Liquid chromatographic determination of diastereomeric glutathione conjugates and further derivatives of alpha-bromoisovalerylurea in rat bile and urine by electrochemically generated bromine

To study the glutathione conjugation of alpha-bromoisovalerylurea in the rat in vivo, a reversed-phase liquid chromatographic assay of the thioether metabolites in bile and urine was developed. Since alpha-bromoisovalerylurea has a chiral centre, two diastereomeric glutathione conjugates (in bile) and two diastereomeric mercapturates (in urine) can be expected. The separation characteristics of these metabolites and the corresponding cysteine conjugates were investigated. Whereas all thioether metabolites could be separated in one run, optimal separation of the diastereomers required different mobile phases for the glutathione conjugates (in bile) and the mercapturates (in urine). The glutathione conjugates were analysed with the ion-pairing agent sodium decanesulphonate in the mobile phase, but the mercapturates were analysed without an ion-pair-forming agent. For detection, on-line generation of a constant bromine level (100%) was used; bromine-reactive compounds result in a decrease of the amperometric response from the 100% baseline. This technique could be used in continuous automated operation and required little clean-up of the sample. Thus, the diastereomeric glutathione conjugates and mercapturates were quantified in rat bile and urine samples, respectively, by direct injection of the (centrifuged and diluted) samples on the column. The limit of determination of the respective metabolites was 9 and 2.6 ng in bile and urine, respectively. Incubation mixtures of alpha-bromoisovalerylurea with a rat liver cytosolic fraction or with isolated rat hepatocytes were chromatographed after deproteinization with a double volume of methanol. The limit of determination of the diastereomeric glutathione conjugates in the deproteinized incubation samples was 2.0 ng.