The measurement of cytochrome P450 (CYP450) isoenzyme inhibition is often done during evaluation of new chemical entities in drug discovery. Typical assay protocol consists of multiple CYP450 probe substrates incubated with selected drug candidates and CYP450. Results of the assay, the amount of probe substrate metabolite formed with respect to control, are used to determine the level of interaction. Liquid chromatography utilizing columns packed with sub-2-micron particles have been shown to provide up to 8X faster analysis time and 3X increases in sensitivity over traditional high-performance liquid chromatography (HPLC). The work presented here shows the development of a high-throughput, sub-2-micron particle LC method coupled with tandem quadrupole mass spectrometry for the rapid analysis of six CYP450 probe substrate metabolites in 30s.

EE was adequately resolved using an unendcapped high-strength silica C(18) column. The average matrix factor in six sources of plasma was 1.14 with a %CV of 4.48. Standard curves were linear with 1/x weighting and r(2) value of 0.999 over three orders of magnitude. Average accuracy for standard curves and quality control samples was 96%. LOD of 0.001 ng/ml was achieved.

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