Abstract
The quantification of steroid hormones of individual zebrafish (Danio rerio) provides perspective to understand endogenous hormone function. A UPLC–TOF–MS method was developed to provide a reproducible, sensitive, and efficient assay to determine the concentration of steroid hormones, including cortisol, testosterone, androstenedione, 11-deoxycortisol, 11-deoxycorticosterone, and 17-hydroxyprogesterone in whole-body homogenates of each zebrafish. Solid-phase extraction was used to sample matrix clean-up and acquired a recovery from 89.7% to 107.9%. The analytes were separated on an Aquity BEH C18 column using gradient elution. Mass spectrometric analysis was performed by single reaction monitoring (SRM) using positive electrospray ionization mode. The total running time was 6 min, which was greatly shortened compared with a previously reported method. The developed method exhibited excellent linearity for all the analytes, with regression coefficients higher than 0.99. The limit of detection varied between 0.1 and 0.5 ng/L and the limit of quantification was 0.5–1.7 ng/L for all analytes. The precision of the method was assessed on replicate measurements and was found to be in the ranges of 1.9 % to 6.6% and 4.3% to 8.6%, for intra- and inter-day analysis, respectively. This method was validated according to FDA guidance and applied to determine steroid hormone levels in the tissue homogenate of zebrafish acutely treated with caffeine and ethanol.
Highlights
Zebrafish share many developmental aspects with their mammalian counterparts and many features in the endocrine system, including hormones, receptors, and signaling cascades [1,2]
Chromatographic separation is crucial for the performance of an LC–MS analytical system, especially when applied to measure analytes at low concentration ranges, such as steroid hormones in biological matrices
We developed a method based on the solid-phase extraction (SPE) approach, coupled with UPLC–TOF–MS, for the simultaneous quantification of steroid hormones in individual zebrafish homogenates
Summary
Zebrafish share many developmental aspects with their mammalian counterparts and many features in the endocrine system, including hormones, receptors, and signaling cascades [1,2]. The pathway of steroidogenesis has been characterized and a plethora of steroid hormones have been shown to play similar roles compared to those in mammals (Figure 1) [3]. In this species, androgens regulate male sexual differentiation and behavior [5] and estrogens affect anxiety and shoaling behavior [6] in different gender and life stages [7]. The selected six hormones are involved in different putative biosynthesis pathways (Figure 1), which may be helpful in discovering steroid function under acute stress
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