Multidimensional Separations of Intact Phase II Steroid Metabolites Utilizing LC-Ion Mobility-HRMS.

The detection and unambiguous identification of anabolic-androgenic steroid metabolites are essential in clinical, forensic, and antidoping analyses. Recently, sulfate phase II steroid metabolites have received increased attention in steroid metabolism and drug testing. In large part, this is because phase II steroid metabolites are excreted for an extended time, making them a potential long-term chemical marker of choice for tracking steroid misuse in sports. Comprehensive analytical methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), have been used to detect and identify glucuronide and sulfate steroids in human urine with high sensitivity and reliability. However, LC-MS/MS identification strategies can be hindered by the fact that phase II steroid metabolites generate nonselective ion fragments across the different metabolite markers, limiting the confidence in metabolite identifications that rely on exact mass measurement and MS/MS information. Additionally, liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is sometimes insufficient at fully resolving the analyte peaks from the sample matrix (commonly urine) chemical noise, further complicating accurate identification efforts. Therefore, we developed a liquid chromatography-ion mobility-high resolution mass spectrometry (LC-IM-HRMS) method to increase the peak capacity and utilize the IM-derived collision cross section (CCS) values as an additional molecular descriptor for increased selectivity and to improve identifications of intact steroid analyses at low concentrations.

The presence of doping agents in dietary supplements: A glimpse into the Brazilian situation.

Dietary supplements (DS) are intended for healthy people to maintain or improve their overall health. Its consumption is widespread in large part of the general population and at all levels of athletes. Nevertheless, DS use can also pose health risks to individuals and, in the case of athletes, may lead to adverse analytical findings (AAFs) due to the possibility of DS contamination or adulteration with doping agents banned by the World Anti-Doping Agency. Although educational initiatives are being performed in Brazil to warn the sports community about inadvertent doping cases, AAFs connected to the DS administration have been increasingly growing. The findings of DS analyzed by the Brazilian Doping Control Laboratory (LBCD), between 2017 and 2022, after Testing Authorities (TAs) analysis requests, showed an alarming number of tainted samples. Diuretics were the most common adulterants found in all supplement types. However, the profile of prohibited substances in manufactured and compounded dietary supplements (MDS and CDS, respectively) were distinct, with stimulants being most prevalent in MDS and anabolic agents in CDS products. Additionally, MDS samples generally presented higher estimated concentrations of banned substances (mg/g) than CDS samples (µg/g). The common practice of DS intake by athletes continues to be of great concern for a doping-free sport, given the high prevalence of prohibited substances detected in the analyzed samples by the LBCD. The current Brazilian scenario reinforces the importance of raising awareness in the sports community of the possible consequences of an unintentional doping case linked to DS use.

No association between psychiatric symptoms and doses of anabolic steroids in a cohort of male and female bodybuilders.

The use of androgenic-anabolic steroids (AAS) can be associated with psychiatric symptoms such as insomnia, anxiety and increased aggressiveness. Although dose-dependent effects have been observed in some controlled studies, this association is not always seen in the ecological use of AAS. This study utilized WADA's steroid profile of suspicious use of AAS, urinary detection of AAS metabolites and measurement of sexual hormones to confirm recent use of AAS in a cohort of 103 bodybuilders (75 males, 28 females). The majority of participants (61.2%) presented symptoms of agitation, insomnia, increased aggressiveness or depression in the last 3 months. About one-third of participants presented scores on the HAM-A anxiety scale equivalent to moderate to severe symptoms of anxiety. A minority of participants (12.6%) presented high to moderate scores on the BPQ aggressiveness scale. The majority of participants (73.8%) presented hyperthymic temperament in the BRIEF-TEMPS scale. There was no significant difference in the presence of psychiatric symptoms between males and females and no association between psychiatric symptoms and estimated weekly doses of AAS. A negative association was observed between scores on the BPQ scale (verbal aggression, anger and total score) and the time of AAS use. We discuss differences of AAS use between male and female bodybuilders and the screening of AAS use in the general population. Our findings highlight the importance of mental health awareness among people using AAS.

Effective treatment and prevention of attempted suicide, anxiety, and aggressiveness with fluoxetine, despite proven use of androgenic anabolic steroids.

The treatment of a man who attempted suicide after experiencing symptoms of anxiety and aggressiveness associated with the use of androgenic-anabolic steroids (AAS) is described. This report includes 30 days of inpatient treatment and a 6-month follow-up. Regular use of fluoxetine apparently prevented the onset of anxiety, depression, aggressiveness, and suicide ideation, even with the concurrent use of AAS. The urinary concentration of androgens, metabolites of AAS, and fluoxetine were monitored through analysis of urinary samples by the Brazilian Laboratory of Doping Control. Our results are congruent with previous findings describing the risk of suicide prompted by AAS use as well as the efficacy of fluoxetine in the treatment of mood disorders associated with the use of anabolic steroids.

