Abstract
Capillary electrophoresis and UV-visible absorbance detection are used with sample stacking to achieve detection limits ranging from 0.2 to 2 ng/mL (0.8 to 6 nM) for steroids. Stacking is accomplished using negatively charged cyclodextrin steroid-carrier molecules at a discrete pH interface between the reconstituted sample and the separation electrolyte. Steroids are then separated in under 5 min using capillary electrophoresis that incorporates secondary equilibria via sodium dodecyl sulfate and cyclodextrin. The effectiveness of the method for measurements of multiple steroids in limited sample volumes is demonstrated in individual female fish with total circulating blood volumes of 5 μL or less. Steroid recoveries from plasma following a sample processing method developed with commercial extraction cartridges range from 81 to 109 % for 17α,20β-dihydroxy-pregn-4-en-3-one, testosterone, 11-ketotestosterone, estrone, 17β-estradiol, and 17α-ethinyl estradiol. When applied to reproductively active female zebrafish, changes were detected in the levels of circulating steroids as a result of exposure to different solvents and 17β-estradiol.Graphical abstractSteroids are measured in individual zebrafish subject to chemical exposureElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-015-8785-0) contains supplementary material, which is available to authorized users.
Highlights
Endocrine-disrupting chemicals mimic natural hormones and lead to impaired reproduction and adverse health outcomes [1]
This ensured that the maximum number of carboxymethyl β-cyclodextrin carriers accumulated at the interface of the basic stacking buffer and acidic separation buffer
The reported capillary electrophoresis method is amenable to the limited sample volume generated by individual zebrafish and provides a rapid means to measure multiple steroids in the large number of samples required for toxicity testing
Summary
Endocrine-disrupting chemicals mimic natural hormones and lead to impaired reproduction and adverse health outcomes [1]. Studies with zebrafish are integral to assessing the effects of endocrine-disrupting chemicals on human health because genes, development, and the hypothalamic-pituitary-gonadal axis are similar to those of humans [2]. Chemical biomarkers in the fish, such as steroid hormones and proteins, are measured to evaluate endocrine disruption [3]. Circulating steroids are effective to study both genomic and non-genomic mechanisms of action of the endocrine system, because it is well established that reproduction involves synchronized changes in steroidal hormones [4]. Endocrine-disrupting chemicals impact levels of multiple circulating steroidal hormones because they are regulated through interrelated and complex biological pathways. A set of steroid hormones must be monitored in an individual to elucidate the physiological response to toxicants because pooling plasma samples disproportionally normalizes the surges in steroid levels from outliers
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