Analytical and Bioanalytical Chemistry

, Volume 400, Issue 8, pp 2371–2381

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

  • Karin Ardjomand-Woelkart
  • Manfred Kollroser
  • Li Li
  • Hartmut Derendorf
  • Veronika Butterweck
  • Rudolf Bauer
Original Paper

DOI: 10.1007/s00216-010-4618-3

Cite this article as:
Ardjomand-Woelkart, K., Kollroser, M., Li, L. et al. Anal Bioanal Chem (2011) 400: 2371. doi:10.1007/s00216-010-4618-3

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.

https://static-content.springer.com/image/art%3A10.1007%2Fs00216-010-4618-3/MediaObjects/216_2010_4618_Figa_HTML.gif
Figure

The new used 96 well Hybrid-SPE™-precipitation plate for plasma extraction.

Keywords

Hybrid-SPE™ precipitationValidationPhospholipidsCYP450

Supplementary material

216_2010_4618_MOESM1_ESM.pdf (829 kb)
ESM 1(PDF 828 kb)

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Karin Ardjomand-Woelkart
    • 1
  • Manfred Kollroser
    • 2
  • Li Li
    • 3
  • Hartmut Derendorf
    • 3
  • Veronika Butterweck
    • 3
  • Rudolf Bauer
    • 1
  1. 1.Institute of Pharmaceutical Sciences, Department of PharmacognosyKarl-Franzens-University GrazGrazAustria
  2. 2.Institute of Forensic MedicineMedical University of GrazGrazAustria
  3. 3.Department of Pharmaceutics, College of PharmacyUniversity of FloridaGainesvilleUSA