Methasterone is a designer anabolic steroid that is prohibited for athletes and is monitored by anti-doping laboratories. In this work, our objective is to discover new human phase I metabolites, define their excretion kinetics for 30 days and analyze their phase II metabolism (sulfate, cysteine and N-acetylcysteine conjugates). Urine samples from four volunteers were analyzed by chromatographic techniques. Through gas chromatography coupled to mass spectrometry analysis it was possible to detect methasterone and its nine phase I metabolites in the urine samples after glucuronide enzymatic hydrolysis, from which one were previously unreported. These nine compounds were not excreted in free form. The new proposed metabolite is 17β-hydroxy-2α,17α-dimethyl-5β-androstan-3-one, obtained from the epimerization at C5. The 3α-hydroxy metabolite, currently monitored by anti-doping laboratories, was the most abundant and was detected for the longest time. Furthermore, four other long-term metabolites were identified. By ultra-performance liquid chromatography coupled to tandem mass spectrometry, only the drug and a known metabolite were detected after glucuronide hydrolysis, and phase II metabolites were not found. Thus, our results contribute to elucidating methasterone metabolism, including long-term metabolites besides of the 3α-hydroxy in routine doping analysis, which is very important due to variation in human metabolism.

Ephedrines are widely used in therapy. Because of their stimulant properties, these substances are relevant in different forensic fields. At present, the state of the art for ephedrines quantification relay based on a liquid chromatography mass spectrometry, mainly because of the dilute-and-shoot approach. Notwithstanding, several gas chromatography based methods have already been described, all of them include cleanup steps, with the potential disadvantage of incurring errors and increasing the workload. In this paper, a straightforward method for ephedrine quantification based on gas chromatographic mass spectrometry, without cleanup and based on Doehlert matrix optimization is presented. Only 10 µL of a urine sample is necessary and for N-methyl-N-(trimethylsilyl)trifluoroacetamide/N-methyl-bis-trifluoracetamide derivatives, the intermediate precision was 2.77% for ephedrine, 9.20% for cathine, 8.29% for norephedrine and 4.27% for pseudoephedrine. The limit of detection was 20 ng mL-1 for ephedrine, 30 ng mL-1 for cathine and 40 ng mL-1 for norephedrine and pseudoephedrine.

Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry was used for the identification of forty doping agents. The improvement in the specificity was remarkable, allowing the resolution of analytes that could not be done by one-dimensional chromatographic systems. The sensitivity observed for different classes of prohibited substances was clearly below the value required by the World Anti-Doping Agency. In addition time-of-flight mass spectrometry gives full spectrum for all analytes without any interference from the matrix, resulting in selectivity improvements. These results could support the implementation of an exhaustive monitoring approach for hundreds of doping agents in a single injection.