Elixir Department: SOP for Assay of API by HPLC – V 2.0

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Elixir Department: SOP for Assay of API by HPLC – V 2.0

Standard Operating Procedure for Assay of Active Pharmaceutical Ingredient (API) by HPLC in Elixirs

Department Elixir Department
SOP No. SOP/ELX/212/2025
Supersedes SOP/ELX/212/2022
Page No. Page 1 of 10
Issue Date 11/04/2025
Effective Date 15/04/2025
Review Date 11/04/2026

1. Purpose

To describe the procedure for performing quantitative analysis of the Active Pharmaceutical Ingredient (API) in elixir formulations using High-Performance Liquid Chromatography (HPLC), ensuring accuracy, precision, and compliance with pharmacopeial or validated methods.

2. Scope

This SOP applies to the Quality Control (QC) Department responsible for testing elixir samples for API content using HPLC instrumentation as per standard or in-house validated methods.

3. Responsibilities

  • QC Analyst:
    • Prepare samples and standards, operate HPLC, record and interpret results.
  • QC Supervisor:
    • Review chromatograms and calculations for accuracy.
  • QA Officer:
    • Verify compliance with method and documentation.

4. Accountability

The QC Head is accountable for the execution and validity of HPLC assay results. QA Head is responsible for verifying that all assay data complies with GMP requirements.

See also  Elixir Department: SOP for Sampling from Storage Tank - V 2.0

5. Procedure

5.1 Preparation for

HPLC Analysis
  1. Review the current pharmacopeial monograph or validated in-house method for assay conditions.
  2. Ensure HPLC instrument calibration is within due date.
  3. Clean and check the system – purge mobile phase lines and equilibrate the column.
  4. Prepare mobile phase as per method, filter through 0.45 µm filter, and degas.

5.2 Preparation of Standard Solution

  1. Weigh the specified quantity of working standard (e.g., 100 mg) and transfer to a 100 mL volumetric flask.
  2. Dissolve in suitable solvent (e.g., methanol, water) and dilute to volume.
  3. Filter through 0.45 µm syringe filter and transfer to labeled HPLC vial.

5.3 Preparation of Sample Solution

  1. Shake the elixir sample to ensure uniformity.
  2. Transfer the required volume (e.g., 10 mL) into a volumetric flask, dilute as per method, and sonicate if needed.
  3. Filter and transfer the clear solution to an HPLC vial for injection.

5.4 HPLC Conditions (Example Method)

  • Column: C18, 250 mm × 4.6 mm, 5 µm
  • Mobile Phase: Methanol:Water (60:40 v/v)
  • Flow Rate: 1.0 mL/min
  • Detection Wavelength: 254 nm
  • Injection Volume: 20 µL
  • Run Time: As per method (typically 10 min)

5.5 Analysis and Calculation

  1. Inject standard and sample solutions into the HPLC system.
  2. Record retention time and area under curve (AUC) for main API peak.
  3. Calculate API content using the formula:
    API (%) = (Sample Area × Std Concentration × Dilution Factor × 100) / (Standard Area × Sample Concentration)

5.6 System Suitability and Acceptance Criteria

  1. Check system suitability parameters:
    • Theoretical Plates: ≥ 2000
    • Tailing Factor: ≤ 2.0
    • RSD of replicate injections (n=6): ≤ 2.0%
  2. API content must be within 90.0%–110.0% of label claim (or as per specification).

5.7 Documentation

  1. Record sample preparation, instrument conditions, and results in the HPLC Assay Logbook (Annexure-1).
  2. Attach chromatograms, calculations, and method reference to the BMR.

6. Abbreviations

  • SOP: Standard Operating Procedure
  • QA: Quality Assurance
  • QC: Quality Control
  • HPLC: High Performance Liquid Chromatography
  • API: Active Pharmaceutical Ingredient
  • BMR: Batch Manufacturing Record

7. Documents

  1. HPLC Assay Logbook (Annexure-1)
  2. Batch Manufacturing Record (BMR)
  3. Instrument Calibration Records

8. References

  • USP General Chapter <621> – Chromatography
  • ICH Q2(R1) – Validation of Analytical Procedures
  • 21 CFR Part 211.165 – Testing and Release for Distribution

9. SOP Version

Version: 2.0

10. Approval Section

Prepared By Checked By Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: HPLC Assay Logbook

Date Batch No. Sample ID Retention Time Area API Content (%) Tested By Checked By
11/04/2025 ELX-0425-030 API-A 5.36 120345 99.2% Rajesh Kumar Sunita Reddy

Revision History:

Revision Date Revision No. Revision Details Reason for Revision Approved By
01/01/2024 1.0 Initial Release New SOP QA Head
11/04/2025 2.0 Updated with CAPA linkage and new system suitability criteria GMP Enhancement QA Head
Elixers V 2.0 Tags:, , , , , , , , , , , , , , , , , , , , , , , ,