How to Perform the Water BET (Bacterial Endotoxin Test) – Step-by-Step Guide

Introduction

The Bacterial Endotoxin Test (BET) — often performed using Limulus Amebocyte Lysate (LAL) reagents — is a critical quality control assay for pharmaceutical water systems (Purified Water, Water For Injection). This guide explains how to perform a Water BET in a laboratory-appropriate SOP format and provides tips for reliable results and regulatory compliance.

Quick note: Regulatory endotoxin limits and specific acceptance criteria should be taken from your applicable pharmacopeia (USP / BP / EP) or product specifications. Always follow your lab's approved SOPs and safety rules.

Principle

LAL reagents react with bacterial endotoxin (lipopolysaccharide) and produce a measurable response. There are three common LAL methods:

• Gel-clot method — endpoint test where gel formation indicates presence of endotoxin at or above the test sensitivity.
• Kinetic chromogenic — measures color development over time; quantitative and suitable for automation.
• Kinetic turbidimetric — measures increase in turbidity over time; quantitative.

Scope and Intended Use

This SOP covers routine endotoxin testing of laboratory water samples (e.g., Purified Water, WFI) using LAL. Use only validated LAL reagents and follow manufacturer instructions for reagent storage and handling.

Materials and Equipment

• Validated LAL reagent (gel-clot / kinetic chromogenic / kinetic turbidimetric)
• Endotoxin-free glassware and pipette tips
• Calibrated micropipettes (endotoxin-free)**
• Certified endotoxin standards (EU/mL) for curves or verification
• Positive Product Control (PPC) reagents (if applicable)
• Sterile endotoxin-free vials or tubes
• Incubator (37 ± 1 °C) for gel-clot, or plate reader for kinetic assays
• Water samples (collected aseptically from the water system)
• Clean bench or laminar flow hood (if required by lab)

Tip: Use only consumables labeled endotoxin-free. Pre-wet and rinse glassware with endotoxin-free water if reusable.

Reagent Preparation & Storage

• Store LAL reagent per manufacturer instructions (usually refrigerated 2–8 °C).
• Reconstitute lyophilized reagents using reagent-grade, endotoxin-free water following vendor guidelines.
• Prepare standard endotoxin dilutions immediately before use and discard unused standards after the run.

Controls and Validation

• Negative control (reagent blank): Endotoxin-free water processed like samples to confirm reagent and technique cleanliness.
• Positive control: Known concentration of endotoxin to confirm LAL activity and assay sensitivity.
• Positive Product Control (PPC): Spike a sample with known endotoxin to detect inhibition/enhancement caused by matrix.
• Standard curve (kinetic methods): Prepare standard dilutions covering assay range.
• Inhibition/Enhancement test: Required when testing matrices other than water — for water these are usually straightforward but still recommended for new water systems.

Perform and record an inhibition/enhancement check whenever a new reagent lot, new water source, or new analytical method is introduced.

Sampling

1. Collect water samples aseptically in certified endotoxin-free containers.
2. Pre-rinse sampling containers with the water being sampled (if required by your SOP).
3. Record sample ID, source, date/time, and operator.
4. Transport and test samples promptly or store per validated conditions (short cold hold only if validated).

Step-by-Step Procedure — Gel-Clot (example)

Use gel-clot when a simple pass/fail at a defined sensitivity is sufficient.

1. Bring all reagents and samples to room temperature as specified by the reagent manufacturer.
2. Label endotoxin-free tubes for standards, negative control, PPC, and samples.
3. Prepare endotoxin standard dilutions to the required sensitivities (e.g., 0.125 EU/mL, 0.25 EU/mL — according to lab need and reagent sensitivity).
4. Pipette volumes (e.g., 0.1 mL) of sample/standard into separate tubes.
5. Add equal volume of LAL reagent to each tube (follow reagent instructions for volumes and mixing).
6. Gently mix by inversion — avoid vigorous shaking to prevent bubble formation.
7. Incubate tubes at 37 ± 1 °C for recommended time (typical 60 minutes for many gel-clot reagents).
8. At the end of incubation, invert tubes slowly. A firm clot that does not flow indicates a positive reaction at that sensitivity.
9. Record results and interpret against acceptance criteria.

