Simultaneous quantitative analysis of three drugs by high-performance liquid chromatography/electrospray ionization mass spectrometry and its application to cassette in vitro metabolic stability studies

A sensitive and selective liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) method has been developed for the simultaneous quantitative determination of three new chemical entities (NCEs), of the class of aryloxy-substituted aryl piperazines, in rat liver S9 fraction. S9 fraction samples (0.1 mL) were simply extracted with 2% isopropanol in diethyl ether and the extracts analyzed by HPLC with the detection of the analytes in the selective ion recording (SIR) mode. The determination of the analytes was accurate and reproducible, with a limit of quantification of 50 ng/mL for all the analytes in rat liver S9 fraction. The standard calibration curve for the analytes was linear over the concentration range 50-4000 ng/mL. Analysis accuracy and precision over the concentration range were lower than +/-15%. This method offered significant increase in the analytical throughput, which is illustrated by the 'N-in-One' study of metabolic stability of the compounds in rat liver S9 fractions. The quantitative results from the 'N-in-One' procedure correlated well with those obtained from conventional discrete analyses. In addition, the samples were reanalyzed to allow for detection of the metabolites formed during the same incubation. The metabolites were first characterized by nominal mass measurement of the corresponding protonated molecules. Subsequent tandem mass spectrometry allowed confirmation of the detected metabolites.