High-performance liquid chromatographic quantification of 4-methylumbelliferyl-beta-D-glucuronide as a probe for human beta-glucuronidase activity in tissue homogenates

An internally standardized HPLC method to determine the concentration of 4-methylumbelliferone liberated from 4-methylumbelliferyl-beta-D-glucuronide by human beta-glucuronidase was developed. The assay allows the precise and rapid measurement of specific Enzyme activity in human tissue homogenates. Without prior extraction the incubation mixture can be separated using a C8 column followed by fluorescence detection. The assay showed good accuracy and precision with a detection limit of 20 nM and a limit of quantification of 167 nM. The suitability of the method was shown in Enzyme kinetic experiments with human liver homogenates.