SOP Guide for Pharma

Analytical Method Development: Peak Purity Evaluation using PDA – V 2.0

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Analytical Method Development: Peak Purity Evaluation using PDA – V 2.0

SOP for Peak Purity Assessment Using PDA Detector in HPLC Method Development


Department Analytical Method Development
SOP No. SOP/AMD/073/2025
Supersedes SOP/AMD/073/2022
Page No. Page 1 of 14
Issue Date 19/05/2025
Effective Date 20/05/2025
Review Date 19/05/2026

1. Purpose

This SOP describes the procedure for evaluating peak purity using a Photodiode Array (PDA) detector during HPLC method development. Peak purity assessment is essential to confirm that 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

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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.
See also  Analytical Method Development: Reference Standard Qualification for AMD - V 2.0

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

See also  Analytical Method Development: HPLC Method Development for Finished Product - V 2.0

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

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
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