A rapid and sensitive LC–MS/MS-ESI method for the determination of tolvaptan and its two main metabolites in human plasma

• This validated method for tolvaptan and its two main metabolites for use in a human pharmacokinetic study has not previously been reported.
• An RP-HPLC–MS/MS method for tolvaptan and its two main metabolites in vivo was developed and fully validated.
• This method was successfully used to quantify tolvaptan and its two main metabolites in plasma samples from a phase I clinical trial of tolvaptan.
• The specificity and sensitivity, the chromatography conditions, and the sample pre-treatment procedure are reported.
• Compared to previously published methods, this RP-HPLC–MS/MS method is easier to perform, more rapid and has higher sensitivity.

A liquid chromatography–tandem mass spectrometry (LC–MS) method to quantify tolvaptan and its two main metabolites and applied to human study was first developed and validated as a measure of compliance in clinical research. Because of the structure similarity of tolvaptan and its multiple metabolites, the method was optimized to obtain a chromatographic and MS separation of the endogenous interference and isotope ions as well as high analysis throughput. Tolvaptan, its two main metabolites and the internal standard were extracted from human serum (0.1 mL) using solid-phase extraction, separated on a Waters nova-pak C18 column (150 × 3.9 mm, 5 μm) using isocratic elution with a mobile phase composed of acetonitrile, water and formic acid (65:35:0.25, v/v/v). The total run-time was shortened to 3.5 min. The mass transition ranges under positive electrospray ionisation that were monitored for quantitation included m/z 449–252 for tolvaptan, m/z 479–252 for metabolite DM-4103, m/z 481–252 for metabolite DM-4107 and m/z 463–266 for the internal standard (IS). The limit of quantification in plasma for all three analytes was 1 ng/mL. The method was validated over a linear range from 1 to 500 ng/mL for all three analytes with acceptable inter- and intra-assay precision and accuracy. The stability of the analytes was determined to be suitable for routine laboratory practices. The method was successfully applied to samples taken from research volunteers who ingested a 15 mg tolvaptan tablet.