title: An ultra-high performance liquid chromatography-tandem mass spectrometric assay for quantifying 3-ketocholanoic acid: Application to the human liver microsomal CYP3A-dependent lithocholic acid 3-oxidation assay.
abstract: Lithocholic acid (LCA), a hepatotoxic and carcinogenic bile acid, is metabolized to 3-ketocholanoic acid (3-KCA) by cytochrome P450 3A (CYP3A). In the present study, the objectives were to develop and validate an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify 3-KCA and apply it to the human liver microsomal CYP3A-dependent LCA 3-oxidation assay. Chromatographic separation was achieved on a Waters ACQUITY™ UPLC C18 column (50×2.1mm, 1.7μm) with a gradient system consisting of 0.1% v/v formic acid in water (solvent A) and 0.1% v/v formic acid in acetonitrile (solvent B). The retention time was 3.73min for 3-KCA and 2.73min for cortisol (internal standard). Positive electrospray ionization with multiple reaction monitoring (MRM) mode was used to quantify 3-KCA (m/z 375.4→135.2) and cortisol (m/z 363.5→121.0). The limit of detection of 3-KCA was 10μM, the lower limit of quantification was 33.3μM, and the calibration curve was linear from 0.05-10μM with r(2)>0.99. Intra-day and inter-day accuracy and precision were <13.7%. The quality control samples were stable when assessed after 4h at room temperature, 24h at 4°C, 14days at -20°C, and three freeze-thaw cycles. The liver microsomal matrix did not affect 3-KCA quantification. The amount of KCA formed in the human liver microsomal LCA 3-oxidation assay was linear with respect to the amount of microsomal protein (up to 40μg) and incubation time (5-30min). Enzyme kinetics experiment indicated that LCA 3-oxidation followed the Michaelis-Menten model with an apparent Km of 26±7μM and Vmax of 303±50pmol/min/mg protein. This novel UPLC-MS/MS method for quantifying 3-KCA offers a specific, sensitive, and fast approach to determine liver microsomal LCA 3-oxidation.
authors.affiliation: Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore.
authors.forename: Sumit
authors.initials: S
authors.lastname: Bansal
authors.affiliation: Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore.
authors.forename: Swee Fen
authors.initials: SF
authors.lastname: Chai
authors.affiliation: Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore; Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Electronic address: aikjiang.lau@nus.edu.sg.
authors.forename: Aik Jiang
authors.initials: AJ
authors.lastname: Lau
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D008095:Lithocholic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism*; D010084:Oxidation-Reduction; D015203:Reproducibility of Results
chemical_list: C545376:3-ketocholanoic acid; D008095:Lithocholic Acid; D051544:Cytochrome P-450 CYP3A
keywords: 3-Ketocholanoic acid; 3-Ketolithocholic acid; CYP3A; Human liver microsomes; Lithocholic acid; Ultra-high performance liquid chromatography–tandem mass spectrometry