Determination by high resolution liquid chromatography of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal) and intermediates of its synthesis

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