degradant identification.

2. Scope

This SOP applies to all analytical activities in the Analytical Method Development (AMD) laboratory that require LC-MS or GC-MS-based peak identification using m/z data, including unknown peak investigation in stability and stress studies.

3. Responsibilities

• Analytical Scientist: Executes m/z-based analysis, matches theoretical masses, and interprets fragmentation patterns.
• Mass Spectrometry Specialist: Configures high-resolution MS and assists in isotopic distribution interpretation.
• QA Officer: Verifies data quality, traceability, and compliance.
• Head – AMD: Approves final identification reports and associated annexures.

4. Accountability

The Head of Analytical Method Development is accountable for ensuring scientifically justified, reproducible, and regulatory-aligned identification of unknown peaks by mass spectrometric analysis.

5. Procedure

5.1 Sample and Standard Preparation

1. Prepare API, formulation, and stress-degraded samples in appropriate solvent (e.g., methanol or ACN:water 50:50).
2. Use known reference standards wherever available.
3. Filter through 0.22 µm PTFE filter.
4. Document preparation details in Annexure-1: Sample Log Sheet.

5.2 Data Acquisition

1. Set LC-MS method to high-resolution full scan (100–1000 m/z) on Q-TOF or Orbitrap system.
2. Run in both positive and negative ion modes as applicable.
3. Record MS and MS/MS spectra at different collision energies (10–40 eV).
4. Acquire Total Ion Chromatograms (TIC) and Extracted Ion Chromatograms (EIC).
5. Record parameters in Annexure-2: Acquisition Details.

5.3 m/z-Based Peak Identification

1. Locate unknown peaks in TIC/EIC and determine exact m/z value.
2. Compare observed m/z with theoretical m/z values of expected compounds and known impurities using:
   • Molecular formula calculators
   • Compound libraries
   • Software prediction tools
3. Calculate mass error in ppm:
   Mass Error (ppm) = [(Measured m/z – Theoretical m/z) / Theoretical m/z] × 10⁶
4. Accept identification if error ≤ 5 ppm and isotopic pattern matches.
5. Log observations in Annexure-3: m/z Matching Sheet.

5.4 Fragmentation and Structural Confirmation

1. Use MS/MS spectra to confirm fragmentation pathways of selected m/z peaks.
2. Propose bond cleavage logic and fragment assignments.
3. Draw proposed structure with fragmentation arrows for reporting.
4. Refer to known fragmentation libraries (e.g., MassBank, mzCloud).
5. Record structural proposals in Annexure-4: Fragmentation Interpretation Sheet.

5.5 Reporting and Finalization

1. Compile:
   • RT, m/z, theoretical vs measured values
   • Isotopic match (%)
   • Fragmentation evidence
   • Final peak assignment (known, probable, unknown)
2. Review report with QA and mass spectrometry team.
3. Finalize report and archive spectra, chromatograms, and annotations.
4. Summarize in Annexure-5: Peak Identification Summary Log.

6. Abbreviations

• m/z: Mass-to-charge ratio
• RT: Retention Time
• TIC: Total Ion Chromatogram
• EIC: Extracted Ion Chromatogram
• ppm: Parts per million
• Q-TOF: Quadrupole Time-of-Flight
• MS/MS: Tandem Mass Spectrometry

7. Documents

1. Sample Log Sheet – Annexure-1
2. Acquisition Details – Annexure-2
3. m/z Matching Sheet – Annexure-3
4. Fragmentation Interpretation Sheet – Annexure-4
5. Peak Identification Summary Log – Annexure-5

8. References

• ICH Q3A(R2) – Impurities in New Drug Substances
• FDA Guidance for Industry: Analytical Procedures and Methods Validation
• MassBank of North America (MoNA)

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 Log Sheet

Sample ID	Matrix	Solvent	Prepared By	Date
FDS-110-API	API	ACN:Water	Rajesh Kumar	18/05/2025

Annexure-2: Acquisition Details

Instrument	Ion Mode	Range	Resolution	CE
Q-TOF	Positive	100–1000	High	20–35 eV

Annexure-3: m/z Matching Sheet

Peak RT	Observed m/z	Theoretical m/z	Mass Error (ppm)	Status
12.8	345.1542	345.1537	1.45	Confirmed

Annexure-4: Fragmentation Interpretation Sheet

MS/MS spectra for m/z 345.1542 showed fragments at m/z 287.1, 243.1, and 121.0. Fragmentation suggests neutral losses of C₂H₄O and amide cleavage. Proposed structure aligns with known degradant DP-3.

Annexure-5: Peak Identification Summary Log

RT	m/z	Identity	Evidence Type	Conclusion
12.8	345.1542	DP-3	MS, MS/MS, Library	Confirmed
14.2	362.1608	Unknown	MS only	Unidentified

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added ppm calculation, fragmentation annexure, isotopic matching logic	Annual Review