In vitro and in vivo metabolism studies of dimethazine.

The use of anabolic steroids is prohibited in sports. Effective control is done by monitoring their metabolites in urine samples collected from athletes. Ethical objections however restrict the use of designer steroids in human administration studies. To overcome these problems alternative in vitro and in vivo models were developed to identify metabolites and to assure a fast response by anti-doping laboratories to evolutions on the steroid market. In this study human liver microsomes and an uPA(+/+) -SCID chimeric mouse model were used to elucidate the metabolism of a steroid product called 'Xtreme DMZ'. This product contains the designer steroid dimethazine (DMZ), which consists of two methasterone molecules linked by an azine group. In the performed stability study, degradation from dimethazine to methasterone was observed. By a combination of LC-High Resolution Mass Spectrometry (HRMS) and GC-MS(/MS) analysis methasterone and six other dimethazine metabolites (M1-M6), which are all methasterone metabolites, could be detected besides the parent compound in both models. The phase II metabolism of dimethazine was also investigated in the mouse urine samples. Only metabolites M1 and M2 were exclusively detected in the glucuro-conjugated fraction; all other compounds were also found in the free fraction. For effective control of DMZ misuse in doping control samples, screening for methasterone and methasterone metabolites should be sufficient. Copyright © 2016 John Wiley & Sons, Ltd.

In Vitro Metabolic Studies of REV-ERB Agonists SR9009 and SR9011.

SR9009 and SR9011 are attractive as performance-enhancing substances due to their REV-ERB agonist effects and thus circadian rhythm modulation activity. Although no pharmaceutical preparations are available yet, illicit use of SR9009 and SR9011 for doping purposes can be anticipated, especially since SR9009 is marketed in illicit products. Therefore, the aim was to identify potential diagnostic metabolites via in vitro metabolic studies to ensure effective (doping) control. The presence of SR9009 could be demonstrated in a black market product purchased over the Internet. Via human liver microsomal metabolic assays, eight metabolites were detected for SR9009 and fourteen metabolites for SR9011 by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Structure elucidation was performed for all metabolites by LC-HRMS product ion scans in both positive and negative ionization mode. Retrospective data analysis was applied to 1511 doping control samples previously analyzed by a full-scan LC-HRMS screening method to verify the presence of SR9009, SR9011 and their metabolites. So far, the presence of neither the parent compound nor the metabolites could be detected in routine urine samples. However, to further discourage use of these potentially harmful compounds, incorporation of SR9009 and SR9011 into screening methods is highly recommended.

In vitro models for metabolic studies of small peptide hormones in sport drug testing.

Peptide hormones represent an emerging class of potential doping agents. Detection of their misuse is difficult due to their short half-life in plasma and rapid elimination. Therefore, investigating their metabolism can improve detectability. Unfortunately, pharmacokinetic studies with human volunteers are often not allowed because of ethical constraints, and therefore alternative models are needed. This study was performed in order to evaluate in vitro models (human liver microsomes and S9 fraction) for the prediction of the metabolism of peptidic doping agents and to compare them with the established models. The peptides that were investigated include desmopressin, TB-500, GHRP-2, GHRP-6, hexarelin, LHRH and leuprolide. Several metabolites were detected for each peptide after incubation with human liver microsomes, S9 fraction, and serum, which all showed endopeptidase and exopeptidase activity. In vitro models from different organs (liver vs. kidney) were compared, but no significant differences were recorded. Deamidation was not observed in any of the models and was therefore evaluated by incubation with α-chymotrypsin. In conclusion, in vitro models are useful tools for forensic and clinical analysts to detect peptidic metabolic markers in biological fluids.

Metabolism of methylstenbolone studied with human liver microsomes and the uPA⁺/⁺-SCID chimeric mouse model.

