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.
Methods: A robust LC-MS/MS method was developed to measure low levels of bilirubin glucuronides
accurately from in vitro incubations, as well as stabilizing the analytes during sample preparation and
analysis. The metabolites were quantified using a qualitative/quantitative approach utilizing UV to MS
correction, thereby eliminating the need for synthetic standards.
Results: The method was sensitive enough to quantify mono- and di-glucuronides as low as 3 nM from
in vitro incubations, and kinetic data was determined for total glucuronide formation. The Km and Vmax
values for total bilirubin glucuronide formations were determined to be 0.05 ± 0.01 μM and 181.9 ± 5.3
pmol/min/mg-protein, respectively, in human recombinant UGT1A1, and 0.23 ± 0.05 μM and 875 ± 45
pmol/min/mg protein in human liver microsomes (HLM).
Conclusion: We have developed a sensitive LC-MS/MS based method for the quantitation of bilirubin
and its glucuronides from in vitro incubations. This method was successfully utilized to determine bilirubin
glucuronidation kinetics in HLM and human rUGT1A1.