Development and validation of a microsomal online cytochrome P450 bioreactor coupled to solid-phase extraction and reversed-phase liquid chromatography.
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.
LACDR-Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HVAmsterdam, The Netherlands.
Sebastiaan M
SM
van Liempd
Jeroen
J
Kool
Jelle
J
Reinen
Tim
T
Schenk
John H N a
JH
Meerman
Hubertus
H
Irth
Nico P E
NP
Vermeulen
D000818:Animals; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D015203:Reproducibility of Results
D019363:Cytochrome P-450 CYP1A1