Anti-doping laboratories need to be aware of evolutions on the steroid market and elucidate steroid metabolism to identify markers of misuse. Owing to ethical considerations, in vivo and in vitro models are preferred to human excretion for nonpharmaceutical grade substances. In this study the chimeric mouse model and human liver microsomes (HLM) were used to elucidate the phase I metabolism of a new steroid product containing, according to the label, methylstenbolone. Analysis revealed the presence of both methylstenbolone and methasterone, a structurally closely related steroid. Via HPLC fraction collection, methylstenbolone was isolated and studied with both models. Using HLM, 10 mono-hydroxylated derivatives (U1-U10) and a still unidentified derivative of methylstenbolone (U13) were detected. In chimeric mouse urine only di-hydroxylated metabolites (U11-U12) were identified. Although closely related, neither methasterone nor its metabolites were detected after administration of isolated methylstenbolone. Administration of the steroid product resulted mainly in the detection of methasterone metabolites, which were similar to those already described in the literature. Methylstenbolone metabolites previously described were not detected. A GC-MS/MS multiple reaction monitoring method was developed to detect methylstenbolone misuse. In one out of three samples, previously tested positive for methasterone, methylstenbolone and U13 were additionally detected, indicating the applicability of the method.

An improved gas chromatography screening method for doping substances using triple quadrupole mass spectrometry, with an emphasis on quality assurance.

A GC-QqQ-MS method was developed for the detection of over 150 compounds from different classes (steroids, narcotics, stimulants, ß-blockers, ß-2-agonists and hormone antagonists) in a qualitative way. In the quantitative part, the traditional steroid profile with the most important endogenous steroids is expanded with six minor metabolites, which further improves the detection and identification of endogenous steroid abuse. In addition to these, norandrosterone, salbutamol and the major metabolite of cannabis are also quantified. Methods developed for anti-doping purposes should be subjected to the highest level of quality. Here, the addition of a combination of (deuterated) internal standards allows for an accurate quality control of every single step of the methodology: hydrolysis efficiency, derivatization efficiency and microbiological degradation are monitored in every single sample. Additionally, special attention is paid to the relationships between parameters indicating degradation by micro-organisms and the reliability of the steroid profile. The impact of the degradation is studied by evaluation of the quantities and percentages of 5α-androstane-3,17-dione and 5ß-androstane-3,17-dione. The concept of measurement uncertainty was introduced for the evaluation of relative abundances of mass-to-charge ratios and the obtained ranges were compared with the World Anti-Doping Agency regulations on tolerance windows for relative ion intensities. The results indicate that the approaches are similar.

Identification and characterization of novel long-term metabolites of oxymesterone and mesterolone in human urine by application of selected reaction monitoring GC-CI-MS/MS.

The search for metabolites with longer detection times remains an important task in, for example, toxicology and doping control. The impact of these long-term metabolites is highlighted by the high number of positive cases after reanalysis of samples that were stored for several years, e.g. samples of previous Olympic Games. A substantial number of previously alleged negative samples have now been declared positive due to the detection of various long-term steroid metabolites the existence of which was unknown during the Olympic Games of 2008 and 2012. In this work, the metabolism of oxymesterone and mesterolone, two anabolic androgenic steroids (AAS), was investigated by application of a selected reaction monitoring gas chromatography-chemical ionization-triple quadrupole mass spectrometry (GC-CI-MS/MS) protocol for metabolite detection and identification. Correlations between AAS structure and GC-CI-MS/MS fragmentation behaviour enabled the search for previously unknown but expected AAS metabolites by selection of theoretical transitions for expected metabolites. Use of different hydrolysis protocols allowed for evaluation of the detection window of both phase I and phase II metabolites. For oxymesterone, a new metabolite, 18-nor-17ß-hydroxymethyl-17α-methyl-4-hydroxy-androst-4,13-diene-3-one, was identified. It was detectable up to 46 days by using GC-CI-MS/MS, whereas with a traditional screening (detection of metabolite 17-epioxymesterone with electron ionization GC-MS/MS) oxymesterone administration was only detectable for 3.5 days. A new metabolite was also found for mesterolone. It was identified as 1α-methyl-5α-androstan-3,6,16-triol-17-one and its sulfate form after hydrolysis with Helix pomatia resulted in a prolonged detection time (up to 15 days) for mesterolone abuse. Copyright © 2017 John Wiley & Sons, Ltd.

In vitro metabolism study of a black market product containing SARM LGD-4033.

