Phase II metabolism study of stanozolol through Zebrafish Water Tank (ZWT) experimental setup and liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS/MS)

Abstract

The growth-promoting anabolic-androgenic steroid stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. Thus, its extensive metabolism has been exhaustively studied. Its misuse in humans is mainly detected by monitoring intact phase II metabolites as conjugated with glucuronic acid or sulfate moiety by liquid chromatography-tandem mass spectrometry. Metabolic studies are a crucial element in forensic toxicology that aids the better understanding of biological processes, increasing the detection windows through additional biomarkers. However, administration studies in humans of non-approved substances face an essential ethical bottleneck that has been circumvented using in vitro and in vivo models. In this way, the zebrafish (Danio rerio) water tank has been emerging as an in vivo model for studying non-approved drugs' metabolism mainly because zebrafish’s genome, which has already been sequenced, presents substantial homology with mammals. The Zebrafish Water Tank (ZWT) experimental setup can produce phase I STAN metabolites. In the present study, the in vivo phase II metabolism of STAN was investigated through the ZWT model to determine whether the ZWT produces metabolites relevant for doping control. To achieve that, STAN was added to a 200 mL recipient containing eight fish at 32 ± 1°C. The noninvasive samples (recipient water) were analyzed both with and without pretreatment using Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC-HRMS/MS) in positive and negative ionization modes. As a result, four hydroxylated-sulfate and four hydroxylated-glycoconjugate metabolites were formed, two of the last ones being 3’OH-STAN-Glucuronide and 16β-OH-STAN-Glucuronide. Additionally, two STANGlucuronide derivatives were produced: one was confirmed to be STAN-N11 Glucuronide, and the other was presumed to be STAN-O-Glucuronide. After eight hours of the experiment, STAN-O-Glucuronide was the most intense phase II metabolite produced. The accumulation curves suggest that high concentrations of fish and substrate in water are required to form phase II metabolites. In addition, it is possible to predict by the bioaccumulation curves that by increasing the experiment time, the biosynthesis of long-excreted metabolites will also increase. Thus, from the results obtained here, further studies aiming at obtaining metabolites of long steroid excretion may be elaborated. It is also possible to infer that to understand the fragmentation profile of phase II metabolites it is necessary to use low and high collision energies in order to obtain a comprehensive fragmentation profile.