Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
7616034 | Journal of Chromatography B | 2016 | 8 Pages |
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 Ã 2.1 mm, 1.7 μm) 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 3.73 min for 3-KCA and 2.73 min for cortisol (internal standard). Positive electrospray ionization with multiple reaction monitoring (MRM) mode was used to quantify 3-KCA (m/z 375.4 â 135.2) and cortisol (m/z 363.5 â 121.0). The limit of detection of 3-KCA was 10 μM, the lower limit of quantification was 33.3 μM, and the calibration curve was linear from 0.05-10 μM with r2 > 0.99. Intra-day and inter-day accuracy and precision wereâ<13.7%. The quality control samples were stable when assessed after 4 h at room temperature, 24 h at 4 °C, 14 days at â20 °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 40 μg) and incubation time (5-30 min). Enzyme kinetics experiment indicated that LCA 3-oxidation followed the Michaelis-Menten model with an apparent Km of 26 ± 7 μM and Vmax of 303 ± 50 pmol/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.
Keywords
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Sumit Bansal, Swee Fen Chai, Aik Jiang Lau,