Important: Follow your LAL reagent manufacturer's specific volumes, incubation times and temperatures — they vary by product.

Step-by-Step Procedure — Kinetic Methods (summary)

Kinetic methods produce numerical endotoxin concentrations using a standard curve.

1. Prepare standard curve dilutions in endotoxin-free water.
2. Dispense standards, controls and samples into a microplate in duplicate/triplicate.
3. Add LAL reagent and immediately start the plate reader program to record absorbance/turbidity change over time at the specified wavelength.
4. Use instrument software to calculate endotoxin concentrations from the standard curve.

Calculations and Reporting

Kinetic assays return endotoxin concentration (EU/mL) directly from the standard curve. For gel-clot, reporting is typically "Pass at X EU/mL" or "Fail at X EU/mL". Always apply any dilution factors used.

// Example calculation (if sample diluted 1:10)
Measured concentration (from assay) = 0.05 EU/mL
Dilution factor = 10
Actual sample concentration = 0.05 x 10 = 0.5 EU/mL

Reminder: Do not publish or apply numeric limits until you reference the applicable pharmacopeial limit or product specification.

Acceptance Criteria

Acceptance criteria depend on the product specification or pharmacopeial chapter. For water systems, labs commonly reference USP/BP/EP monographs or internal limits. Always document the acceptance criteria used for each sample in the test report.

Deviations, Out-of-Specification (OOS) and Investigations

• If negative controls show endotoxin, discard run and investigate contamination sources (reagents, consumables, environment, operator technique).
• If PPC or standard results are out-of-range, repeat entire assay and perform root cause analysis.
• Follow the lab's deviation and CAPA procedures for any OOS result.

Troubleshooting — Common Problems & Fixes

• Unexpected positive in blank: Check reagent lot, glassware cleaning, operator technique, and environmental contamination.
• Inhibition (false negatives): Perform dilution series or PPC to detect matrix effects. Use validated recovery criteria.
• High variability between replicates: Check pipettes, mixing technique, reagent handling and plate reader calibration.
• Standards not linear (kinetic): Prepare fresh standards and verify standard stock concentration.

Best Practices

• Use dedicated endotoxin-free consumables and dedicated pipettes for BET work.
• Document reagent lot numbers, expiry dates and storage conditions for traceability.
• Run internal QC samples daily or per lab schedule to monitor performance.
• Validate method performance (accuracy, precision, specificity, linearity, detection limit) before use for release testing.

Appendix: Template Logbook Entries & Example Report

Include: sample ID, sampling location, date/time, operator, reagent lot numbers, reagent preparation details, standard curve data, raw results, calculated concentrations, pass/fail, signatures.

Sample ID: PW-2025-08-01-01
Location: PW loop - sampling port A
Collected: 2025-08-01 09:10
Operator: S. Siva
LAL reagent lot: LAL-1234 (expiry 2026-02-01)
Method: Kinetic chromogenic
STD curve: 0.05 - 5.0 EU/mL R2=0.998
Result (after dilution): 0.12 EU/mL (pass/fail per spec)

References & Further Reading

Consult the latest pharmacopeia (USP Biological Reactivity / BET chapters), reagent manufacturer manuals, and your laboratory's quality documents when implementing or modifying BET procedures.

💬 About the Author

Siva Sankar is a Pharmaceutical Microbiology Consultant and Auditor with extensive experience in sterility testing, validation, and GMP compliance. He provides consultancy, training, and documentation services for pharmaceutical microbiology and cleanroom practices.

📧 Contact: siva17092@gmail.com
Mobile: 09505626106

📱 Disclaimer: This article is for educational purposes and does not replace your laboratory’s SOPs or regulatory guidance. Always follow validated methods and manufacturer instructions.