An enantiomeric quantitative LC-MS/MS method for tolvaptan and its monohydroxylates in human plasma using a reversed-phase separation procedure

Abstract

Racemic tolvaptan possessing an asymmetric carbon is metabolized to three pairs of monohydroxylate enantiomers of diol form with V2 receptor antagonistic activity via CYP3A. This study aimed to develop a simultaneous quantitative liquid chromatography-tandem mass spectrometry method for 5R- and 5S-tolvaptan and their monohydroxylate enantiomers in human plasma and to apply it to patient samples. Deproteinized plasma specimens were separated using a polysaccharide derivative chiral column in a reversed-phase elution mode. The mass spectrometer was run in positive ion electrospray ionization mode. The chromatographic peaks of tolvaptan monohydroxylate enantiomers were identified by the recombinant CYP3A4/5 digestion of 5R- and 5S-tolvaptan. The calibration curves ranged over the plasma concentrations of 0.25–125 ng/mL for 5R- and 5S-tolvaptan, 0.025–12.5 ng/mL for 4R5R- and 4S5S-diols, and 0.025–38.15 ng/mL for 4S5R-, 4R5S-, 3S5R-, and 3R5S-diols with a large variation. Their pre-treatment recovery rates and matrix factors in human plasma were 85.2–112.9 % and 86.9–113.1 %, respectively. The intra- and inter-day accuracy and imprecision were 92.3–113.8 % and 3.5–14.6 % for all analytes, respectively. The plasma concentration ranges of 5R- and 5S-tolvaptan, 4R5R-, 4S5S-, 4S5R-, 4R5S-, 3S5R-, and 3R5S-diols in heart failure patients with a 5-fold dilution procedure were 0.634–28.4, 2.37–131, 0.525–15.4, 0.0970–4.08, 6.82–108, 0.271–6.49, 0.394–4.18, and 4.81–39.8 ng/mL, respectively. In conclusion, the present method has an acceptable analytical performance level and can be helpful for characterization of the plasma 5R- and 5S-tolvaptan and their monohydroxylate enantiomers in heart failure patients.