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A novel method for determining the testosterone/epitestosterone concentration ratio in human urine was established by capillary electrophoresis with diode-array detector. The urine samples were firstly purified by the solid extraction. The optimal experimental conditions were: running buffer pH = 4.74, 15.0 mmol L -1 HAc-NaAc, separation voltage 25 kV, temperature 25 °C, sample injection pressure 3.43 × 10 3  Pa, and duration 10 s. The testosterone and epitestosterone linear range were determined as 8.0-960.0 ng mL -1 , respectively. The testosterone and epitestosterone detection limits were determined as 4.6 and 4.5 ng mL -1 , respectively. The relative standard deviation was less than 0.36%., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Testosterone is one of the most abused pseudo-endogenous anabolic steroids in sport doping. The current method adopted to detect the abuse of testosterone and other pseudo-endogenous steroids (endogenous steroids when administered exogenously) is first based on the longitudinal monitoring of several urinary biomarkers, which constitute the so called "steroidal module" of the Athlete Biological Passport (ABP): atypical samples undergo a confirmation analysis based on the measurement of the 13 C/ 12 C isotopic ratio of selected target compounds, to distinguish their endogenous or exogenous origin. At the same time, testosterone administration can be allowed in athletes diagnosed with hypogonadism, provided they are granted a therapeutic use exemption by the relevant medical authority. In this pilot study we have investigated whether the approach based on the preliminary determination of the urinary steroid profile, in the format considered in the steroidal module of the ABP, also integrated with the inclusion of the sulfo-conjugates and of additional target steroids, can retain its validity also in the case of hypogonadal athletes. We have studied the effects of a single low dose (40 mg) of testosterone gel (T-gel) on the urinary concentration of the markers of steroidal module of the ABP, as well as on some additional steroid markers. The study was based on the analysis of urinary samples from 19 non-hospitalized hypogonadal men, 10 of them with late-onset hypogonadism (LOH), collected before, after 4 h and after 24 h the transdermal self-administration of 40 mg of T-gel. None of the patient had any co-morbidities possibly affecting the urinary excretion of the steroidal markers. The steroidal markers were quantified by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) after the enzymatic hydrolysis of the respective glucuro-conjugates and the chemical hydrolysis of the respective sulfo-conjugates. Targeted GC-MS/MS analysis was carried out operating in electron impact (EI) ionization mode, with acquisition in multiple reaction monitoring (MRM) mode. Our preliminary results show that, as expected, the treatment with T-gel leads, in all hypogonadal men, to an increase of the urinary concentration of the glucuro-conjugate metabolites of testosterone and its main metabolites, with special relevance to those with 5α-reduction. Furthermore, samples collected from non-LOH hypogonadal men showed an increase also in the levels of epitestosterone glucuronide, testosterone sulfate and epitestosterone sulfate. Apart from their biochemical and pharmacological relevance, these outcomes could be leveraged to refine the analytical strategy currently followed in the antidoping field for the analysis of the urinary steroidal markers, with potential implications also in other forensic and/or clinical investigations., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This article studies the genetic influence of polymorphism of the UGT2B17 gen on the urinary steroid profile and its implications for the anti-doping field. The study presents the results of a triple-blind randomized placebo-controlled crossover trial with healthy athletes submitted to a single dose of 250 mg of testosterone cypionate. Forty urine samples were collected from each participant. Mass spectrometry-based techniques commonly used in Anti-Doping laboratories, were employed to measure the urinary concentration and the Δδ 13 C values of a selection of target compounds for testosterone (T) administration together with LH. Twelve volunteers were included in the study; the polymorphism was evenly distributed among them. After T administration, the most meaningful change affected the Testosterone/Epitestosterone ratio (T/E) and the urinary concentration of LH. In relation with T/E, the wild type homozygous (ins/ins) group there was a mean relative increase of 30 (CI 95%: 25.2 to 36.7); in the heterozygous mutant (del/ins) group it was 19.8 (CI 95%:15.9 to 24.7); and in the homozygous mutant (del/del) group it was 19.7 (CI 95% 14.9 to 26.2). In the case of LH, it́s observed how LH values decrease significantly after the administration of Testex homogeneously among the three groups. The main outcome was related to the (del/del) group (homozygous mutant), where due to the depressed basal level of the steroid profile, if the longitudinal steroid profile of the athlete was not available, the analysis by GC/MS would not produce an "atypical" result according to the WADA TD2016EAAS despite the T administration. However, the genotyping of the UGT2B17 polymorphism, the follow up of LH and the use of GC-C-IRMS makes it possible to identify most of these samples as Adverse., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chronic and heavy alcohol consumption disrupts lipid metabolism and hormonal balance including testosterone levels. However, studies doubt the relationship between moderate alcohol intake and sex hormone levels. Therefore, the aim of the present investigation was to establish the direct impact of chronic and moderate alcohol intake on cholesterol homeostasis and steroid hormone synthesis. Asymptomatic chronic and moderate alcoholics (n=12) without chronic liver disease and healthy volunteers (n=14) were selected for the study. Furthermore, effects of standardized water extract of Tinospora cordifolia (Willd) Mier. (Menispermaceae) (TCJ), a well reported anti-alcoholic herbal drug, on urinary steroids was studied. This study included four groups, i.e. a) healthy; b) healthy+TCJ; c) alcoholic; d) alcoholic+TCJ. The blood and urine samples from each group were collected on day 0 and 14 of the post-treatment with TCJ and analyzed. Alcoholic blood samples showed the significantly higher values of traditional biomarkers γ-GT and MCV along with cholesterol, LDL, TGL and urinary methylglucuronide compared to healthy. Qualitative analysis of steroids showed that moderate alcohol intake in a chronic manner increased the cholesterol synthesis and directed its flow toward C-21 steroids; shown by increased levels of corticosterone (2.456 fold) and cortisol (3.7 fold). Moreover, alcohol intake also increased the synthesis of estradiol and clearance rate of other steroids through the formation of glucuronides. Therefore, it decreased the synthesis and increased the clearance rate of testosterone (T) and androstenedione (A). Quantitative analysis confirmed decreased T/A ratio from 2.31 to 1.59 in plasma and 2.47 to 1.51 in urine samples of alcoholics. TCJ intervention normalized the levels of steroids and significantly improved the T:A ratio to 2.0 and 2.12 in plasma and urine. The study revealed that TCJ modulated lipid metabolism by inhibiting cholesterol and glucuronides synthesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)