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

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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.

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Acknowledgments

This study was supported by NIEHS (R01 ES002710, P42 ES004699), NIOSH (PHS OH07550) and the German Academic Exchange Service. B.D.H is as senior fellow of the American Asthma Society. We acknowledge Lionel Blanchet for his help with data management.

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Authors and Affiliations

  1. Department of BioMolecular Analysis, VU University Amsterdam, Amsterdam, The Netherlands

    David Falck, Setyo Prihatiningtyas, Jiawen Zhang, Ferry Heus, Jeroen Kool & Wilfried M. A. Niessen

  2. Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis, CA, USA

    Nils Helge Schebb, Christophe Morisseau & Bruce D. Hammock

  3. Institute for Food Toxicology and Chemical Analysis, University of Veterinary Medicine Hannover, Hannover, Germany

    Nils Helge Schebb

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  1. David Falck
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  2. Nils Helge Schebb
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  3. Setyo Prihatiningtyas
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  4. Jiawen Zhang
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  5. Ferry Heus
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  6. Christophe Morisseau
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  7. Jeroen Kool
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  8. Bruce D. Hammock
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  9. Wilfried M. A. Niessen
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Corresponding author

Correspondence to Wilfried M. A. Niessen.

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Falck, D., Schebb, N.H., Prihatiningtyas, S. et al. Development of On-line Liquid Chromatography-Biochemical Detection for Soluble Epoxide Hydrolase Inhibitors in Mixtures. Chromatographia 76, 13–21 (2013). https://doi.org/10.1007/s10337-012-2343-0

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  • DOI: https://doi.org/10.1007/s10337-012-2343-0

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