manufactured at [Company Name] that require endotoxin testing prior to batch release.

3. Responsibilities

• Microbiology Analyst: Conducts the BET and documents the results.
• QC Reviewer: Verifies results and checks calculations.
• QA Personnel: Reviews test reports and ensures compliance with applicable specifications.

4. Accountability

The Head – Quality Control shall be accountable for ensuring implementation and compliance of the BET procedures.

5. Procedure

5.1 Preparation

1. Ensure all glassware and equipment used are depyrogenated at 250°C for 30 minutes.
2. Confirm that the LAL reagent, water for BET, and standard endotoxin are within expiry.
3. Record lot numbers and preparation details in the Endotoxin Test Log (Annexure-1).

5.2 Sample Preparation

1. Withdraw sample under aseptic conditions using pyrogen-free apparatus.
2. Dilute sample appropriately based on Maximum Valid Dilution (MVD) and label test tubes.

5.3 Gel Clot Method

1. Add 0.1 mL of sample or standard endotoxin to 0.1 mL LAL reagent in LAL test tubes.
2. Include positive product control (PPC), negative product control (NPC), positive control (PC), and negative control (NC).
3. Incubate tubes at 37±1°C for 60±2 minutes in a dry block incubator.
4. After incubation, invert each tube gently. Presence of firm gel indicates a positive result.

5.4 Chromogenic or Turbidimetric Method (Kinetic)

1. Prepare microplate with required standards, samples, PPCs, and controls in duplicate.
2. Add LAL reagent and read reaction kinetics in a validated microplate reader at specified wavelength.
3. Use standard curve to calculate endotoxin concentration in sample.

5.5 Acceptance Criteria

• Results must be below specified endotoxin limit (EU/mL) as per monograph or calculated using:
  Endotoxin Limit = K/M, where:
  • K = Threshold pyrogenic dose of endotoxin (generally 5 EU/kg for IV)
  • M = Maximum dose/kg/hour

5.6 Documentation

1. Record observations in Annexure-1 and calculate results using Annexure-2.
2. Report results in Annexure-3 and attach raw data printouts if applicable.

6. Abbreviations

• SOP: Standard Operating Procedure
• LAL: Limulus Amebocyte Lysate
• BET: Bacterial Endotoxin Test
• EU: Endotoxin Unit
• MVD: Maximum Valid Dilution

7. Documents

1. Endotoxin Test Log – Annexure-1
2. Endotoxin Limit Calculation Sheet – Annexure-2
3. Endotoxin Test Report – Annexure-3

8. References

• USP <85> Bacterial Endotoxins Test
• EP 2.6.14 Bacterial Endotoxins
• ICH Q6A: Specifications

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation	QC Analyst	QC Manager	Head QA
Department	Quality Control	Quality Control	Quality Assurance

11. Annexures

Annexure-1: Endotoxin Test Log

Date	Batch No.	Product	Method	Analyst
17/06/2025	IVINF0021	Inj. Meropenem	Gel Clot	Pratik Jha

Annexure-2: Endotoxin Limit Calculation Sheet

Product	Max Dose (mL/kg/hr)	K	Limit (EU/mL)	MVD
Inj. Piperacillin	20	5 EU/kg	0.25	100

Annexure-3: Endotoxin Test Report

Product	Batch	Test Date	Result (EU/mL)	Status	Reviewed By
Inj. Amikacin	AMK-055	15/06/2025	0.18	Complies	Shraddha Desai

Revision History

Revision Date	Revision No.	Details	Reason	Approved By
01/01/2022	1.0	Initial version	New SOP	QA Head
18/06/2025	2.0	Included kinetic methods and calculation format	Annual Review	QA Head