(APIs) and impurities.

2. Scope

This SOP is applicable to all 1D and 2D NMR spectral interpretation activities performed in the Analytical Method Development (AMD) department, covering 1H, 13C, COSY, HSQC, and HMBC spectra.

3. Responsibilities

• NMR Analyst: Analyzes raw data, processes spectra, and performs chemical shift and coupling analysis.
• Method Developer: Correlates NMR findings with known structures and proposes identification or confirmation.
• QA Officer: Ensures compliance of documentation and interpretation with SOPs and regulatory expectations.
• Head – AMD: Reviews and approves final interpretation reports.

4. Accountability

The Head of Analytical Method Development is accountable for the quality and compliance of spectral interpretation reports used for structural confirmation and impurity identification.

5. Procedure

5.1 Raw Data Review and Baseline Assessment

1. Open the acquired NMR data file using the validated NMR processing software.
2. Assess:
   • Baseline correction
   • Phase correction (manual or auto)
   • Signal-to-noise ratio
   • Solvent peak position and suppression effectiveness
3. Mark all peaks for further interpretation and note any unusual baseline noise.
4. Log this information in Annexure-1: Baseline Review Checklist.

5.2 Chemical Shift Assignment

1. Calibrate chemical shifts using standard solvent signals:
   • CDCl₃: 7.26 ppm (1H), 77.0 ppm (13C)
   • DMSO-d₆: 2.50 ppm (1H), 39.5 ppm (13C)
2. Use integration, multiplicity, and coupling constants to assign each signal.
3. Document chemical shift table in Annexure-2: Shift Assignment Table.

5.3 Multiplicity and Coupling Analysis

1. Classify signals as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), or broad (br).
2. Determine J-values (Hz) between coupled protons using line separation.
3. Correlate splitting patterns with structural groups (e.g., aromatic systems, methyl groups, CH₂, CH₃).
4. Cross-reference expected vs observed patterns.
5. Record in Annexure-3: Coupling and Multiplicity Log.

5.4 Integration and Proton Count Verification

1. Confirm total proton count by comparing relative integrations to known structure.
2. Ensure the total integration matches theoretical proton number within ±10% tolerance.
3. Highlight discrepancies and investigate possible overlapping or exchangeable protons.
4. Document in Annexure-4: Proton Count Verification Sheet.

5.5 Structural Correlation

1. Map each assigned signal to respective atoms in the proposed chemical structure.
2. Use correlation diagrams to represent assignments visually.
3. In case of multiple tautomers or stereoisomers, identify possible assignments using 2D spectra (if available).
4. Attach chemical structure with annotated shifts to final report.
5. Log in Annexure-5: Structural Correlation Map.

5.6 Optional 2D Spectral Interpretation

1. If available, analyze COSY, HSQC, or HMBC spectra to confirm:
   • Through-bond (scalar) couplings
   • Carbon–proton connectivity
   • Long-range heteronuclear interactions
2. Overlay 2D maps with 1D data for signal confirmation.
3. Document observations in Annexure-6: 2D Spectra Interpretation Log.

5.7 Final Review and Reporting

1. Compile all annexures and attach processed spectra, tables, and annotated structures.
2. Ensure:
   • Signal-to-noise ≥ 10:1 for key peaks
   • No missing or extra peaks unaccounted for
   • Consistency across replicates
3. Review report internally, then submit for QA approval and Head–AMD sign-off.

6. Abbreviations

• NMR: Nuclear Magnetic Resonance
• ppm: Parts Per Million
• J: Coupling Constant
• HSQC: Heteronuclear Single Quantum Coherence
• HMBC: Heteronuclear Multiple Bond Correlation
• SOP: Standard Operating Procedure

7. Documents

1. Baseline Review Checklist – Annexure-1
2. Shift Assignment Table – Annexure-2
3. Coupling and Multiplicity Log – Annexure-3
4. Proton Count Verification Sheet – Annexure-4
5. Structural Correlation Map – Annexure-5
6. 2D Spectra Interpretation Log – Annexure-6

8. References

• ICH Q6A – Specifications
• USP <761> – Nuclear Magnetic Resonance
• Textbook: Organic Structure Determination Using NMR Spectroscopy

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Baseline Review Checklist

Sample ID	Solvent	Baseline Corrected	Phase Adjusted	Comments
NMR-112-A	CDCl3	Yes	Yes	Minor noise near solvent peak

Annexure-2: Shift Assignment Table

δ (ppm)	Assignment	Multiplicity	J (Hz)	Integration
7.26	Aromatic-H	m	8.2	1H
2.35	CH3	s	–	3H

Annexure-3: Coupling and Multiplicity Log

CH₃ singlet confirms isolation; aromatic multiplet split over 4 protons indicating substitution pattern consistent with proposed structure.

Annexure-4: Proton Count Verification Sheet

The total observed integral matches theoretical 8 protons for target molecule within 5% deviation.

Annexure-5: Structural Correlation Map

Annotated structure attached with atom labels and assigned δ values confirming identity.

Annexure-6: 2D Spectra Interpretation Log

HSQC confirmed one-to-one C–H connectivity. HMBC cross-peaks supported quinoline core and methylene linkage.

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added 2D NMR correlation and annexure templates	Annual SOP Update