Reversed-Phase Liquid Chromatography Coupled On-Line to Receptor Affinity Detection Based on the Human Estrogen Receptor

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Abstract
On-line coupling of reversed-phase liquid chromatography to a biochemical detection system based on receptor-ligand interactions is described. The receptor affinity detection is performed using a postcolumn reaction detection system with open-tubular reaction coils. In the first step, a solution containing the steroid binding domain of the human estrogen receptor is added to the LC effluent via a mixing union, and the mixture is allowed to react. In the second step, a fluorescent estrogenic ligand, coumestrol, is added to saturate the remaining free binding sites of the estrogen receptor. Both heterogeneous and homogeneous detection systems have been developed. In the former case, free and receptor-bound coumestrol were separated prior to detection by means of a short column containing a restricted-access reversed-phase support. Since free and receptor-bound coumestrol differ significantly in fluorescence intensity, the system can also be operated in the homogeneous mode without requiring a separation step. Using 5 nmol/L of human estrogen receptor, a detection limit of 5 nmol/L was obtained for compounds possessing a high affinity for the estrogen receptor, such as 17 beta-estradiol and diethylstilbestrol. Detection limits for weaker ligands amount to 20 nmol/L. Non-estrogenic steroids such as methyltestosterone or progesterone are not detected at all. The selectivity of the present method is demonstrated for the analysis of urine samples.