An improved method for simultaneous analysis of steroid and phenolic endocrine disrupting chemicals in biological samples

Abstract

An improved method was developed for the simultaneous determination of eight steroid and phenolic endocrine disrupting chemicals, such as oestrone (E1), 17β-oestradiol (E2), oestriol (E3), 17α-ethynylestradoil (EE2), 4-nonylphenol (4-NP), bisphenol A (BPA), 4-tert-octylphenol (4-t-OP) and 4-cumylphenol (4-CP), in biological samples. The optimal extraction and clean-up procedures were investigated using microwave-assisted extraction (MAE), automated gel permeation chromatography (GPC) and solid phase extraction (SPE). As a consequence, the most efficient extraction was achieved by using MAE with methanol as solvent at an extraction temperature of 110°C for 20 min. The clean-up of extracts was carried out by GPC on a Biobeads S-X3 column with cyclohexane/ethyl acetate (1:1, v/v) as mobile phase. Target compounds were eluted in the fraction from 7–14 min retention time. Moreover, the cleanest extracts were obtained by solid phase extraction with C-18 cartridges after the elution with 15 mL ethyl acetate. The final sample extracts were derivatised using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (1% trimethylchlorosilane, TMCS) as derivatisation reagent with pyridine as the solvent. Quantification was performed by gas chromatography-mass spectrometry (GC-MS) with electron ionisation (EI) and selected ion monitoring (SIM) mode. The method was validated by spiked samples which showed good recovery and reproducibility. The overall recoveries ranged between 55.1% and 100.6%, with relative standard deviations (RSD) of 2.3–12.7% for the entire procedure. Method detection limits (MDL) ranged from 0.3 to 0.7 ng g⁻¹ dry weight (dw). Performance of the method was demonstrated by its application on tissues from fish exposed to high concentration of EDCs in the laboratory. The developed method is a promising approach for the analysis of steroid and phenolic endocrine disrupting chemicals in various biological samples.

Keywords:

• endocrine disrupting chemicals
• biological samples
• microwave-assisted extraction
• gel permeation chromatography
• solid phase extraction
• gas chromatography-mass spectrometry

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 20767002), Environmental Protection Commonwealth Scientific Research Foundation of China (No. 200809136), Yunnan Provincial Natural Science Foundation (No. 2007B035M) and State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (No. KF2008-09).