and unknown impurities at trace levels.

2. Scope

This SOP applies to the impurity evaluation of pharmaceutical APIs and formulations during development, stability testing, and stress degradation studies in the Analytical Method Development (AMD) department.

3. Responsibilities

• Analytical Scientist: Prepares samples, develops LC-MS method, runs analyses, and interprets results.
• Mass Spectrometry Specialist: Supports method optimization and structural elucidation using MS/MS.
• QA Officer: Reviews profiling data for regulatory compliance.
• Head – AMD: Approves impurity reports and ensures method alignment with ICH guidelines.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring the scientific integrity, traceability, and compliance of impurity profiling activities using LC-MS.

5. Procedure

5.1 Sample Preparation

1. Weigh appropriate quantity of sample (API/formulation) and dissolve in suitable diluent (ACN:Water, 50:50, or per solubility).
2. Filter through 0.22 µm syringe filter before LC-MS analysis.
3. Prepare placebo and blank samples for baseline comparison.
4. Document sample prep in Annexure-1: Sample Preparation Log.

5.2 LC Conditions Setup

1. Choose appropriate column (e.g., C18, 150×4.6 mm, 3.5 µm).
2. Select mobile phase:
   • A: Water with 0.1% formic acid
   • B: Acetonitrile with 0.1% formic acid
3. Gradient: 5% to 95% B over 30 minutes.
4. Flow rate: 0.6 mL/min; Column temperature: 35°C; Injection volume: 10 µL.
5. Log parameters in Annexure-2: LC Setup Sheet.

5.3 MS Conditions Setup

1. Use ESI in positive and/or negative ionization mode.
2. Scan range: m/z 100–1000.
3. Capillary voltage: 3500–4000 V; Desolvation temperature: 350°C.
4. Enable auto MS/MS for unknown impurity fragmentation.
5. Record in Annexure-3: MS Setup Log.

5.4 Data Acquisition

1. Inject blank, placebo, sample, and spiked standards.
2. Acquire full-scan data and perform data-dependent MS/MS for unknown peaks.
3. Ensure repeat injections (n=3) for reproducibility.
4. Overlay chromatograms to identify additional impurity peaks.
5. Log observations in Annexure-4: LC-MS Acquisition Log.

5.5 Data Analysis

1. Use software to deconvolute spectra and identify:
   • Retention time
   • m/z of parent and fragment ions
   • Mass accuracy and isotopic pattern
2. Compare against mass libraries, databases, or in-house MS records.
3. For unknowns, propose structure using fragmentation data and molecular formula calculator.
4. Prepare impurity table in Annexure-5: Impurity Summary Sheet.

5.6 Reporting and Documentation

1. Include:
   • Total ion chromatograms (TIC)
   • Extracted ion chromatograms (EIC)
   • MS and MS/MS spectra
   • Impurity table with RRT, RT, m/z, identity (if known), and proposed formula
2. Summarize data trends in stressed vs unstressed samples (if applicable).
3. Submit complete report for QA review and final approval by Head – AMD.

6. Abbreviations

• LC-MS: Liquid Chromatography–Mass Spectrometry
• MS/MS: Tandem Mass Spectrometry
• RRT: Relative Retention Time
• EIC: Extracted Ion Chromatogram
• TIC: Total Ion Chromatogram
• SOP: Standard Operating Procedure

7. Documents

1. Sample Preparation Log – Annexure-1
2. LC Setup Sheet – Annexure-2
3. MS Setup Log – Annexure-3
4. LC-MS Acquisition Log – Annexure-4
5. Impurity Summary Sheet – Annexure-5

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 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: Sample Preparation Log

Sample Name	Type	Solvent	Concentration	Date	Prepared By
API-B	Stressed	MeOH:Water (50:50)	0.5 mg/mL	17/05/2025	Sunita Reddy

Annexure-2: LC Setup Sheet

Column	Flow Rate	Gradient	Temp	Injection Vol
C18, 150 mm	0.6 mL/min	5–95% B in 30 min	35°C	10 µL

Annexure-3: MS Setup Log

Ion Source	Polarity	Scan Range	Collision Energy
ESI	Positive	100–1000 m/z	20–35 eV

Annexure-4: LC-MS Acquisition Log

Sample	Injection No.	TIC Observations	Comment
Formulation F1	1	6 additional peaks vs blank	Requires identification

Annexure-5: Impurity Summary Sheet

RT (min)	RRT	m/z	Proposed Identity	Status
13.2	1.12	345.1	Oxidized Degradant	Unconfirmed
15.8	1.34	321.0	Related Compound C	Confirmed

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Expanded scope and annexures for impurity tables	Annual SOP Review