Abstract
A rapid and selective high-throughput HESI-LC-MS/MS method for determining eight cytochrome P450 probe drugs in one-step extraction and single run was developed and validated. The four specific probe substrates midazolam, dextromethorphan, tolbutamide, theophylline and their metabolites 1-hydroxymidazolam, dextrorphan, hydroxyl(methyl)tolbutamide, 1,3-dimethyluric acid, together with the deuterated internal standards, were extracted from rat plasma using a novel 96-well Hybrid-SPE™-precipitation technique. The bioanalytical assay was based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using selected reaction monitoring for drug (-metabolite) quantification. All analytes were separated simultaneously in a single run that lasted less than 11 min. The intra- and inter-day precisions for all eight substrates/metabolites were 1.62–12.81% and 2.09–13.02%, respectively, and the relative errors (accuracy) for the eight compounds ranged from −9.62% to 7.48% and −13.84% to 8.82%. Hence, the present method provides a robust, fast and reproducible analytical tool for the evaluation of four major drug metabolising cytochrome P450 (3A4, 2C9, 1A2 and 2D6) activities with a cocktail approach in rats to clarify herb–drug interactions. The method can be used as a basic common validated high-throughput analytical assay for in vivo interaction studies.
The new used 96 well Hybrid-SPE™-precipitation plate for plasma extraction.
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Acknowledgement
The Austrian Science Fund (FWF) is thanked for the award of an Erwin-Schroedinger scholarship to K. Ardjomand-Woelkart (J2754-B05).
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Published in the special issue Analytical Sciences in Austria with Guest Editors G. Allmaier, W. Buchberger and K. Francesconi.
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Ardjomand-Woelkart, K., Kollroser, M., Li, L. et al. Development and validation of a LC-MS/MS method based on a new 96-well Hybrid-SPE™-precipitation technique for quantification of CYP450 substrates/metabolites in rat plasma. Anal Bioanal Chem 400, 2371–2381 (2011). https://doi.org/10.1007/s00216-010-4618-3
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DOI: https://doi.org/10.1007/s00216-010-4618-3