Determination by Liquid Chromatography High Resolution 5-bromo-4-chloro-3-indolyl-b -D-galactopyranoside (Xgal) and the intermediaries of its synthesis

Abstract

In microbiology, microorganisms markers have been found to be useful in the quality control of different
products such as water and food, in which the presence of coliforms, especially E. coli has been identified. Several methods
for detection and quantification of enterobacteria have been developed by using chromogenic substrates. Among
them, indole derivatives such as 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal), have come out. This indole
derivative is widely employed as a constituent of microbiological diagnostic media because in the presence of coliforms
(β-galactosidase producers), it forms a blue-indigo color. In this paper, the development and the validation, of an HPLC
method for the quality control of Xgal in the presence of its intermediates obtained in each step of the synthesis is reported.
These compounds were separated by using a RP-18 column and gradient elution with a mobile phase consisting
of triethylammonium phosphate solution at pH 3(A) and acetonitrile (B). The detection and quantitation limits of the
method was between 0.2 – 0.5 % and 1.0 – 2.0 % respectively, which is acceptable if we consider that Xgal is not used as a
drug. The proposed method is simple, selective, linear, precise and accurate and it can be used for the quality control of
each of the intermediates and the final product, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.