and finished products.

2. Scope

This SOP applies to the Analytical Method Development (AMD) laboratory and is intended for complexometric titration techniques using EDTA or other ligands as titrants for the determination of calcium, magnesium, zinc, copper, and other metal ions.

3. Responsibilities

• Analytical Chemist: Conducts titration trials, prepares reagents, performs method validation, and records results.
• Reviewer: Reviews accuracy, reproducibility, and calculation records for compliance and completeness.
• QA Representative: Ensures that the SOP follows GMP principles and supports regulatory expectations.
• Head – AMD: Approves developed methods for QC use and regulatory filing.

4. Accountability

The Head of AMD is accountable for ensuring scientifically justified, accurate, and validated complexometric titration methods are implemented for metal ion determination in pharmaceutical substances.

5. Procedure

5.1 Selection of Complexometric Reaction

1. Identify metal ions present in the test substance or formulation.
2. Select appropriate chelating agent (commonly EDTA) and endpoint indicator.
3. Examples:
   • Calcium & Magnesium – EDTA with Eriochrome Black T (EBT) at pH 10
   • Zinc – EDTA with Xylenol Orange at pH 5–6
   • Copper – EDTA with PAN indicator
4. Document selection in Annexure-1: Reaction Justification Sheet.

5.2 Preparation and Standardization of Reagents

1. Prepare 0.05 M or 0.1 M EDTA solution using disodium salt of EDTA.
2. Standardize EDTA solution using known concentration of calcium carbonate or magnesium sulfate.
3. Prepare buffer solutions:
   • Ammonia-Ammonium Chloride buffer (pH 10) for Ca/Mg
   • Acetate buffer (pH 5–6) for Zn/Cu
4. Prepare and filter indicators as per required conditions (e.g., 0.5% EBT in ethanol).
5. Record all preparation steps in Annexure-2: Reagent Preparation Log.

5.3 Sample Preparation

1. Accurately weigh the sample containing the metal ion to be determined.
2. Dissolve in suitable solvent (e.g., distilled water, dilute HCl).
3. If necessary, remove interfering ions by masking agents (e.g., cyanide, triethanolamine).
4. Adjust pH using appropriate buffer.
5. Document all steps in Annexure-3: Sample Preparation Record.

5.4 Execution of Titration

1. Transfer sample solution to a clean conical flask.
2. Add buffer solution and indicator (e.g., EBT turns wine red in presence of Mg/Ca).
3. Titrate with EDTA solution until endpoint is reached (color changes to blue in EBT).
4. Perform blank and standard titrations for comparison.
5. Repeat titrations in triplicate for statistical consistency.
6. Record endpoint volumes and calculations in Annexure-4: Titration Worksheet.

5.5 Calculation

Metal content (%) = (V × M × Eq. Wt × 100) / W × 1000
Where:
V = Volume of EDTA used (mL)
M = Molarity of EDTA
Eq. Wt = Equivalent weight of analyte (e.g., Ca = 20.04)
W = Sample weight (mg)

5.6 Method Optimization

1. Evaluate:
   • Buffer pH impact on endpoint sharpness
   • Indicator concentration and color transition range
   • Interference from co-existing ions
2. Conduct trials using known standards and observe endpoint stability.
3. Document observations in Annexure-5: Optimization Record.

5.7 Method Validation

1. Specificity: Demonstrate absence of interference from excipients or other metal ions.
2. Linearity: Analyze standards at 50–150% concentration; R² ≥ 0.999.
3. Accuracy: Recovery from spiked samples should be within 98–102%.
4. Precision:
   • Repeatability: RSD ≤ 2.0% for six replicates
   • Intermediate Precision: Reproducibility on different days/analysts
5. Robustness: Test minor variations in pH, temperature, and buffer strength.
6. Record outcomes in Annexure-6: Validation Summary Report.

6. Abbreviations

• SOP: Standard Operating Procedure
• EDTA: Ethylenediaminetetraacetic Acid
• RSD: Relative Standard Deviation
• EBT: Eriochrome Black T
• AMD: Analytical Method Development

7. Documents

1. Reaction Justification Sheet – Annexure-1
2. Reagent Preparation Log – Annexure-2
3. Sample Preparation Record – Annexure-3
4. Titration Worksheet – Annexure-4
5. Optimization Record – Annexure-5
6. Validation Summary Report – Annexure-6

8. References

• USP <541> – Titrimetry
• IP Appendix 3.5 – Complexometric Methods
• Ph. Eur. 2.2.20 – Potentiometric Titrations
• 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: Reaction Justification Sheet

Metal Ion	Chelating Agent	Indicator	Buffer	Analyst
Calcium	EDTA	EBT	pH 10 (NH₄OH buffer)	Sunita Reddy

Annexure-2: Reagent Preparation Log

Reagent	Strength	Standardized Against	Date
EDTA	0.05 M	Calcium Carbonate	10/05/2025

Annexure-3: Sample Preparation Record

Sample	Weight	Solvent	pH Adjusted
Calcium Gluconate	500 mg	Water	10

Annexure-4: Titration Worksheet

Trial	EDTA Volume (mL)	Calculation	% Assay
1	20.35	Formula Applied	99.4%

Annexure-5: Optimization Record

Parameter	Variation	Observation	Conclusion
pH	9.5	Less sharp endpoint	Rejected

Annexure-6: Validation Summary Report

Parameter	Criteria	Result	Status
Accuracy	98–102%	99.3%	Pass
Precision	RSD ≤ 2%	0.84%	Pass
Linearity	R² ≥ 0.999	0.9991	Pass

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded validation criteria and included masking strategies	Annual Review