Method development for effect-directed analysis of endocrine disrupting compounds in human amniotic fluid

The developing fetus represents a highly sensitive period of exposure to endocrine disrupting compounds (EDCs). However, risk assessment of EDCs is hampered by the lack of data on direct in utero exposure. In this study, we developed a robust analytical methodology for the identification of a wide range of known and unknown EDCs in full-term amniotic fluid (AF). First, a method for extraction and fractionation of a broad range of polar and nonpolar EDCs was developed and validated. Maximal recoveries of reference compounds and minimal interference from the matrix were achieved with a combination of solid phase extraction and dispersive liquid/liquid extraction. Bioassay analysis using cell-based reporter gene assays revealed estrogenic, androgenic, and dioxin-like activity in AF extract corresponding to 1.4 nmol EEQ/L, 76.6 pmol DHT-EQ/L, and 10.1 pmol TEQ/L, respectively. Targeted analysis revealed 13 xenobiotics, phytoestrogens, and endogenous hormones in the AF extract that partly contributed to the bioassay activity. Separation of the complex mixture of chemicals in the AF extract with reversed-phase chromatographic fractionation and subsequent bioassay analysis revealed activity in fractions over a wide range of polarity, indicating diverse (unidentified) substances with potential ED activity. The method developed here represents the first methodological step in an effect-directed analysis approach to identify unknown biologically active compounds in the fetal environment.