Analytical Strategy for Identification and Quantification of 13 Steroids in Sole (solea Senegalensis) Tissues, Eggs, and Larvae for Application in Aquaculture Studies of Reproduction

Knowing the levels of steroids in fish is crucial for understanding the reproductive processes during fish development and for reproductive success in aquaculture facilities. Although some of these compounds are present at very low concentrations, they play a relevant role in reproduction processes. Therefore, a very sensitive and reliable analytical methodology is required for their accurate determination. In this work, ultra-high-performance liquid chromatography coupled to tandem mass spectrometry with a triple quadrupole has been optimized for the identification and quantification of 13 steroids in plasma, muscle, and ovaries of sole (Solea senegalensis) and, for the first time, in fish eggs and larvae. This is noteworthy because studying fish development in its early stages could be the key to solving the reproductive problems observed in farmed specimens. Different strategies have been applied to solve analytical challenges such as matrix effects or the small amount of sample available. Compounds selected include progestagens (progesterone, 17 alpha,20 beta-dihydroxypregnenone, and 17 alpha,20 beta,21-trihydroxypregnenone), androgens (androstenedione, 11 beta-hydroxyandrostenedione, 11-ketoandrostenedione, testosterone, 5 alpha-dihydrotestosterone, and 11-ketotestosterone), estrogens (estrone and estradiol), and corticosteroids (cortisol and 11-cleoxycortisol). The methodology was validated in terms of accuracy (between 73% and 131%) and precision (relative standard deviations of <24%), at concentrations as low as 0.1 ng/mL for plasma, 0.1 ng/g for muscle and eggs, and 0.5 ng/g for ovaries and larvae. The application of the developed methodology to the analysis of samples from captive fish has allowed the identification and quantification of steroids at sub-nanogram per milliner and subnanogram per gram levels.