Investigation of bioaccumulation profile of oestrogens in zebrafish liver by hollow fibre protected liquid phase microextraction with gas chromatography-mass spectrometric detection.

The applicability of hollow fibre protected liquid phase microextraction (HF-LPME) for the determination of three oestrogens, namely estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) from individual zebrafish liver samples, in a bioaccumulation study on these organisms, is reported. The oestrogens were extracted from single, mechanically crushed and minced livers from fish that were heaved in tubes containing water spiked at low concentration of the analytes. Extraction was performed with ∼3 μL of toluene contained in the hollow fibre. In order to achieve high extraction efficiency, the parameters that could affect the effectiveness of HF-LPME were optimized, i.e. the extracting organic solvent, extraction time, stirring speed and pH of the aqueous phase. For gas chromatography/mass spectrometry (GC/MS) analysis, injection port derivatization of the oestrogens with bis(trimethylsilyl)trifluoroacetamide was conducted. Under the most favourable extraction and derivatization conditions, enrichment factors of 158-279 were obtained. Linearity of the HF-LPME-GC/MS method was evaluated from 1 to 50 μg/L and the coefficient of determination (r²) ranged from 0.9687 to 0.9926. The LODs were between 0.017 and 0.033 μg/L (at a signal to noise ratio of 3) with relative standard deviations (RSDs, analytes spiked at 5 μg/L) of between 15 and 17% (n=3).