title: 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.
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.
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Pharmacognosy, Karl-Franzens-University Graz, Graz, Austria.
authors.forename: Karin
authors.initials: K
authors.lastname: Ardjomand-Woelkart
authors.forename: Manfred
authors.initials: M
authors.lastname: Kollroser
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Hartmut
authors.initials: H
authors.lastname: Derendorf
authors.forename: Veronika
authors.initials: V
authors.lastname: Butterweck
authors.forename: Rudolf
authors.initials: R
authors.lastname: Bauer
mesh_terms: D000818:Animals; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000097:blood* / Q000378:metabolism; D008297:Male; D015394:Molecular Structure; D004364:Pharmaceutical Preparations / Q000097:blood* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D052616:Solid Phase Extraction; D013237:Stereoisomerism; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D004364:Pharmaceutical Preparations; D003577:Cytochrome P-450 Enzyme System