Anabolic agents are often used by athletes to enhance their performance. However, use of steroids leads to considerable side effects. Non-steroidal selective androgen receptor modulators (SARMs) are a novel class of substances that have not been approved so far but seem to have a more favourable anabolic/androgenic ratio than steroids and produce fewer side effects. Therefore the use of SARMs has been prohibited since 2008 by the World Anti-Doping Agency (WADA). Several of these SARMs have been detected on the black market. Metabolism studies are essential to identify the best urinary markers to ensure effective control of emerging substances by doping control laboratories. As black market products often contain non-pharmaceutical-grade substances, alternatives for human excretion studies are needed to elucidate the metabolism. A black market product labelled to contain the SARM LGD-4033 was purchased over the Internet. Purity verification of the black market product led to the detection of LGD-4033, without other contaminants. Human liver microsomes and S9 liver fractions were used to perform phase I and phase II (glucuronidation) metabolism studies. The samples of the in vitro metabolism studies were analyzed by gas chromatography-(tandem) mass spectrometry (GC-MS(/MS)), liquid chromatography-high resolution-tandem mass spectrometry (LC-(HR)MS/MS). LC-HRMS product ion scans allowed to identify typical fragment ions for the parent compound and to further determine metabolite structures. In total five metabolites were detected, all modified in the pyrrolidine ring of LGD-4033. The metabolic modifications ranged from hydroxylation combined with keto-formation (M1) or cleavage of the pyrrolidine ring (M2), hydroxylation and methylation (M3/M4) and dihydroxylation (M5). The parent compound and M2 were also detected as glucuronide-conjugates. Copyright © 2016 John Wiley & Sons, Ltd.

Metabolic studies of prostanozol with the uPA-SCID chimeric mouse model and human liver microsomes.

Anabolic androgenic steroids are prohibited by the World Anti-Doping Agency because of their adverse health and performance enhancing effects. Effective control of their misuse by detection in urine requires knowledge about their metabolism. In case of designer steroids, ethical objections limit the use of human volunteers to perform excretion studies. Therefore the suitability of alternative models needs to be investigated. In this study pooled human liver microsomes (HLM) and an uPA(+/+)-SCID chimeric mouse model were used to examine the metabolism of the designer steroid prostanozol as a reference standard. Metabolites were detected by GC-MS (full scan) and LC-MS/MS (precursor ion scan). In total twenty-four prostanozol metabolites were detected with the in vitro and in vivo metabolism studies, which could be grouped into two broad classes, those with a 17-hydroxy- and those with a 17-keto-substituent. Major first phase metabolic sites were tentatively identified as C-3'; C-4 and C-16. Moreover, 3'- and 16ß-hydroxy-17-ketoprostanozol could be unequivocally identified, since authentic reference material was available, in both models. Comparison with published data from humans showed a good correlation, except for phase II metabolism. As metabolites were in contrast to the human studies predominantly present in the free fraction. Two types of metabolites ((di)hydroxylated prostanozol metabolites) that have not been described before could be confirmed in a real positive doping control sample. Hence, the results provide further evidence for the applicability of chimeric mice and HLM to perform metabolism studies of designer steroids.

Urinary detection of conjugated and unconjugated anabolic steroids by dilute-and-shoot liquid chromatography-high resolution mass spectrometry.

Anabolic androgenic steroids (AAS) are an important class of doping agents. The metabolism of these substances is generally very extensive and includes phase-I and phase-II pathways. In this work, a comprehensive detection of these metabolites is described using a 2-fold dilution of urine and subsequent analysis by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The method was applied to study 32 different metabolites, excreted free or conjugated (glucuronide or sulfate), which permit the detection of misuse of at least 21 anabolic steroids. The method has been fully validated for 21 target compounds (8 glucuronide, 1 sulfate and 12 free steroids) and 18 out of 21 compounds had detection limits in the range of 1-10 ng mL(-1) in urine. For the conjugated compounds, for which no reference standards are available, metabolites were synthesized in vitro or excretion studies were investigated. The detection limits for these compounds ranged between 0.5 and 18 ng mL(-1) in urine. The simple and straightforward methodology complements the traditional methods based on hydrolysis, liquid-liquid extraction, derivatization and analysis by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS).