SOP for Development of Limit Test for Chloride and Sulphate in AMD Laboratory

Department	Analytical Method Development
SOP No.	SOP/AMD/052/2025
Supersedes	SOP/AMD/052/2022
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 defines the process for developing, standardizing, and validating limit tests for detecting chloride and sulphate impurities in APIs and excipients using visual comparison techniques with standard turbidity suspensions, in accordance with IP, USP, and Ph. Eur. guidelines.

2. Scope

This procedure applies to the Analytical Method Development (AMD) laboratory for substances where chloride and sulphate are controlled as impurity limits, typically in milligram per liter (ppm) or percentage ranges.

3. Responsibilities

• Analytical Chemist: Develops the method, prepares standard and test solutions, and performs visual comparisons.
• Method Reviewer: Evaluates accuracy, repeatability, and visual consistency of turbidity.
• QA Personnel: Ensures documentation meets regulatory and GMP standards.
• Head – AMD: Approves final method and ensures compliance with regulatory expectations.

4. Accountability

The Head of AMD is accountable for ensuring that qualitative limit tests for chloride and sulphate are developed and validated per pharmacopeial guidelines and are suitable for regulatory submissions and quality control operations.

5. Procedure

5.1 Preliminary Method Review

1. Refer to official pharmacopeial methods for limit tests:
   • IP – Appendix III A and III B
   • USP <221> – Chloride and Sulfate Tests
   • Ph. Eur. 2.4.4 (Chloride) and 2.4.5 (Sulphates)
2. Determine the applicable impurity limit for the test substance.
3. Record source and applicable method details in Annexure-1: Method Review Log.

5.2 Preparation of Reagents and Apparatus

1. Prepare standard reagents as per pharmacopeia:
   • Chloride Standard Solution: 10 ppm NaCl in distilled water
   • Sulphate Standard Solution: 10 ppm K₂SO₄ in distilled water
   • Reagents: Nitric acid, barium chloride solution (10%), silver nitrate, etc.
2. Use matched Nessler cylinders (50 mL capacity) for visual comparison.
3. Ensure all glassware is chloride/sulphate-free (rinse with distilled water).

5.3 Standardization and Blank Preparation

1. Prepare blank solution using the same volume of reagents without analyte.
2. Prepare standard turbidity solution by adding chloride or sulphate ion solution to test volume with appropriate reagents.
3. Incubate for 5 minutes to allow turbidity to develop fully.
4. Observe turbidity against a black background and record visual clarity.
5. Use this as reference standard for test comparison.

5.4 Test Sample Preparation

1. Dissolve appropriate quantity of sample (as per monograph) in distilled water.
2. Filter or centrifuge if sample is not clear.
3. Transfer specified volume into Nessler cylinder.
4. Add acidifying agent (e.g., nitric acid) followed by turbidity reagent (e.g., barium chloride for sulphate).
5. Incubate for specified duration (usually 5 minutes).
6. Compare turbidity visually against standard solution side-by-side under identical lighting conditions.
7. Document comparison results in Annexure-2: Visual Comparison Record.

5.5 Visual Comparison Criteria

1. Pass Criteria: Turbidity in test sample must not be more intense than in standard solution.
2. If the turbidity is equal or less than the standard, record as “Complies.”
3. If higher, repeat test with another aliquot or diluted sample.
4. Any color interference or haziness shall be reported and investigated.

5.6 Method Optimization

1. Perform multiple trials using known chloride/sulphate concentrations near the specification limit.
2. Evaluate:
   • Reagent freshness and batch variability
   • Reaction time and stability of turbidity
   • Effect of light and temperature on visual comparison
3. Record observations in Annexure-3: Optimization Log.

5.7 Method Validation (Visual Tests)

1. As these are qualitative limit tests, full quantitative validation is not required.
2. Perform robustness by changing reagent volumes (±10%), pH (±0.5), and incubation time (±2 mins).
3. Perform specificity check using blank and placebo matrix.
4. Test detection limit by preparing serial dilutions around the limit concentration.
5. Ensure operator reproducibility with blind testing across three analysts.
6. Summarize all validation exercises in Annexure-4: Validation Summary Report.

6. Abbreviations

• SOP: Standard Operating Procedure
• IP: Indian Pharmacopoeia
• USP: United States Pharmacopeia
• Ph. Eur.: European Pharmacopoeia
• QA: Quality Assurance
• AMD: Analytical Method Development

7. Documents

1. Method Review Log – Annexure-1
2. Visual Comparison Record – Annexure-2
3. Optimization Log – Annexure-3
4. Validation Summary Report – Annexure-4

8. References

• USP <221> – Chloride and Sulphate Limit Tests
• IP Appendix III – Limit Tests for Inorganic Impurities
• Ph. Eur. 2.4.4 & 2.4.5 – Limit Tests for Chloride and Sulphate
• ICH Q2(R1) – Validation of Analytical Procedures

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Method Review Log

Analyte	Pharmacopeial Reference	Limit	Notes	Prepared By
Chloride	USP <221>	≤ 0.014%	Limit Test Method	Sunita Reddy

Annexure-2: Visual Comparison Record

Sample ID	Test Solution Clarity	Standard Solution Clarity	Result	Analyst
API-23	Faint Turbidity	Same	Complies	Ajay Mehra

Annexure-3: Optimization Log

Trial	Parameter Changed	Observation	Conclusion
1	+10% Barium Chloride	Increased turbidity	Not acceptable

Annexure-4: Validation Summary Report

Validation Parameter	Criteria	Outcome	Status
Specificity	No interference	Confirmed	Pass
Reproducibility	3 analysts same result	Confirmed	Pass
Robustness	±10% reagent	Stable turbidity	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded validation and added operator variability checks	Audit Preparedness