💊 Bacterial endotoxins test (LAL)

Pharma Ph.Eur. 2.6.14

Overview

STATUS (as of 03.11.2026). General chapter Ph. Eur. 2.6.14 “Bacterial endotoxins” applies in the wording of the 12th edition of the European Pharmacopoeia (version 12.3, in force from 1.07.2026). The text is harmonised in the Pharmacopoeial Discussion Group with chapter JP 4.01 and USP <85>; according to guideline ICH Q4B Annex 14 (Step 4, 18.10.2012), the texts of the three pharmacopoeias can be used interchangeably, and their endotoxin reference standards — calibrated against the WHO International Standard — are considered equivalent. The PDG sign-off cover sheet of April 2026 adds to the European text — as a non-harmonised part — method G: a fluorimetric end-point method using recombinant factor C (rFC). In Issue 13.1 (in force from 1.01.2027), chapter 2.6.14 is revised, and chapter 2.6.32 on the test for bacterial endotoxins using recombinant factor C is suppressed as of that date. The description below gives the harmonised content of the chapter (PDG version of April 2026).

PRINCIPLE AND TECHNIQUES. The test detects or quantifies endotoxins from Gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). The gel-clot technique is based on gel formation, the turbidimetric technique on the development of turbidity after cleavage of an endogenous substrate, and the chromogenic technique on the development of colour after cleavage of a synthetic peptide–chromogen complex. Any of the three techniques can be used; in the event of doubt or dispute, the gel-clot limit test decides, unless the product monograph indicates otherwise. Endotoxin is expressed in endotoxin units (EU); one International Unit (IU) of endotoxin equals 1 EU. The lysate also reacts to some β-glucans — in the presence of glucans, lysates without the G factor or with an inhibited G factor reacting system can be used.

APPARATUS AND REAGENTS (clauses 1–5). Glassware and other heat-stable materials are depyrogenated in a hot-air oven by a validated process — commonly at least 30 min at 250 °C; plastic apparatus (microplates, pipette tips) must be free of detectable endotoxin and must not interfere with the test. The test is carried out in a manner that avoids endotoxin contamination. The lysate is dissolved by gentle stirring in water for bacterial endotoxins test or in a buffer recommended by the manufacturer; the water for the test is water for injections or other water that shows no reaction with the lysate employed at the detection limit of the reagent. The standard endotoxin stock solution is prepared from an endotoxin reference standard calibrated against the current WHO International Standard, and dilutions are made in water for the test and used as soon as possible. The pH of the mixture of lysate and sample solution must fall within the range specified by the lysate manufacturer, usually 6.0–8.0.

MAXIMUM VALID DILUTION AND LIMIT (clause 6). MVD = endotoxin limit × concentration of sample solution / λ, where λ is the labelled lysate sensitivity in the gel-clot technique or the lowest concentration of the standard curve in the turbidimetric and chromogenic techniques. The endotoxin limit for parenteral drugs, defined on the basis of dose, equals K/M: K is the threshold pyrogenic dose of endotoxin per kilogram of body weight, M is the maximum recommended bolus dose of product per kilogram of body weight and, when the product is injected at frequent intervals or infused continuously, the maximum total dose administered in a single hour. The limit for a product is given in the individual monograph, e.g. in EU/ml or EU/mg.

GEL-CLOT TECHNIQUE (clause 7). The labelled lysate sensitivity λ is confirmed in four replicates with solutions of 2λ, λ, 0.5λ and 0.25λ for each new lot of lysate and whenever conditions change; equal volumes of lysate solution and standard solution (e.g. 0.1 ml each) are incubated usually for 60 ± 2 min at 37 ± 1 °C avoiding vibration, and the tube is inverted through about 180° in one smooth motion — a firm gel is a positive result. The sensitivity is confirmed when the geometric mean of the end-point concentrations lies within 0.5λ–2λ. The test for interfering factors (solutions A–D of Table 1) is performed on a sample free of detectable endotoxins at a dilution less than the MVD; interference is overcome by a greater dilution not exceeding the MVD, a more sensitive lysate or a treatment (filtration, neutralisation, dialysis, heating), checking that the treatment does not cause loss of endotoxins. In the limit test, the sample complies when both replicates of solution A are negative, with positive controls B and C and a negative control D; a discordant result requires a repeat test. The quantitative test determines endotoxins by titration to an end point in a dilution series of 1, 1/2, 1/4 and 1/8.

