A Novel Liquid Chromatography Tandem Mass Spectrometry Method for the Estimation of Bilirubin Glucuronides and its Application to In Vitro Enzyme Assays

Background: Bilirubin is a toxic waste product of metabolism, eliminated mainly through UGT1A1 mediated conjugation to mono- and di-glucuronides. Due to the potentially low Km value of bilirubin glucuronidation, the quantitative sensitivity obtained with most UV/visible light detection methods are not sufficient to accurately calculate UGT1A1 enzyme kinetics at low bilirubin concentrations. In addition, bilirubin, as well as its metabolites, are unstable during sample preparation and bioanalysis. This necessitates the need for a rapid, sensitive and robust assay to measure bilirubin glucuronides. Keywords: Bilirubin glucuronidation, enzyme kinetics, hyperbilirubinemia, LC-MS/MS method, UGT1A1.