Introduction

The method for the detection of Gram-negative bacterial endotoxins is based on the gelation of a lysate of amoebocytes (limulus amoebocyte lysate, LAL) from the horseshoe crab, Limulus polyphemus or Limulus tachypleus. The addition of a solution containing endotoxins to a solution of the lysate produces turbidity, precipitation, or gelation of the mixture.

The rate of reaction depends on the concentration of endotoxin, the pH, and the temperature. The reaction requires the presence of certain divalent cations, an enzyme system, and protein capable of clotting, which are provided by the lysate.

The bacterial endotoxin test (BET) is carried out in a manner that avoids microbial contamination.

Before carrying out the test on the preparation to be examined, it is necessary to verify:

- that the equipment used does not adsorb endotoxins;

- the sensitivity of the lysate;

- the absence of interfering factors.

All equipment used must be free of endotoxins.

Recommended procedure

• Unless otherwise prescribed, the solutions and dilutions used in the test are prepared using water BET.

Pre-test. Adjust the solution to be examined to pH 6.5-7.5 using hydrochloric acid (0.1 mol/l) BET, sodium hydroxide (0.1 mol/l) BET, or a suitable buffer, if necessary. To each of the requisite number of chosen receptacles (for example, slides or tubes) add a volume of the lysate and maintain the temperature at 37 ± 1°C. Then add to each receptacle an equal volume of the solution to be examined, mixing immediately and gently with the lysate. Incubate the reaction mixture without vibration, avoiding loss of water by evaporation, for a set period of time that has been determined under experimental conditions (usually 20-60 minutes), and check the results.

A positive result is indicated by the formation of a firm gel and no disintegration when the receptacle is gently inverted. If no such gel is formed the result is negative.

Validation. A cross-validation will need to be performed if the lysate used is different to the ones described in the "Introduction".

Sensitivity of the lysate. Prepare not fewer than four replicate series of two-fold dilutions of endotoxin RS to give concentrations of 2λ, 1λ, 0.5λ, and 0.25λ, where λ is the stated sensitivity of the lysate used. The final dilution in each series must at the minimum give a negative result. Examine the dilutions and a negative control solution consisting of water BET. Calculate the average of the logarithms of the lowest concentration of endotoxin in each series of dilutions giving a positive result. The antilogarithm of this average gives the estimated lysate sensitivity. If the latter does not differ by more than a factor of 2 from the stated sensitivity, the sensitivity is confirmed and is used for all tests performed using the same lysate.

Interfering factors. Prepare as described under "Sensitivity of the lysate", but use untreated specimens of the preparation to be examined in which no endotoxins are detectable to prepare the dilutions of endotoxin RS. Use these specimens at the maximum valid dilution (MVD) calculated from the expression:

both values being expressed in International Units (IU) of endotoxin per millilitre.

When the endotoxin limit concentration is specified in individual monographs in terms of IU of endotoxin per mg or per IU of product, multiply the endotoxin limit by the concentration of the product in the solution tested (in mg or IU of the product per ml of solution) to obtain the endotoxin limit concentration in IU of endotoxin per ml of solution tested. Where relevant, the multiplication applies to a reconstituted solution of the product as stated on the label.

The preparation to be examined may need to be treated if it contains interfering factors and acts as an inhibitor or an activator as determined under experimental conditions. Suitable treatments are dilution, filtration, neutralization, dialysis, or addition of substances that displace adsorbed endotoxins. The sensitivity of the lysate in the presence of the preparation to be examined should not differ by more than a factor of 2 from the sensitivity of the lysate alone. More sensitive lysates permit a greater dilution of the preparation to be examined and may contribute to the elimination of interference.

Interfering factors passing through a filter with a nominal separation limit corresponding to a relative molecular mass of 10000 to 20000 may be separated adequately by ultrafiltration. Asymmetric membrane filters of cellulose triacetate may be used. The presence of components causing false positive results must be determined. The material containing the endotoxins that is retained on the filter is rinsed with water BET or a suitable buffer, and the endotoxins are recovered in the water BET or the buffer. The test volume and the final volume used for the recovery of the endotoxins are determined for each preparation to be examined.

To establish if interfering factors have been eliminated without removing endotoxins, repeat the test using the preparation to be examined, add endotoxin RS, and submit it to the chosen treatment.

Preparation to be examined. Prepare in duplicate as described under "Pre-test", using the maximum valid dilution of the preparation to be examined, this having been treated if necessary for the elimination of interfering factors. At the same time examine a negative control consisting of water BET and two positive controls, both of which contain endotoxin RS at a concentration corresponding to twice the stated sensitivity of the lysate and one of which contains the preparation to be examined at the same concentration as in the test (if necessary treated for the elimination of interfering factors after the addition of the endotoxin standard).

The test is valid if the negative and both positive controls give the appropriate result. The endotoxin limit concentrations of the preparations are given in the individual monograph. The product conforms if it complies with the endotoxin limit concentration. However, compliance with this requirement can only be demonstrated by showing that the endotoxin concentration of the product is less than the endotoxin limit concentration.

The preparation to be examined does not comply with the test if a positive result is found for both test mixtures. If a positive result is found for one test mixture and a negative result for the other, repeat the test.

The preparation to be examined complies with the test if a negative result is found for both test mixtures.