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Publication Date: 2016-Jul-25
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A Bacterial Endotoxin Test (BET) method has been developed using gel clot method for alkaline substances and is applicable for highly alkaline substances wherein the BET limits are as low as 0.0025 EU/mg.

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Disclosed herein is a Bacterial Endotoxin Test (BET) method for alkaline substances using gel clot method. The method is simple, cost effective and is applicable for highly alkaline substances wherein the BET limits are as low as < 0.0025 EU/mg.

Kinetic turbidometry assay and kinetic chromogenic assay are amongst the usual methods that are followed for BET, especially for substances having low BET limits of the order of < 0.0025 EU/mg. However, these methods necessitate high investment costs.

Gel clot method, on the other hand, is an easy and cost effective but poses serious limitations for alkaline substances having low BET limits, due to the constraints of maximum valid dilution (MVD) limits.

The procedure for Bacterial Endotoxin Test (BET) for alkaline substances using gel clot method described herein is applicable to inorganic alkaline substrates such as sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate as well organic substrates like Tromethamine.

The method has following advantages

a) The present method is simple and cost efficient as compared to high investment methods such as kinetic turbidometry assay or kinetic chromogenic assay.

b) The method offers full recovery of bacterial endotoxins for substrates having low BET limits and accordingly very low MVD limits.

c) The method which involves controlled dilution but avoids decomposition provides for complete recovery of endotoxins.

d) Various organic and inorganic alkaline substrates having pH in the range of 11-12 can be tested for BET using above method.

Example 1: Bacterial Endotoxin Test for Sodium carbonate Requirements:

Control Standard Endotoxin (CSE)

Limulus amoebocyte Lysate (Lysate)

LAL Reagent water (LRW)
200 µl micropipette (range 20 to 200 µl) with pyrogen free tips 1000 µl micropipette (range 200 to 1000 µl) with pyrogen free tips Depyrogenated glass tubes (10 x 75 mm and 16 x 100 mm) Vortex mixer

Heating block incubator set at 37°C  1°C


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Sterilized and depyrogenated glassware Procedure:

A.Reconstitution of LAL reagent

Remove aluminum seal of the Lysate vial without opening rubber stopper and carefully remove stopper. Reconstitute the lyophilized powder using required quantity of LAL reagent water (LRW) with the help of micropipettes with pyrogen free tips. Stopper the reconstituted vial and mix gently without formation of air bubble. Store reconstituted LAL reagent vial at 2 to 8°C.

B.Reconstitution of Control Standard Endotoxin (CSE)

Reconstitute the lyophilized control standard Endotoxin (E. coli O55:B5) with LRW as per manufacturer's instructions. Vortex intermittently for 15 minutes and use this concentrate for making appropriate serial dilutions as described under "Preparation of Standard CSE Dilution".

C.Preparation of Standard CSE Dilution

Mix vigorously the reconsti...