UPLC–TOF–MS Method for Simultaneous Quantification of Steroid Hormones in Tissue Homogenates of Zebrafish with Solid-Phase Extraction

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.

Spatially Resolved Metabolomics Method for Mapping the Global Molecular Landscape of Whole-Body Zebrafish (Danio rerio) Using Ambient Mass Spectrometry Imaging

“…The genome reference sequence of zebrafish has been acquired, providing a high-quality sequence assembly generated following methods used for human and mouse genome sequencing. , Several studies have also mapped the global proteomic profiles of zebrafish adults and embryos, providing an important proteome resource for understanding developmental processes and recapitulating disease mechanisms. − In recent years, research on the combination of the zebrafish model and metabolomics has increased. , However, the majority focused on lipid metabolites due to the significant connection between lipid metabolism and disease processes. And for adult zebrafish, only individually dissected tissues were analyzed. − In addition, metabolomics applying platforms such as liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR) do not provide in situ distribution information of metabolites.…”

Epidermal oxysterols function as alarm substances in zebrafish

“…The method developed and validated by our laboratory was used to extract and determine the whole-body cortisol of zebrafish. 64 Briefly, the fish were euthanized using tricaine at a concentration of 500 mg/L in the ice bath, removed of excessive water using paper towels, frozen in liquid nitrogen, and stored at –80°C. To process the samples, the fish were thawed, weighed, diced, and then homogenized at 60 Hz for 90 seconds.…”

“…The UPLC-MS method validated following FDA guidelines was used to accurately measure cortisol concentration in whole-body homogenates of zebrafish. 64 …”

Novel Epidermal Oxysterols Function as Alarm Substances in Zebrafish

“…The method developed and validated by our laboratory was used to extract and determine the whole-body cortisol of zebrafish 59 . Briefly, the fish were euthanized using tricaine at a concentration of 500 mg/L in the ice bath, removed of excessive water using paper towels, frozen in liquid nitrogen, and stored at -80°C.…”

“…The concentrated eluates were then reconstituted in 100 μL of methanol/H 2 O (55:45, v:v) for UPLC-MS analysis. The UPLC-MS method validated following FDA guidelines was used to accurately measure cortisol concentration in whole-body homogenates of zebrafish 59 .…”