Validation of Analytical Procedures
• USP <857> – Ultraviolet-Visible Spectroscopy
• FDA Guidance for Industry – Analytical Procedures and Methods Validation
• Instrument Manufacturer Guidelines

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Standard Preparation Log

Sample ID	Weight (mg)	Solvent	Final Volume (mL)	Prepared By
STD-095	100.0	Methanol	100	Sunita Reddy

Annexure-2: Dilution Worksheet

Level (%)	Concentration (µg/mL)	Dilution	Final Volume (mL)
50%	10	1 mL stock + 9 mL diluent	10
150%	30	3 mL stock + 7 mL diluent	10

Annexure-3: Absorbance Record Sheet

Concentration (µg/mL)	Replicate 1	Replicate 2	Replicate 3	Mean
10	0.325	0.326	0.324	0.325

Annexure-4: Calibration Curve Summary

Curve Equation	R²	Slope	Intercept
y = 0.032x + 0.005	0.9998	0.032	0.005

Attach printed curve and Excel regression output here.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added dilution calculation examples and R² criteria update	Annual SOP Review