Development and validation of a LC-MS/MS method based on a new 96-well HybridSPETM-precipitation technique for quantification of CYP450 substrates/metabolites in rat plasma

A rapid and selective high throughput HESI-LC-MS/MS method for determining eight cytochrome P450 (CYP) 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^TM-precipitation technique. This novel technology combines the simplicity of precipitation with the selectivity of SPE and hence leads to much cleaner extracts than with conventional procedures. The bioanalytical assay was based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using selected reaction monitoring (SRM) for drug (-metabolite) quantification. All analytes were separated simultaneously in a single run that lasted less than 11min. 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 –9.62–7.48% and –13.84–8.82%. Hence, the present method provides a robust, fast and reproducible analytical tool. The method enables the determination of four major drug metabolising cytochrome P450 (3A4, 2C9, 1A2, and 2D6) enzymes and can be used as a common high throughput analytical assay for in-vivo herb-drug interaction studies.