Biomaterial Database

Utility of a column-switching LC/MS/MS method in cytochrome P450 inhibition assays using human liver microsomes. 2013

Dayanidhi Behera, and Rambabu Pattem, and M Siva Selva Kumar, and Girish S Gudi

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS Similar column-switching methods may be used for other isoforms as well and offer a convenient increased analytical throughput in CYP inhibition assays.

UI	MeSH Term	Description	Entries
D008862	Microsomes, Liver	Closed vesicles of fragmented endoplasmic reticulum created when liver cells or tissue are disrupted by homogenization. They may be smooth or rough.	Liver Microsomes,Liver Microsome,Microsome, Liver
D002853	Chromatography, Liquid	Chromatographic techniques in which the mobile phase is a liquid.	Liquid Chromatography
D004791	Enzyme Inhibitors	Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction.	Enzyme Inhibitor,Inhibitor, Enzyme,Inhibitors, Enzyme
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D013058	Mass Spectrometry	An analytical method used in determining the identity of a chemical based on its mass using mass analyzers/mass spectrometers.	Mass Spectroscopy,Spectrometry, Mass,Spectroscopy, Mass,Spectrum Analysis, Mass,Analysis, Mass Spectrum,Mass Spectrum Analysis,Analyses, Mass Spectrum,Mass Spectrum Analyses,Spectrum Analyses, Mass
D013250	Steroid Hydroxylases	Cytochrome P-450 monooxygenases (MIXED FUNCTION OXYGENASES) that are important in steroid biosynthesis and metabolism.	Steroid Hydroxylase,Steroid Monooxygenases,Hydroxylase, Steroid,Hydroxylases, Steroid,Monooxygenases, Steroid
D053719	Tandem Mass Spectrometry	A mass spectrometry technique using two (MS/MS) or more mass analyzers. With two in tandem, the precursor ions are mass-selected by a first mass analyzer, and focused into a collision region where they are then fragmented into product ions which are then characterized by a second mass analyzer. A variety of techniques are used to separate the compounds, ionize them, and introduce them to the first mass analyzer. For example, for in GC-MS/MS, GAS CHROMATOGRAPHY-MASS SPECTROMETRY is involved in separating relatively small compounds by GAS CHROMATOGRAPHY prior to injecting them into an ionization chamber for the mass selection.	Mass Spectrometry-Mass Spectrometry,Mass Spectrometry Mass Spectrometry,Mass Spectrometry, Tandem
D020128	Inhibitory Concentration 50	The concentration of a compound needed to reduce population growth of organisms, including eukaryotic cells, by 50% in vitro. Though often expressed to denote in vitro antibacterial activity, it is also used as a benchmark for cytotoxicity to eukaryotic cells in culture.	IC50,Concentration 50, Inhibitory
D065607	Cytochrome P-450 Enzyme Inhibitors	Drugs and compounds which inhibit or antagonize the biosynthesis or actions of CYTOCHROME P-450 ENZYMES.	Cytochrome P-450 Inhibitors,Cytochrome P-450 Monooxygenase Inhibitors,Cytochrome P-450 Oxygenase Inhibitors,Cytochrome P-450-Dependent Monooxygenase Inhibitors,P-450 Enzyme Inhibitors,P450 Enzyme Inhibitors,Cytochrome P 450 Dependent Monooxygenase Inhibitors,Cytochrome P 450 Enzyme Inhibitors,Cytochrome P 450 Inhibitors,Cytochrome P 450 Monooxygenase Inhibitors,Cytochrome P 450 Oxygenase Inhibitors,Enzyme Inhibitors, P-450,Enzyme Inhibitors, P450,Inhibitors, Cytochrome P-450,Inhibitors, P-450 Enzyme,Inhibitors, P450 Enzyme,P 450 Enzyme Inhibitors,P-450 Inhibitors, Cytochrome