setting, and regulatory compliance.

2. Scope

This procedure applies to the Analytical Method Development (AMD) department and is intended for unknown impurities present above the identification threshold as per ICH Q3A and Q3B guidelines.

3. Responsibilities

• Analytical Scientist: Responsible for sample preparation, impurity isolation, and applying characterization techniques.
• Instrument Operator: Operates LC-MS, NMR, and FTIR equipment used in impurity elucidation.
• QA Officer: Reviews the records and ensures proper documentation and regulatory alignment.
• Head – AMD: Verifies data and approves the impurity report for submission to regulatory affairs.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring all unknown impurities above the threshold are scientifically characterized and documented as per regulatory guidelines.

5. Procedure

5.1 Selection of Samples

1. Select drug product/API batches that exhibit unknown peaks above identification thresholds (e.g., >0.10% for APIs).
2. Use samples from stability studies, forced degradation studies, or scale-up batches.
3. Log selected samples in Annexure-1: Impurity Selection Log.

5.2 Impurity Isolation Techniques

1. Choose the appropriate isolation strategy based on impurity level and nature:
   • Preparative HPLC or UPLC for high-precision separation
   • Liquid-liquid extraction or SPE for preliminary enrichment
   • Fraction collection based on UV or MS detection
2. Evaporate collected fractions under nitrogen or lyophilize if thermally sensitive.
3. Re-check purity using analytical HPLC before structural studies.

5.3 Structural Characterization

1. Use a combination of spectroscopic tools:
   • LC-MS/MS: Identify molecular weight and fragmentation patterns
   • 1H and 13C NMR: Determine carbon-hydrogen framework
   • FTIR: Identify functional groups
   • UV-Vis: Compare λ_max values
2. Interpret spectral data in collaboration with senior scientists.
3. Fill Annexure-2: Spectral Data Interpretation Sheet.

5.4 Data Compilation and Reporting

1. Compare results against literature or known degradation pathways.
2. Assign a unique impurity code (e.g., IMP-UNK-01) and update impurity tracker.
3. Document:
   • Isolation strategy
   • Spectroscopic confirmation
   • Proposed chemical structure
4. Prepare Annexure-3: Unknown Impurity Characterization Report.

5.5 Decision on Qualification

1. Determine if the unknown impurity:
   • Has structural alerts for genotoxicity
   • Exceeds the qualification threshold
2. If applicable, initiate safety qualification studies or justify based on TTC.

6. Abbreviations

• API: Active Pharmaceutical Ingredient
• LC-MS: Liquid Chromatography-Mass Spectrometry
• NMR: Nuclear Magnetic Resonance
• FTIR: Fourier Transform Infrared Spectroscopy
• SPE: Solid Phase Extraction
• SOP: Standard Operating Procedure

7. Documents

1. Impurity Selection Log – Annexure-1
2. Spectral Data Interpretation Sheet – Annexure-2
3. Unknown Impurity Characterization Report – Annexure-3

8. References

• ICH Q3A(R2) – Impurities in New Drug Substances
• ICH Q3B(R2) – Impurities in New Drug Products
• ICH M7 – Assessment and Control of DNA Reactive (Mutagenic) Impurities

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 Selection Log

Date	Sample ID	RT (min)	Area %	Selected By
18/05/2025	API-X23	9.88	0.26%	Sunita Reddy

Annexure-2: Spectral Data Interpretation Sheet

Technique	Observation	Interpretation
LC-MS	m/z = 315	Likely sulfonated by-product
1H NMR	δ = 8.1–8.5 ppm	Aromatic protons near carbonyl
FTIR	1675 cm-1	Amide C=O stretch

Annexure-3: Unknown Impurity Characterization Report

IMP-UNK-01 was isolated via preparative HPLC from a degradation study. LC-MS and NMR revealed a sulfonated degradation product. No genotoxic alerts. Qualification not required under ICH Q3A.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded isolation strategies and ICH M7 reference included	Annual SOP Update