Development and validation of a microsomal online cytochrome P450 bioreactor coupled to solid-phase extraction and reversed-phase liquid chromatography

S.M. van Liempd, J. Kool, J. Reinen, T. Schenk, J.H.N. Meerman, H. Irth, N.P.E. Vermeulen

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

The development and validation of an online cytochrome P450 (CYP)-based bioreactor coupled to automated solid-phase extraction (SPE) and gradient HPLC separation is described. The analytical method was checked on intra- and inter-day repeatability of the ethoxyresorufin-O-demethylation (EROD) reaction with CYP 1Al/1A2 containing beta-NF induced rat liver microsomes as an enzyme source. These experiments showed that CYP activity was linearly decreased with 16% over an 11 h period. Inter-day measurements had a CV of 9.1%. Furthermore, Km and Vmax values of the EROD reaction, measured with the bioreactor, were 2.72 +/- 0.46 microM and 7.9 +/- 0.5 nmol/min/mg protein, respectively. These were in good correspondence with Km and Vmax values, measured with standard batch assay, which amounted 0.66 +/- 0.08 microM and 6.4 +/- 0.2 nmol/min/mg protein respectively. In conclusion the newly developed analytical method can be used effectively and at a microliter scale for online generation, extraction and separation of metabolites.

Original languageEnglish
Pages (from-to)205-212
Number of pages8
JournalJournal of Chromatography A
Volume1075
Issue number1-2
Early online date20 Apr 2005
DOIs
Publication statusPublished - 20 May 2005

Keywords

  • Animals
  • Cytochrome P-450 CYP1A1
  • Kinetics
  • Microsomes, Liver
  • Rats
  • Reproducibility of Results
  • Journal Article
  • Validation Studies

Fingerprint

Dive into the research topics of 'Development and validation of a microsomal online cytochrome P450 bioreactor coupled to solid-phase extraction and reversed-phase liquid chromatography'. Together they form a unique fingerprint.

Cite this