Elixir Department: SOP for Endotoxin Testing – V 2.0

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Elixir Department: SOP for Endotoxin Testing – V 2.0

Standard Operating Procedure for Endotoxin Testing in Elixir Formulations

Department Elixir Department
SOP No. SOP/ELX/229/2025
Supersedes SOP/ELX/229/2022
Page No. Page 1 of 8
Issue Date 11/04/2025
Effective Date 15/04/2025
Review Date 11/04/2026

1. Purpose

To establish a validated and standardized procedure for performing endotoxin testing on elixir formulations using the Limulus Amebocyte Lysate (LAL) test to ensure compliance with pharmacopoeial and regulatory requirements.

2. Scope

This SOP applies to the Quality Control Department and is intended for use during batch release, in-process verification, and stability testing for elixirs where low endotoxin levels are required, especially for oral solutions administered to immunocompromised patients or products labeled “pyrogen-free.”

3. Responsibilities

  • QC Analyst:
    • Perform endotoxin testing as per the validated method and ensure the sample is free from interference.
    • Ensure all observations are recorded promptly and accurately.
  • QC Supervisor:
    • Verify compliance with reagent, instrument, and method controls.
    • Review results and approve data entry.
  • QA Officer:
    • Ensure regulatory compliance and sign off for batch release or deviation if limits are exceeded.
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4. Accountability

The QC Head is accountable for proper execution and integrity of the endotoxin

test. The QA Head is responsible for ensuring regulatory and GMP compliance for product release.

5. Procedure

5.1 Equipment and Materials

  • LAL test kit (gel clot, chromogenic, or turbidimetric type)
  • Depyrogenated glassware and instruments
  • Endotoxin-free water
  • Positive control endotoxin standard
  • Water bath or incubator at 37±1°C

5.2 Sample Handling

  1. Ensure samples and equipment are handled in an endotoxin-free environment (e.g., LAF cabinet).
  2. Prepare appropriate dilutions using LAL reagent water to minimize interference from sample matrix (e.g., alcohol, preservatives, colors).

5.3 Method Overview

  1. Gel Clot Method: Preferred for qualitative analysis. Results are based on clot formation at a specified sensitivity (e.g., 0.25 EU/mL).
  2. Chromogenic/Turbidimetric Methods: Used for quantitative results. Requires a kinetic reader and calibration curve.
  3. Follow manufacturer’s instructions for the LAL kit in use.

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5.4 Controls and Validity

  1. Include positive product control (PPC), negative product control (NPC), positive control (CSE), and reagent blank.
  2. The test is valid only if:
    • Blank shows no reaction.
    • PPC gives accurate spike recovery (50–200%).
    • Standard endotoxin response falls within the acceptable range.

5.5 Interpretation of Results

  1. Gel Clot: Presence of firm clot indicates positive result (fails limit); absence of clot = negative (passes).
  2. Quantitative Methods: Results are expressed in EU/mL or EU/g. Product must comply with labeled or pharmacopeial limit (e.g., NMT 0.5 EU/mL).

5.6 Acceptance Criteria

  1. Result must be below the endotoxin limit specified in the product monograph or validated specification (e.g., NMT 0.5 EU/mL for oral liquids if specified).
  2. Out-of-specification (OOS) results must be investigated as per deviation SOP.

5.7 Documentation

  1. Record all raw data, including dilution, lot numbers of kits, and observations in the Endotoxin Test Logbook (Annexure-1).
  2. Maintain calibration certificates for LAL reader and qualification reports.

6. Abbreviations

  • SOP: Standard Operating Procedure
  • LAL: Limulus Amebocyte Lysate
  • EU: Endotoxin Units
  • PPC: Positive Product Control
  • NPC: Negative Product Control
  • CSE: Control Standard Endotoxin
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7. Documents

  1. Endotoxin Test Logbook (Annexure-1)
  2. Batch Manufacturing Record (BMR)
  3. Instrument Calibration and Qualification Records

8. References

  • USP Chapter <85> – Bacterial Endotoxins Test
  • ICH Q6A – Specifications: Test Procedures and Acceptance Criteria
  • 21 CFR Part 211.160 – Laboratory Controls

9. SOP Version

Version: 2.0

10. Approval Section

Prepared By Checked By Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Endotoxin Test Logbook

Date Batch No. Dilution Method Result (EU/mL) Limit Pass/Fail Tested By Verified By
11/04/2025 ELX-0425-047 1:10 Gel Clot <0.25 0.5 Pass Rajesh Kumar Sunita Reddy

Revision History:

Revision Date Revision No. Revision Details Reason for Revision Approved By
01/01/2024 1.0 Initial SOP Release New SOP Creation QA Head
11/04/2025 2.0 Included quantitative LAL methods and acceptance criteria Regulatory Alignment QA Head
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