title: Fast and sensitive quantification of human liver cytosolic lithocholic acid sulfation using ultra-high performance liquid chromatography-tandem mass spectrometry.
abstract: Detoxification of lithocholic acid (LCA) to lithocholic acid sulfate (LCA-S) is catalyzed by sulfotransferases, mainly SULT2A1. We developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify human liver cytosolic-dependent LCA sulfation. Chromatographic separation was achieved on an UPLC C18 column (2.1×50mm, 1.7μm) and a gradient elution of 0.1% formic acid in water and acetonitrile. Negative electrospray ionization with multiple reaction monitoring (MRM) mode was used to quantify LCA-S (455.3→97.0) and cholic acid (407.2→343.3; internal standard). The retention time was 3.51min for LCA-S and 3.08min for cholic acid. The lower limit of quantification of LCA-S was 0.5nM (or 0.23ng/ml in 400μl total volume) and the assay was linear from 0.2 to 200pmol. Intra-day and inter-day accuracy and precision were <14%. The quality control samples were stable at room temperature for 4h, 4°C for 24h, -20°C for 14 days, and after three freeze-thaw cycles. The matrix (20-100μg cytosolic protein) did not affect LCA-S quantification. This is the first UPLC-MS/MS method applied to optimization of the human liver cytosolic LCA sulfation assay. The optimal levels of MgCl2 and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) cofactor were 2.5mM and 20μM, respectively. Addition of reducing agents (2-mercaptoethanol and DL-dithiothreitol) did not affect LCA-S formation. Human liver cytosolic LCA sulfation was linear with 20-100μg of cytosolic protein and 5-30min incubation time. This UPLC-MS/MS approach offers a specific, sensitive, fast, and direct approach for quantifying human liver cytosolic LCA sulfation.
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; 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*; D003600:Cytosol / Q000737:chemistry*; D004355:Drug Stability; D006801:Humans; D016014:Linear Models; D008095:Lithocholic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D008099:Liver / Q000166:cytology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C014202:sulfolithocholic acid; D008095:Lithocholic Acid
keywords: Human liver cytosol; Lithocholic acid; Lithocholic acid sulfate; Sulfotransferase; Sulfotransferase 2A1; Ultra-high performance liquid chromatography–tandem mass spectrometry