Improved application of recombinant yeast assays on environmental samples by size exclusion chromatography

The utility of HPLC with a size exclusion chromatography (SEC) column for the fractionation of environmental samples was examined to expand the applicability of recombinant yeast assays (RYAs) for the measurement of estrogenic activity in environmental samples. The elution of steroidal hormone standards through SEC was tested, and river water and sediment samples were subjected to SEC fractionation. The retention times of all estrogen standards were longer than those of the major components with absorbance at 230 nm of the environmental sample matrix. The estrogenic activity of SEC fractions of environmental samples from highly polluted sites was measured using RYA and revealed the existence of estrogenic compounds that could not be detected or quantified in extracted samples before fractionation. The fractions from environmental samples that corresponded to the retention time of estrone, 17[small beta]-estradiol, and estriol were analyzed with LC/MS/MS. These three estrogens were separated into three different fractions, and the concentration of estrone coincided with the estrogenic activity of the fraction in which it was detected. The profile of estrogenic activity of SEC fractions indicated the molecular size of the estrogenic compounds and the yeast growth inhibitors in samples.