Fast and sensitive quantification of human liver cytosolic lithocholic acid sulfation using ultra-high performance liquid chromatography–tandem mass spectrometry

•UPLC–MS/MS analysis of human liver cytosolic LCA sulfation.•The matrix (human liver cytosol) did not interfere with the assay.•The assay was optimized for Mg, DTT, 2-mercaptoethanol, and PAPS concentrations.•The assay was linear with respect to amount of enzyme and incubation time.•Faster and more sensitive than existing LC–MS or radiometric methods.

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 × 50 mm, 1.7 μm) 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 3.51 min for LCA-S and 3.08 min for cholic acid. The lower limit of quantification of LCA-S was 0.5 nM (or 0.23 ng/ml in 400 μl total volume) and the assay was linear from 0.2 to 200 pmol. Intra-day and inter-day accuracy and precision were <14%. The quality control samples were stable at room temperature for 4 h, 4 °C for 24 h, −20 °C for 14 days, and after three freeze–thaw cycles. The matrix (20–100 μg 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 2.5 mM and 20 μM, respectively. Addition of reducing agents (2-mercaptoethanol and DL-dithiothreitol) did not affect LCA-S formation. Human liver cytosolic LCA sulfation was linear with 20–100 μg of cytosolic protein and 5–30 min incubation time. This UPLC–MS/MS approach offers a specific, sensitive, fast, and direct approach for quantifying human liver cytosolic LCA sulfation.