Utility of a column-switching LC/MS/MS method in cytochrome P450 inhibition assays using human liver microsomes

Abstract

Liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based in vitro cytochrome P450 (CYP) inhibition assays in pooled human liver microsomes using therapeutically relevant probe drugs are recommended by the US Food and Drug Administration to assess the potential for drug-drug interactions. As these assays are used routinely in pharmaceutical drug discovery screening of new chemical entities for drug interaction liabilities, there is a need to have higher analytical throughput. Column-switching methods may offer increased chromatographic throughput while maintaining the quality of data generated. In this study, the CYP3A4 inhibition assay was used as a potential application to demonstrate the performance of a dual-column parallel chromatographic system in a column-switching mode. Testosterone 6β-hydroxylation was monitored and IC50 values of known CYP3A4 inhibitors were determined using conventional as well as column-switching LC/MS/MS methods. Mean IC50 values of ketoconazole, itraconazole and verapamil were 0.056, 0.061 and 23 μM (conventional method) compared to 0.05, 0.057 and 26 μM (column-switching method), respectively. The two different chromatographic methods resulted in IC50 values that were not statistically different and were within a twofold range, demonstrating reproducibility of results. Further, the column-switching method saved nearly 50% of analytical time in comparison to the conventional chromatographic method, indicating increased throughput leading to better utilization of mass spectrometer time without compromising the quality of data. Similar column-switching methods may be used for other isoforms as well and offer a convenient increased analytical throughput in CYP inhibition assays.