Analysis of exemestane and 17ß-hydroxyexemestane in human urine by gas chromatography/mass spectrometry: development and validation of a method using MO-TMS derivatives.

Trimethylsilylation of anabolic agents and their metabolites is frequently achieved by using the derivatization mixture N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)/NH(4)I/2-mercaptoethanol. Nevertheless, artifacts were formed when this mixture was employed in the monitoring of exemestane and its main metabolite 17ß-hydroxyexemestane prior to GC-MS analysis. These artifacts were identified as the N-methyltrifluoroacetamide (MTFA) and trimethylsiloxyethylmercapto products of the respective trimethylsilyl (TMS) derivatives. Furthermore, artifact formation was evaluated taking the structure (1,4-diene-3-keto-6-exomethylene) of the compounds into account. Although these artifacts are relevant for investigations regarding the derivatization process and may be of interest in many fields, they are detrimental to cope with the requirements of the World Anti-Doping Agency (WADA) in terms of the limits of detection (LODs) required. To overcome this issue, a method using an alternative derivatization was proposed: formation of methyloxime-TMS derivatives through double derivatization using O-methylhydroxylamine/pyridine and MSTFA/TMS imidazole after enzymatic hydrolysis and liquid-liquid extraction. Samples from an excretion study after administration of exemestane to healthy volunteers were analyzed by the proposed method and detection of both exemestane and its main metabolite was possible. This method showed excellent results for both analytes meeting the LODs required for antiestrogenic agents (50 ng/mL) established by WADA. The method was validated for the main metabolite, it was robust and cost-effective for qualitative and quantitative purposes, with LOD and LOQ of 10 ng/mL and 25 ng/mL, respectively.

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.

Increased atherothrombotic markers and endothelial dysfunction in steroid users.

BACKGROUND: The use of androgenic anabolic steroids (AAS) may be associated with changes in atherothrombotic markers and endothelial function. The purpose of this study was to compare atherothrombotic markers and endothelial function of AAS users and non-users. DESIGN: Cross-sectional study. METHODS: Ten athletes who were users of AAS (confirmed by urine analysis) and 12 non-user athletes were evaluated. Body weight, blood pressure, exercise load (hours/week), complete blood count (CBC), platelets, fibrinogen, lipids, high-sensitivity C-reactive protein (hs-CRP), follicle-stimulating hormone, testosterone and estradiol were measured. Endothelium-dependent and independent functions were assessed by brachial artery ultrasound. RESULTS: AAS users had higher body mass and blood pressure (p < 0.05). Platelet count was higher whereas HDL-cholesterol was lower in AAS users compared with non-users (p < 0.05). Levels of hs-CRP were higher in AAS users (p < 0.001). Follicle-stimulating hormone was suppressed in all users and not suppressed in non-users (p < 0.001). Compared with non-users, flow-mediated dilation was significantly reduced in AAS users (p = 0.03), whereas endothelium-independent function was similar in both groups. Additionally, flow-mediated dilation was positively associated with levels of HDL- cholesterol (r = 0.49, p = 0.03). CONCLUSIONS: AAS users present important changes in blood lipids as well as in inflammatory markers, which are compatible with increased cardiovascular risk. Furthermore, this profile is accompanied by a reduction in the endothelial function.

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.

Study of the endogenous steroid profile of male athletes from the Brazilian National Soccer Championship 2009.

Changes in the endogenous profile of androgenic anabolic steroids (AAS) may be interpreted as markers of doping. The objective of this study was to evaluate the endogenous profile of AAS in male athletes of the 2009 Brazilian National Soccer Championship, in normal conditions, particularly in the light of the revision of World Anti-Doping Agency's (WADA) Technical Document on the Interpretation of Endogenous AAS in athletes for doping control drafted in that year, as well as comparing these results to profiles already published in the literature. The upper limit of the 95% central reference interval of the following parameters for the studied population were estimated to be significantly higher than WADA's criteria, with a confidence of 90%: DHEA (about 2.3 times higher), Adiol (1.2 times higher), Bdiol (2.7 times higher), and Adiol/E (6 times higher). These findings seem to imply that WADA's criteria proposed in 2009 for DHEA, Adiol, Bdiol, and Adiol/E may not have been applicable to the studied population. Moreover, their comparison to previously published studies pointed to the need to evaluate in detail the appropriateness of adopting these criteria as universal, since there seems to be variations among different populations of athletes.