Simultaneous determination of UDCA and its major metabolites in human plasma with surrogate matrix by a rapid and specific LC-MS/MS method

A rapid, convenient, and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of ursodeoxycholic acid (UDCA), and its major metabolites, glycoursodeoxycholic acid (GUDCA) and tauroursodeoxycholic acid (TUDCA) in human plasma. Methanol was chosen as surrogate matrix for preparation the calibrators to establish calibration curves. Isotope internal standard was used for each analyte. After plasma samples were deproteinized with methanol, the post-treatment samples were analyzed on a ZORBAX SB-C18 column (2.1 × 50 mm, 1.8 μm) with 2 mM ammonium acetate and acetonitrile as mobile phase at a flow rate of 0.5 mL/min. Detection was performed on a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) employing negative ESI interface using API5500 triple quadrupole tandem mass spectrometer system, with the transitions set at m/z 391.4 → m/z 391.4, m/z 448.3 → m/z 73.9, m/z 498.4 → m/z 80.1, m/z 395.3 → m/z 395.3, m/z 453.3 → m/z 74.0, and m/z 503.2 → m/z 79.9 for UDCA, GUDCA, TUDCA, UDCA-d4, GUDCA-d5, and TUDCA-d5, respectively. The calibration curve ranges were 5.00-2500 ng/mL for UDCA and GUDCA and 0.500-250 ng/mL for TUDCA. The intra- and inter-day precision was within 7.00% in terms of relative standard deviation (RSD%) and the accuracy within 11.75% in terms of relative error. The selectivity, sensitivity, extraction recovery, matrix effect, dilution reliability, and stability were within the acceptable range. The method was successfully applied to a pharmacokinetic study in 12 healthy Chinese volunteers after oral administration of 250 mg UDCA.