Simultaneous quantitative cassette analysis of drugs and detection of their metabolites by high performance liquid chromatography/ion trap mass spectrometry

A method using high performance liquid chromatography (HPLC) coupled with ion trap mass spectrometry (MS) for simultaneous quantification of multiple drugs and detection of their metabolites is described. The new approach offers a significant increase in analytical throughput and is illustrated with analysis of the in vitro metabolism of 19 alpha-1a receptor antagonists. The compounds were separated into four cassette groups by using a computer program as well as by manual examination. The samples from incubation with dog liver microsomes were pooled into the designed cassette groups and analyzed by HPLC/electrospray (ESI) ion trap MS in full-scan mode. The metabolic stability of the drugs was determined by comparing their signals after incubation for 0 and 60 min, respectively. The quantitative results from the cassette analysis procedure agreed well with those obtained from conventional discrete analysis. In addition, the technique allowed simultaneous detection of metabolites formed during the same incubation without having to reanalyze the samples. The metabolites were first characterized by nominal mass measurement of the corresponding protonated molecules. Subsequent multi-stage tandem mass spectrometry (MS(n)) on the ion trap instrument allowed confirmation of the detected metabolites.