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

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

Volume: 10 Issue: 4

Author(s): Siva P. Putluru*, Murali K. Matta, Deepak Ahire, Murali Subramanian, Michael Sinz and Sandhya Mandlekar

Affiliation:

• Pharmaceutical candidate optimization, Biocon BMS R&D center, Syngene international Limited, Biocon Park, Bangalore-560099,India

Keywords: Bilirubin glucuronidation, enzyme kinetics, hyperbilirubinemia, LC-MS/MS method, UGT1A1.

Abstract: 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.

Cite this article as:

Putluru P. Siva*, Matta K. Murali, Ahire Deepak, Subramanian Murali, Sinz Michael and Mandlekar Sandhya, A Novel Liquid Chromatography Tandem Mass Spectrometry Method for the Estimation of Bilirubin Glucuronides and its Application to In Vitro Enzyme Assays, Drug Metabolism Letters 2016; 10 (4) . https://dx.doi.org/10.2174/1872312810666161124143522

DOI https://dx.doi.org/10.2174/1872312810666161124143522	Print ISSN 1872-3128
Publisher Name Bentham Science Publisher	Online ISSN 1874-0758