PHOTOMETRIC TECHNIQUES (clause 8). The turbidimetric and chromogenic techniques, each in an end-point or kinetic variant (time needed to reach a predetermined absorbance, or rate of change), usually at 37 ± 1 °C. For each lot of lysate, the standard curve is generated from at least three concentrations in at least three replicates, and the absolute value of the correlation coefficient must be at least 0.980; if the range is greater than two logs in the kinetic methods, additional standards are included. The recovery of endotoxin added to the sample must be 50–200 %. The sample complies when the mean endotoxin concentration of solution A, after correction for dilution, is less than the limit for the product.

WHAT WE DO NOT GIVE HERE. The lettering of the methods in the European text and the content of its non-harmonised part (method G with recombinant factor C, chapters 5.1.10 and 5.1.13) — we did not have the text of the Ph. Eur. The values of K for individual routes of administration and the endotoxin limits for specific products, including water for injections — they are given in pharmacopoeial monographs and guidelines whose text we did not have.

Method principle

Endotoxins from Gram-negative bacteria activate the horseshoe crab amoebocyte lysate. In the gel-clot technique the lysate clots, in the turbidimetric technique turbidity develops after cleavage of an endogenous substrate, and in the chromogenic technique colour develops after cleavage of a synthetic peptide–chromogen complex. The result is compared with the labelled lysate sensitivity or with a standard curve of an endotoxin standard calibrated against the WHO International Standard; in case of dispute, the gel-clot limit test decides.

Applications

• Detection or quantification of endotoxins from Gram-negative bacteria in medicinal products (introduction of the chapter)
• Medicinal products for parenteral administration — endotoxin limit K/M set on the basis of dose and given in the individual monograph (clause 6)
• Choice of the sample dilution up to the maximum valid dilution (MVD) and test for interfering factors before the test (clauses 6–8)

Key parameters

Standard

Standard number

European Pharmacopoeia 2.6.14

Title (PL)

Endotoksyny bakteryjne

Title (EN)

Bacterial endotoxins

Step-by-step procedure

1. Preparation of apparatus

   Depyrogenate glassware by a validated process (commonly at least 30 min at 250 °C); use only plastic apparatus free of detectable endotoxin (clause 1).

2. Sample solution and MVD

   Dissolve or dilute the sample with water for the test; if necessary, adjust the pH so that the mixture with the lysate has the pH recommended by the manufacturer (usually 6.0–8.0); calculate MVD = limit × concentration of sample solution / λ (clauses 5, 6).

3. Confirmation of lysate sensitivity

   For each new lot of lysate and whenever conditions change, test solutions of 2λ, λ, 0.5λ and 0.25λ in four replicates; the geometric mean of the end points must lie within 0.5λ–2λ (clause 7).

4. Interfering factors

   Test solutions A–D (Table 1 or 4) on a sample diluted not beyond the MVD; overcome interference by a greater dilution, a more sensitive lysate or a treatment, and check that the treatment does not cause loss of endotoxins (clauses 7, 8).

5. Test

   Gel-clot technique: equal volumes of lysate and solution, usually 60 ± 2 min at 37 ± 1 °C, then invert the tube through about 180°. Photometric techniques: according to the lysate instructions, with a standard curve of |r| ≥ 0.980 and a recovery of 50–200 % (clauses 7, 8).

6. Evaluation

   Limit test: both replicates of solution A negative — the sample complies; both positive — it does not comply; discordant — repeat the test. Quantitative techniques: the mean concentration in solution A after correction for dilution must be less than the limit for the product (clauses 7, 8).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The test is carried out in a manner that avoids endotoxin contamination (introduction of the chapter)
• The available text of the chapter contains no health and safety requirements — rules for safe work with reagents and samples are set by the laboratory

Frequently asked questions