The development of an optimized sample preparation for trace level detection of 17α-ethinylestradiol and estrone in whole fish tissue.

The purpose of this study was to develop an optimized method for the extraction and determination of 17α-ethinylestradiol (EE2) and estrone (E1) in whole fish tissues at ng/g levels. The optimized procedure for sample preparation includes extraction of tissue by accelerated solvent extraction (ASE-200), lipid removal by gel permeation chromatography (GPC), and a cleanup step by acetonitrile precipitation followed by a hexane wash. Analysis was performed by gas chromatography/mass spectrometry (GC/MS) in negative chemical ionization (NCI) mode after samples were derivatized with pentafluorobenzoyl chloride (PFBCl). The method was developed using high lipid content wild fish that were exposed to the tested analytes. The whole procedure recoveries ranged from 74.5 to 93.7% with relative standard deviation (RSD) of 2.3-6.2% for EE2 and 64.8 to 91.6% with RSD of 9.46-0.18% for E1. The method detection limits were 0.67 ng/g for EE2 and 0.68 ng/g for E1 dry weight. The method was applied to determine EE2 levels in male goldfish (Carrasius auratus) after a 72 h dietary exposure. All samples contained EE2 averaging 1.7ng/g (±0.29 standard deviation, n=5). This is the first optimized protocol for EE2 extraction from whole fish tissue at environmentally relevant concentrations. Due to high sensitivity and recovery, the developed method will improve our knowledge about the environmental fate and uptake of synthetic steroidal estrogens in fish populations.