title: A Novel Liquid Chromatography Tandem Mass Spectrometry Method for the Estimation of Bilirubin Glucuronides and its Application to In Vitro Enzyme Assays.
abstract: 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.
  
  
  
  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.
  
  
  
  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).
  
  
  
  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.
authors.affiliation: Pharmaceutical candidate optimization, Biocon BMS R&D center, Syngene international Limited, Biocon Park, Bangalore-560099. India.
authors.forename: Siva P
authors.initials: SP
authors.lastname: Putluru
authors.affiliation: Current affiliations: Center for Drug Evaluation and Research, Office of Clinical Pharmacology, United States Food and Drug Administration, Silver Spring, MD. United States.
authors.forename: Murali K
authors.initials: MK
authors.lastname: Matta
authors.affiliation: Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center, Syngene International Ltd., Bangalore. India.
authors.forename: Deepak
authors.initials: D
authors.lastname: Ahire
authors.affiliation: Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center, Syngene International Ltd., Bangalore. India.
authors.forename: Murali
authors.initials: M
authors.lastname: Subramanian
authors.affiliation: Department of Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., 5 Research Parkway, Wallingford, CT 06492. United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Sinz
authors.affiliation: Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center, Bristol-Myers Squibb India Pvt. Ltd, Bangalore. India.
authors.forename: Sandhya
authors.initials: S
authors.lastname: Mandlekar
mesh_terms: D001663:Bilirubin / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000099148:UGT1A1 Enzyme
chemical_list: D001663:Bilirubin; D014453:Glucuronosyltransferase; D011994:Recombinant Proteins; D000099148:UGT1A1 Enzyme; C016266:bilirubin glucuronate
keywords: Bilirubin glucuronidation; LC-MS/MS method; UGT1A1; enzyme kinetics; hyperbilirubinemia