Chromatographia

, Volume 76, Issue 1–2, pp 13–21 | Cite as

Development of On-line Liquid Chromatography-Biochemical Detection for Soluble Epoxide Hydrolase Inhibitors in Mixtures

  • David Falck
  • Nils Helge Schebb
  • Setyo Prihatiningtyas
  • Jiawen Zhang
  • Ferry Heus
  • Christophe Morisseau
  • Jeroen Kool
  • Bruce D. Hammock
  • Wilfried M. A. Niessen
Original

Abstract

In this study, an end-point-based fluorescence assay for soluble epoxide hydrolase (sEH) was transformed into an on-line continuous-flow format. The on-line biochemical detection system (BCD) was coupled on-line to liquid chromatography (LC) to allow mixture analysis. The on-line BCD was based on a flow system wherein sEH activity was detected by competition of analytes with the substrate hydrolysis. The reaction product was measured by fluorescence detection. In parallel to the BCD data, UV and MS data were obtained through post-column splitting of the LC effluent. The buffer system and reagent concentrations were optimized resulting in a stable on-line BCD with a good assay window and good sensitivity (S/N > 60). The potency of known sEH inhibitors (sEHis) obtained by LC–BCD correlates well with published values. The LC–BCD system was applied to test how oxidative microsomal metabolism affects the potency of three sEHis. After incubation with pig liver microsomes, several metabolites of sEHis were characterized by MS, while their individual potencies were measured by BCD. For all compounds tested, active metabolites were observed. The developed method allows for the first time the detection of sEHis in mixtures providing new opportunities in the development of drug candidates.

Keywords

Column liquid chromatography On-line screening Bioassays Enzyme inhibition Soluble epoxide hydrolase Metabolism 

Supplementary material

10337_2012_2343_MOESM1_ESM.pdf (83 kb)
Supplementary material 1 (PDF 84 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • David Falck
    • 1
  • Nils Helge Schebb
    • 2
    • 3
  • Setyo Prihatiningtyas
    • 1
  • Jiawen Zhang
    • 1
  • Ferry Heus
    • 1
  • Christophe Morisseau
    • 2
  • Jeroen Kool
    • 1
  • Bruce D. Hammock
    • 2
  • Wilfried M. A. Niessen
    • 1
  1. 1.Department of BioMolecular AnalysisVU University AmsterdamAmsterdamThe Netherlands
  2. 2.Department of Entomology and UC Davis Comprehensive Cancer CenterUniversity of CaliforniaDavisUSA
  3. 3.Institute for Food Toxicology and Chemical Analysis, University of Veterinary Medicine HannoverHannoverGermany

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