Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization.
We describe the development and validation of a high-resolution screening (HRS) platform which couples gradient reversed-phase high-performance liquid chromatography (RP-HPLC) on-line to estrogen receptor alpha (ERalpha) affinity detection using fluorescence polarization (FP). FP, which allows detection at high wavelengths, limits the occurrence of interference from the autofluorescence of test compounds in the bioassay. A fluorescein-labeled estradiol derivative (E2-F) was synthesized and a binding assay was optimized in platereader format. After subsequent optimization in flow-injection analysis (FIA) mode, the optimized parameters were translated to the on-line HRS bioassay. Proof of principle was demonstrated by separating a mixture of five compounds known to be estrogenic (17beta-estradiol, 17alpha-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone), followed by post-column bioaffinity screening of the individual affinities for ERalpha. Using the HRS-based FP setup, we were able to screen affinities of off-line-generated metabolites of zearalenone for ERalpha. It is concluded that the on-line FP-based bioassay can be used to screen for the affinity of compounds without the disturbing occurrence of autofluorescence.
Reinen
Jelle
J
Department of Chemistry and Pharmaceutical Sciences, LACDR-Division of Molecular Toxicology, Vrije Universiteit, De Boelelaan 1083, 1081 HV, Amsterdam, The Netherlands.
Kool
Jeroen
J
Vermeulen
Nico P E
NP
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D004958:Estradiol / Q000032:analysis / Q000737:chemistry; D047628:Estrogen Receptor alpha / Q000737:chemistry*; D004967:Estrogens / Q000032:analysis*; D017022:Flow Injection Analysis / Q000295:instrumentation / Q000379:methods; D019793:Fluorescein / Q000737:chemistry; D005454:Fluorescence Polarization / Q000295:instrumentation / Q000379:methods; D008024:Ligands; D008862:Microsomes, Liver / Q000737:chemistry; D008968:Molecular Conformation; D015336:Molecular Probe Techniques / Q000295:instrumentation; D010455:Peptides / Q000737:chemistry; D051381:Rats; D015203:Reproducibility of Results; D013237:Stereoisomerism
D047628:Estrogen Receptor alpha; D004967:Estrogens; D008024:Ligands; D010455:Peptides; D004958:Estradiol; D019793:Fluorescein