an analyte peak is not co-eluted with impurities or degradants and that it represents a single chemical entity.

2. Scope

This SOP applies to the Analytical Method Development (AMD) department and is applicable to the evaluation of drug substances, finished dosage forms, degradation studies, and stability samples using PDA-equipped HPLC systems.

3. Responsibilities

• Analytical Chemist: Performs PDA data acquisition, spectral comparison, and documentation of purity results.
• Reviewer: Validates spectral homogeneity and confirms peak purity match parameters are within acceptance criteria.
• QA Officer: Ensures that chromatographic data including PDA spectra and reports meet regulatory and data integrity requirements.
• Head – AMD: Reviews and approves the finalized peak purity reports and method suitability for validation.

4. Accountability

The Head of Analytical Method Development is accountable for verifying that peak purity results are scientifically sound and used to support method specificity and selectivity claims.

5. Procedure

5.1 Instrument and Software Requirements

1. Use a qualified HPLC system equipped with a PDA detector and validated chromatography software (e.g., Empower, Chromeleon).
2. Ensure baseline noise and drift are within specification before sample analysis.
3. Verify detector calibration using holmium oxide filter or certified wavelength standards.

5.2 Sample Preparation

1. Prepare standard API solution and test sample in appropriate diluent at a concentration that ensures optimal peak intensity without saturation.
2. Filter through 0.45 µm or 0.22 µm PVDF or nylon syringe filters.
3. Document all preparations in Annexure-1: Sample Preparation Log.

5.3 Chromatographic Conditions

1. Inject blank, standard, and sample under the selected method conditions.
2. Set PDA acquisition parameters:
   • Wavelength range: 200–400 nm
   • Sampling rate: ≥ 10 Hz
   • Resolution: 1.2 nm or better
3. Ensure the PDA trace is enabled and active on chromatographic method file.
4. Perform triplicate injections for peak reproducibility and purity confirmation.

5.4 Spectral Evaluation

1. Use chromatography software to compare UV spectra across:
   • Peak apex
   • Peak leading edge (start)
   • Peak trailing edge (end)
2. Calculate match angle or similarity index using software algorithm (e.g., Apex vs Start and Apex vs End).
3. Acceptance criteria:
   • Purity angle must be less than purity threshold
   • Similarity index > 990 (or > 99%) depending on software
   • No major spectral deviation across the peak
4. Document observations in Annexure-2: Peak Purity Evaluation Log.

5.5 Interference and Co-Elution Check

1. Overlay UV spectra of the analyte peak and known impurities.
2. Inject placebo and degradation samples to verify spectral uniqueness.
3. Confirm that no shoulders, split peaks, or non-matching spectra exist under method conditions.
4. Document and attach PDA spectral overlays in Annexure-3: PDA Overlay Report.

5.6 Reporting and Interpretation

1. If peak passes spectral similarity test and purity angle is below threshold, report as “Spectrally Pure”.
2. If results fail, investigate possible co-elution, matrix interference, or system suitability issues.
3. Use peak purity result to support specificity data in validation report or regulatory filing.
4. Final interpretation to be entered in Annexure-4: Peak Purity Certificate.

6. Abbreviations

• PDA: Photodiode Array
• HPLC: High-Performance Liquid Chromatography
• API: Active Pharmaceutical Ingredient
• UV: Ultraviolet
• SOP: Standard Operating Procedure

7. Documents

1. Sample Preparation Log – Annexure-1
2. Peak Purity Evaluation Log – Annexure-2
3. PDA Overlay Report – Annexure-3
4. Peak Purity Certificate – Annexure-4

8. References

• ICH Q2(R1) – Validation of Analytical Procedures
• USP General Chapter <621> – Chromatography
• FDA Guidance on Analytical Procedures and Method Validation
• Software Manuals for Empower / Chromeleon / OpenLab

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 Code	Concentration	Diluent	Filtered	Prepared By
XYZ-API	100 µg/mL	Mobile Phase	Yes	Sunita Reddy

Annexure-2: Peak Purity Evaluation Log

Injection	Match Angle	Purity Threshold	Result	Status
STD-01	2.45	3.10	Pass	Pure

Annexure-3: PDA Overlay Report

Overlay ID	Comparison Type	Observation	Status
PDA-001	Apex vs End	Similarity: 99.5%	Pass

Annexure-4: Peak Purity Certificate

Sample ID	Peak RT (min)	Detector	Status	Analyst
XYZ-API	3.24	PDA	Spectrally Pure	Rajesh Kumar

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Added overlay comparison and PDA threshold parameters	Annual Review Update