Standard Operating Procedure for Identification of Impurities in Active Pharmaceutical Ingredients (API)

Department	Analytical Method Development
SOP No.	SOP/AMD/134/2025
Supersedes	SOP/AMD/134/2022
Page No.	Page 1 of 14
Issue Date	19/05/2025
Effective Date	20/05/2025
Review Date	19/05/2026

1. Purpose

To outline a standardized procedure for the identification and characterization of impurities in active pharmaceutical ingredients (API) using validated analytical techniques, ensuring compliance with ICH and regulatory expectations.

2. Scope

This SOP applies to all analytical activities performed in the Analytical Method Development (AMD) laboratory related to impurity profiling, including known, unknown, process-related, and degradation impurities in APIs during development and validation stages.

3. Responsibilities

• Analytical Scientist: Responsible for detecting and confirming the identity of impurities using appropriate instrumentation.
• QC Chemist: Supports impurity trend evaluation and documentation of findings.
• QA Officer: Reviews data and ensures traceability, integrity, and regulatory alignment.
• Head – AMD: Approves reports, ensures compliance with ICH Q3A/Q3B guidelines, and facilitates regulatory submission readiness.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring the scientific soundness and compliance of the impurity identification process within the AMD lab.

5. Procedure

5.1 Sample Selection

1. Choose API batches subjected to stress studies, stability trials, or scale-up manufacturing where impurity levels approach qualification thresholds.
2. Ensure sample integrity and store as per API-specific conditions before analysis.

5.2 Initial Impurity Detection

1. Perform HPLC/UPLC analysis using a validated stability-indicating method.
2. Identify unknown peaks by comparing with blank, placebo, and known impurity standards.
3. Record retention time, area %, and peak shape for each impurity in Annexure-1: Impurity Detection Log.

5.3 Isolation of Impurities (if required)

1. Isolate significant unknown impurities via:
   • Preparative HPLC
   • Solid-phase extraction (SPE)
   • Fraction collection from UPLC
2. Dry collected fractions under vacuum or lyophilization as needed.
3. Ensure purity check using analytical HPLC prior to characterization.

5.4 Structural Elucidation

1. Analyze isolated impurity or enriched fraction using:
   • LC-MS or GC-MS: Determine molecular ion (m/z), fragmentation pattern
   • 1H NMR and 13C NMR: Assign functional groups and structural framework
   • FTIR: Verify functional group presence (e.g., -OH, -COOH, NH₂)
   • UV-Vis: Evaluate chromophore regions for comparison
2. Compare spectral data with known impurities and degradation products.
3. Log findings in Annexure-2: Impurity Spectral Interpretation Sheet.

5.5 Classification and Risk Assessment

1. Classify impurity per ICH Q3A:
   • Identified Impurity
   • Unidentified Impurity
   • Specified/Unspecified Impurity
2. Calculate daily exposure and compare with threshold of toxicological concern (TTC).
3. Initiate toxicological qualification or waive based on historical data.

5.6 Documentation and Reporting

1. Compile impurity profile report summarizing:
   • Method used
   • Detection level
   • Structural confirmation
   • Qualification plan (if required)
2. Submit report to regulatory affairs and retain copies in controlled archives.
3. Complete Annexure-3: Impurity Identification Summary Report.

6. Abbreviations

• API: Active Pharmaceutical Ingredient
• HPLC: High-Performance Liquid Chromatography
• GC-MS: Gas Chromatography-Mass Spectrometry
• FTIR: Fourier Transform Infrared Spectroscopy
• NMR: Nuclear Magnetic Resonance
• TTC: Threshold of Toxicological Concern
• SOP: Standard Operating Procedure

7. Documents

1. Impurity Detection Log – Annexure-1
2. Impurity Spectral Interpretation Sheet – Annexure-2
3. Impurity Identification Summary Report – Annexure-3

8. References

• ICH Q3A(R2) – Impurities in New Drug Substances
• ICH Q3B(R2) – Impurities in New Drug Products
• USP General Chapter <1086> – Impurities in Drug Substances and Drug Products

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Impurity Detection Log

Date	Sample ID	Method Used	Impurity RT (min)	Area %
18/05/2025	API-Lot-A25	UPLC 220 nm	7.54	0.23%

Annexure-2: Impurity Spectral Interpretation Sheet

Technique	Result	Interpretation
LC-MS	m/z 245.2	Molecular ion peak
1H NMR	δ 7.2–7.8 ppm	Aromatic protons
FTIR	1710 cm-1	Carbonyl group (C=O)

Annexure-3: Impurity Identification Summary Report

Unknown impurity detected at RT 7.54 min. Isolated via SPE. LC-MS indicated molecular weight 245. NMR and FTIR confirmed the presence of an aldehyde degradation product. Toxicology waiver applicable based on known pathway.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added risk assessment and TTC evaluation per ICH Q3A(R2)	Annual SOP Update