iron and heavy metals in Active Pharmaceutical Ingredients (APIs), excipients, and pharmaceutical formulations. These tests use visual comparison with standard color or turbidity solutions and follow pharmacopeial methods.

2. Scope

This SOP applies to all substances tested for iron or heavy metal impurities using limit test methods in the Analytical Method Development (AMD) department. The tests are based on Indian Pharmacopoeia (IP), United States Pharmacopoeia (USP), and European Pharmacopoeia (Ph. Eur.) guidelines.

3. Responsibilities

• Analytical Chemist: Develops and performs the test, prepares reagents, and documents comparisons.
• Reviewer: Verifies correct visual comparison, reagent performance, and result accuracy.
• QA Officer: Ensures completeness of documentation and compliance with GMP requirements.
• Head – AMD: Approves method finalization and ensures it meets regulatory compliance for routine QC.

4. Accountability

The Head of AMD is accountable for ensuring that validated methods for limit testing of iron and heavy metals are implemented effectively and align with pharmacopeial and regulatory standards.

5. Procedure

5.1 Preliminary Review

1. Consult pharmacopeial monographs for specific limits:
   • Iron: Typically ≤10 ppm as Fe³⁺
   • Heavy Metals: Expressed as Pb equivalents, usually ≤20 ppm
2. Refer to:
   • IP Appendix III B and III C
   • USP <231>, <233> (where applicable)
   • Ph. Eur. 2.4.8 (Heavy Metals) and 2.4.9 (Iron)
3. Record relevant references and monograph details in Annexure-1: Reference Review Sheet.

5.2 Reagents and Apparatus

1. Prepare standard reagents:
   • Iron Standard Solution: 20 ppm ferric ammonium sulfate solution
   • Heavy Metals Standard Solution: 20 ppm lead nitrate in nitric acid
   • Thioglycolic acid reagent (for Iron)
   • Hydrogen sulfide solution or sodium sulfide solution (for Heavy Metals)
2. Use clean, chloride-free Nessler cylinders or color comparison tubes (50 mL capacity).
3. Ensure all glassware is rinsed with distilled water and acid-treated if necessary to remove residual metals.

5.3 Iron Limit Test

1. Test and standard solutions must be compared simultaneously.
2. Standard Preparation: Dilute ferric ammonium sulfate to known limit (e.g., 10 ppm) in water containing thioglycolic acid and ammonia buffer.
3. Test Preparation: Dissolve or extract sample as per monograph, treat similarly with thioglycolic acid and buffer.
4. Allow color to develop for 5 minutes; a pink to purple color indicates presence of iron.
5. Compare the intensity of test vs. standard side-by-side against a white background.
6. Document whether test solution is darker (Fail) or lighter (Pass).
7. Record in Annexure-2: Iron Test Comparison Log.

5.4 Heavy Metals Limit Test

1. Standard Preparation: Lead nitrate solution prepared to specified concentration (usually 20 ppm), adjusted to pH ~3 using acetic acid.
2. Test Preparation: Dissolve sample in water and adjust pH; add hydrogen sulfide or sodium sulfide reagent dropwise.
3. A brown-black precipitate or turbidity indicates presence of metals like Pb, Hg, Bi, Cu, etc.
4. Allow solutions to stand for 5 minutes before comparison.
5. Visually compare test and standard for turbidity/precipitate density.
6. Record in Annexure-3: Heavy Metals Comparison Log.

5.5 Method Optimization

1. Assess visual detectability by preparing a range of iron or heavy metal concentrations near the specification limit (e.g., 5 ppm, 10 ppm, 15 ppm).
2. Evaluate:
   • Effect of pH and reaction time
   • Stability of color or turbidity after preparation
   • Consistency across different reagent batches
3. Optimize lighting conditions (preferably daylight or cool white lamp) and viewing background.
4. Record in Annexure-4: Method Optimization Log.

5.6 Method Validation for Visual Limit Tests

1. As per regulatory guidance, validation should include:
2. Specificity: Absence of color/turbidity interference from sample matrix.
3. Robustness: Tolerance to minor changes in pH, temperature, and reagent concentration.
4. Reproducibility: Visual result reproducibility across three different analysts.
5. Detection Limit Confirmation: Lowest concentration that yields perceptible change.
6. Precision (Visual): Perform three replicates at the limit level and confirm consistent pass/fail results.
7. Summarize all outcomes in Annexure-5: Validation Summary Report.

6. Abbreviations

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

7. Documents

1. Reference Review Sheet – Annexure-1
2. Iron Test Comparison Log – Annexure-2
3. Heavy Metals Comparison Log – Annexure-3
4. Method Optimization Log – Annexure-4
5. Validation Summary Report – Annexure-5

8. References

• USP <231>, <233> – Heavy Metals and Elemental Impurities
• IP Appendix III B & C – Iron and Heavy Metals Tests
• Ph. Eur. 2.4.8, 2.4.9 – Limit Tests
• 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: Reference Review Sheet

Test	Pharmacopeia	Limit	Reagents Used	Prepared By
Iron	IP	≤10 ppm	Thioglycolic Acid, Ammonia	Rajesh Kumar
Heavy Metals	USP	≤20 ppm	Sodium Sulfide, Acetic Acid	Rajesh Kumar

Annexure-2: Iron Test Comparison Log

Sample	Standard Color	Test Color	Result	Analyst
API-X	Light Pink	Fainter	Complies	Sunita Reddy

Annexure-3: Heavy Metals Comparison Log

Sample	Standard Turbidity	Test Turbidity	Result	Analyst
Excip.-A	Moderate	Less	Complies	Ajay Mehra

Annexure-4: Method Optimization Log

Trial	Variable	Observation	Conclusion
1	pH 2.5	Increased false positives	Not suitable
2	pH 3.0	Stable and distinct turbidity	Accepted

Annexure-5: Validation Summary Report

Parameter	Criteria	Result	Status
Specificity	No interference from matrix	Confirmed	Pass
Robustness	±10% reagent volume	No significant impact	Pass
Reproducibility	3 analysts agree	Consistent	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added validation matrix and annexure expansion	Annual SOP Update