Anamorelin, developed for the treatment of cancer cachexia, is an orally active medication that improves appetite and food intake, thereby increasing body mass and physical functioning. It is classified as a growth hormone secretagogue and strictly monitored by the World Anti-Doping Agency (WADA), owing to its anabolic enhancing potential. Identifying anamorelin and/or metabolite biomarkers of consumption is critical in doping controls. However, there are currently no data available on anamorelin human metabolic fate. The aim of this study was to investigate and identify biomarkers characteristic of anamorelin intake using in silico metabolite predictions with GLORYx, in vitro incubation with 10-donor-pooled human hepatocytes, liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) analysis, and data processing with Thermo Scientific’s Compound Discoverer. In silico prediction resulted in N-acetylation at the methylalanyl group as the main transformation (score, 88%). Others including hydroxylation at the indole substructure, and oxidation and N-demethylation at the trimethylhydrazino group were predicted (score, ≤36%). Hepatocyte incubations resulted in 14 phase I metabolites formed through N-demethylation at the trimethylhydrazino group, N-dealkylation at the piperidine ring, and oxidation at the indole and methylalanyl groups; and two phase II glucuronide conjugates occurring at the indole. We propose four metabolites detected as specific biomarkers for toxicological screening.

Ostarine (enobasarm) is a selective androgen receptor modulator with great therapeutic potential. However, it is also used by athletes to promote muscle growth and enhance performances without the typical adverse effects of anabolic steroids. Ostarine popularity increased in recent years, and it is currently the most abused “other anabolic agent” (subclass S1.2. of the “anabolic agents” class S1) from the World Anti-Doping Agency’s (WADA) prohibited list. Several cases of liver toxicity were recently reported in regular users. Detecting ostarine or markers of intake in biological matrices is essential to document ostarine use in doping. Therefore, we sought to investigate ostarine metabolism to identify optimal markers of consumption. The substance was incubated with human hepatocytes, and urine samples from six ostarine-positive cases were screened. Analyses were performed via liquid chromatography–high-resolution tandem mass spectrometry (LC-HRMS/MS) and software-assisted data mining, with in silico metabolite predictions. Ten metabolites were identified with hydroxylation, ether cleavage, dealkylation, O-glucuronidation, and/or sulfation. The production of cyanophenol-sulfate might participate in the mechanism of ostarine liver toxicity. We suggest ostarine-glucuronide (C₂₅H₂₂O₉N₃F₃, diagnostic fragments at m/z 118, 185, and 269) and hydroxybenzonitrile-ostarine-glucuronide (C₂₅H₂₂O₁₀N₃F₃, diagnostic fragments at m/z 134, 185, and 269) in non-hydrolyzed urine and ostarine and hydroxybenzonitrile-ostarine (C₁₉H₁₄O₄N₃F₃, diagnostic fragments at m/z 134, 185, and 269) in hydrolyzed urine as markers to document ostarine intake in doping.

Background: The abuse of psychoactive substances presents challenges in clinical and forensic toxicology. The emergence of novel and potent drugs that pose significant health risks, in particular towards frequent abusers and users unaware of the ingredients, further complicates the situation. Designer benzodiazepines have become a fast-growing subgroup of these new psychoactive substances (NPSs), and their overdose may potentially turn fatal, especially when combined with other central nervous system depressants. In 2021, flubrotizolam, a potent thieno-triazolo designer benzodiazepine, emerged on the illicit market, available online as a “research chemical”. The identification of markers of consumption for this designer benzodiazepine is essential in analytical toxicology, especially in clinical and forensic cases. Methods: We therefore aimed to identify biomarkers of flubrotizolam uptake in ten-donor-pooled human hepatocytes, applying liquid chromatography high-resolution mass spectrometry and software-aided data mining supported by in silico prediction tools. Results: Prediction studies resulted in 10 and 13 first- and second-generation metabolites, respectively, mainly transformed through hydroxylation and sulfation, methylation, and glucuronidation reactions. We identified six metabolites after 3 h human hepatocyte incubation: two hydroxylated metabolites (α- and 6-hydroxy-flubrotizolam), two 6-hydroxy-glucuronides, a reduced-hydroxy-N-glucuronide, and an N-glucuronide. Conclusions: We suggest detecting flubrotizolam and its hydroxylated metabolites as markers of consumption after the glucuronide hydrolysis of biological samples. The results are consistent with the in vivo metabolism of brotizolam, a medically used benzodiazepine and a chloro-phenyl analog of flubrotizolam.