Colorimetric methods use specific reagents to develop color based on chemical reactions, which is then measured spectrophotometrically to infer concentration.

2. Scope

This SOP is applicable to the Analytical Method Development (AMD) laboratory for developing colorimetric procedures for analysis of APIs, excipients, intermediates, and dosage forms where applicable, in compliance with pharmacopeial and ICH guidelines.

3. Responsibilities

• Analytical Chemist: Responsible for developing and executing the method, selecting reagents, measuring absorbance, and recording observations.
• Method Reviewer: Validates the accuracy and repeatability of the developed method, verifies absorbance readings and calculations.
• QA: Ensures the method and documentation comply with GMP, ICH, and pharmacopeial requirements.
• Head – AMD: Provides approval for final method release and oversees its incorporation into regulatory documents.

4. Accountability

The Head of the Analytical Method Development department is accountable for ensuring the suitability, reproducibility, and regulatory compliance of all colorimetric methods developed for pharmaceutical testing.

5. Procedure

5.1 Preliminary Assessment

1. Review available pharmacopeial and scientific literature for existing colorimetric procedures relevant to the analyte or chemical class.
2. Assess the chemical functionality of the target analyte to identify potential reagents that produce color upon reaction.
3. Determine the purpose of the colorimetric method:
   • Limit test (qualitative)
   • Assay or content determination (quantitative)
4. Record findings and justification in Annexure-1: Preliminary Method Review Sheet.

5.2 Reagent Selection and Reaction Optimization

1. Identify reagents known to form chromogenic complexes with the target analyte. Examples include:
   • 1,10-phenanthroline for iron
   • Nessler’s reagent for ammonia
   • Diphenylcarbazone for mercury
2. Prepare multiple test solutions with varying concentrations of analyte and reagent under controlled conditions.
3. Determine the optimal reagent-to-analyte molar ratio based on the intensity of color and reaction stability.
4. Evaluate different reaction times, pH values, and buffer systems.
5. Measure absorbance at multiple wavelengths to identify λ_max.
6. Document the optimized reaction conditions in Annexure-2: Optimization Worksheet.

5.3 Standard Preparation and Calibration Curve

1. Prepare standard stock solutions of the analyte using a certified reference material (CRM).
2. Prepare a series of dilutions (5–7 points) spanning the expected concentration range.
3. React each dilution with the color reagent under optimized conditions.
4. Measure absorbance at the determined λ_max using a UV-Vis spectrophotometer.
5. Plot the absorbance vs concentration to generate the calibration curve.
6. Ensure linearity with correlation coefficient (R²) ≥ 0.999.
7. Record data in Annexure-3: Calibration Curve Log.

5.4 Sample Analysis

1. Prepare test samples according to formulation or API-specific instructions (e.g., dissolution, dilution, filtration).
2. Treat test samples with the same reagents and under identical conditions as standards.
3. Measure the absorbance and determine analyte concentration using the calibration curve.
4. Repeat analysis in triplicate to ensure precision.
5. Document findings in Annexure-4: Sample Analysis Record.

5.5 Method Validation

1. Conduct validation as per ICH Q2(R1):
2. Specificity: Demonstrate that color development is analyte-specific and unaffected by excipients or solvents.
3. Linearity: Confirm R² ≥ 0.999 over working range.
4. Accuracy: Perform spike recovery studies at 80%, 100%, and 120% levels (acceptable recovery 98–102%).
5. Precision: Determine intra-day and inter-day variability (RSD ≤ 2.0%).
6. LOD & LOQ: Calculate based on signal-to-noise ratio of 3.3 and 10 respectively.
7. Robustness: Vary reaction time, reagent concentration, and pH to observe stability of absorbance.
8. Solution Stability: Test absorbance at intervals over 6 hours.
9. Record validation data in Annexure-5: Validation Summary Report.

5.6 Final Method Documentation

1. Prepare detailed method SOP including:
   • Objective and applicability
   • Reagent composition and preparation
   • Sample preparation instructions
   • Reaction procedure
   • Instrumentation details
   • System suitability and acceptance criteria
2. Submit method to QA for review and approval prior to official use.

6. Abbreviations

• SOP: Standard Operating Procedure
• CRM: Certified Reference Material
• UV-Vis: Ultraviolet-Visible Spectrophotometry
• ICH: International Council for Harmonisation
• RSD: Relative Standard Deviation

7. Documents

1. Preliminary Method Review Sheet – Annexure-1
2. Optimization Worksheet – Annexure-2
3. Calibration Curve Log – Annexure-3
4. Sample Analysis Record – Annexure-4
5. Validation Summary Report – Annexure-5

8. References

• USP <851> – Spectrophotometry and Light Scattering
• ICH Q2(R1) – Validation of Analytical Procedures
• IP, Ph. Eur., BP – General Colorimetric Tests
• AOAC Official Methods for Colorimetric Analysis

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Preliminary Method Review Sheet

Analyte	Literature Source	Color Reagent	Remarks	Prepared By
Iron (III)	USP	1,10-Phenanthroline	Forms orange-red complex	Rajesh Kumar

Annexure-2: Optimization Worksheet

Trial	Reagent Volume (mL)	pH	λmax (nm)	Observation
1	2.0	4.5	510	Stable complex

Annexure-3: Calibration Curve Log

Concentration (ppm)	Absorbance
5	0.135
10	0.270
20	0.540
30	0.810
40	1.080

Annexure-4: Sample Analysis Record

Sample	Absorbance	Calculated Conc. (ppm)	Analyst
API-A	0.540	20.0	Sunita Reddy

Annexure-5: Validation Summary Report

Parameter	Criteria	Result	Status
Precision	RSD ≤ 2%	1.2%	Pass
Accuracy	98–102%	99.6%	Pass
Linearity	R² ≥ 0.999	0.9995	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded validation, annexures and buffer optimization data	Audit Preparedness