LC‐MS/MS Method for Probe Drugs in Human Plasma

Abstract

A liquid chromatography‐tandem mass spectrometry method has been developed and validated for the simultaneous determination of probe drugs and their metabolites in human plasma. The analytes include omeprazole and its metabolites omeprazole sulfone and 5‐hydroxyomeprazole; buspirone and its metabolite 1‐ [2‐pyrimidyl] piperazine (1PP); and fexofenadine. These analytes provide indices for the activities of CYPC19, CYP3A4, and OATP1A2. These substrates and metabolites, along with the internal standard lansoprazole, are isolated from plasma using a protein precipitation with acetonitrile. Chromatography was performed using a Luna C18 column eluted with a gradient of 7.5 mM ammonium bicarbonate (pH 8) and acetonitrile. The mass spectrometer was operated in positive ion electrospray mode and multiple reaction monitoring was used for detection and quantification. The method was validated to quantify the concentration ranges 1.0–1000 ng/ml for omeprazole, omeprazole sulfone, 5‐hydroxyomeprazole and fexofenadine, 0.05–100 ng/mL for buspirone, 1.0–100 ng/mL for 1PP. The precision was between 1.2 – 8.2%, and the accuracy ranged from 85.6 – 114% for these analytes. The lower limit of quantification for buspirone was 0.05 ng/mL; whereas the LLOQ for all other analytes was 1 ng/mL. This method provides a fast and highly sensitive analytical tool for quantification of the key analytes from this probe drug cocktail.