Comprehensive Zebrafish Water Tank Experiment for Metabolic Studies of Testolactone.

Testolactone is a potent steroid aromatase (CYP19A1) inhibitor, and its main effect is a reduction in estradiol and estrone and an increase in testosterone and androstenedione levels. In this work, we evaluated a zebrafish water tank (ZWT) as a model to investigate testolactone biotransformation and the possibility to increase knowledge regarding the applicability of the ZWT on steroid hormone elimination research, as well as on the impact of steroid hormones on the endogenous metabolism of zebrafish. High-resolution mass spectrometry combined with SIEVE software was used to discriminate the peaks of interest based on significant changes in the relative signal intensity of the m/z values between different ZWT experiments. The metabolites, 4,5-dihydrotestolactone and 1,2,4,5-tetrahydrotestolactone, the same metabolites as those described in humans, were detected in ZWT, both in quite similar proportions. The presence of testolactone in the ZWT caused a rise in testosterone and androstenedione in the water tank, similar to that in human serum. These data suggest that, while the concentration of testolactone was high enough to inhibit the aromatase enzyme, an accumulation of androgens in the water occurred, indicating that the ZWT can be considered a model to investigate the impact of steroids on live organisms.

Identification of sympathomimetic alkylamine agents in urine using liquid chromatography-mass spectrometry and comparison of derivatization methods for confirmation analyses by gas chromatography-mass spectrometry.

The detection of 11 sympathomimetic alkylamines in urine was presented with a focus on human doping control is proposed using liquid chromatography tandem mass spectrometry (LC-QqQ) and high resolution mass spectrometry (LC-HRMS) as a screening tool after a dilute-and-shoot (DS) approach. For the LC-HRMS analyses, several compounds exhibited better limits of detection (L.O.D.) than the LC-QqQ. However, due to their small differences in structure, co-elution among the alkylamines was observed. Therefore, the chemical conversion of the alkylamines into an appropriate derivative for the confirmation analyses using gas chromatography-mass spectrometry (GC-MS) was evaluated. Five derivatization approaches were evaluated in an attempt to increase the analytical response and the confidence of the identification. The choice of the appropriated derivative for each alkylamine makes their spectra more easily interpretable, fulfills the WADA's rather strict identification criteria and enables the unequivocal identification of alkylamines in urine.