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The ready detectability of synthetic androgens by mass spectrometry (MS)-based antidoping tests has reoriented androgen doping to using testosterone (T), which must be distinguished from its endogenous counterpart making detection of exogenous T harder. We investigated urine and serum steroid and hematological profiling individually and combined to determine the optimal detection model for T administration in women. Twelve healthy females provided six paired blood and urine samples over 2 weeks prior to treatment consisting of 12.5-mg T in a topical transdermal gel applied daily for 7 days. Paired blood and urine samples were then obtained at the end of treatment and Days 1, 2, 4, 7, and 14 days later. Compliance with treatment and sampling was high, and no adverse effects were reported. T treatment significantly increased serum and urine T, serum dihydrotestosterone (DHT), urine 5α-androstane-3α,17β-diol (5α-diol) epitestosterone (E), and urine T/E ratio with a brief window of detection (2-4 days) as well as total and immature (medium and high fluorescence) reticulocytes that remained elevated over the full 14 posttreatment days. Carbon isotope ratio MS and the OFF score and Abnormal Blood Profile score (ABPS) were not discriminatory. The optimal multivariate model to identify T exposure combined serum T, urine T/E ratio with three hematological variables (% high fluorescence reticulocytes, mean corpuscular hemoglobin, and volume) with the five variables providing 93% correct classification (4% false positive, 10% false negatives). Hence, combining select serum and urine steroid MS variables with reticulocyte measures can achieve a high but imperfect detection of T administration to healthy females. more...

Mitotane is a cytotoxic drug used in the treatment of inoperable adrenocortical carcinoma (ACC), it inhibits steroidogenesis as well, and therefore monitoring the level of steroid hormones in patients treated with mitotane is a crucial point of therapy. Hence, we have developed a simple, fast, and efficient electrophoretic method combined with sweeping in reverse polarity (RP-sweeping) as on-line preconcentration technique and dispersive liquid-liquid microextraction (DLLME) for the simultaneous determination of mitotane, its main metabolite DDA and 5 steroid hormones (progesterone, testosterone, epitestosterone, cortisol, corticosterone) in urine samples. In addition, a new sample matrix consisting of β-CD2 SDS1 complexes for a high hydrophobic compounds solubilization was developed. Approach based on the application of β-cyclodextrin (β-CD) and sodium dodecyl sulfate (SDS) complex of a ratio 2: 1 allowed for hydrodynamic injection into the capillary of a solution containing both mitotane and other analytes. The detection limits of the analytes for the RP-sweeping-DLLME method were found to be in the range of 1.5-3 ng/mL, which were approximately 1000 times lower than in the conventional hydrodynamic injection (5 s, 0.5 psi) without any preconcentration procedure. All analytes were completely resolved in less than 13 minutes by uncoated silica capillary with an inner diameter of 75 μm (ID) x 60 cm. Electrophoretic separation was performed in reverse polarity with a voltage of -25 kV with a background electrolyte (BGE) consisting of 100 mM SDS, 25% acetonitrile (ACN), 25 mM phosphate buffer (pH 2.5) and 7mM β-CD. This article is protected by copyright. All rights reserved. more...

This article reports the results obtained from the investigation of the influence of miconazole administration on the physiological fluctuation of the markers of the steroid profile included in the “steroidal module” of the Athlete Biological Passport. Urines collected from male Caucasian subjects before, during, and after either systemic (i.e., oral and buccal) or topical (i.e., dermal) treatment with miconazole were analyzed according to validated procedures based on gas chromatography coupled to tandem mass spectrometry (GC–MS/MS) (to determine the markers of the steroid profile) or liquid chromatography coupled to MS/MS (LC–MS/MS) (to determine miconazole urinary levels). The results indicate that only after systemic administration, the markers of the steroid profile were significantly altered. After oral and buccal administration, we have registered (i) a significant increase of the 5α‐androstane‐3α,17β‐diol/5β‐androstane‐3α,17β‐diol ratio and (ii) a significant decrease of the concentration of androsterone, etiocholanolone, 5β‐androstane‐3α,17β‐diol, and 5α‐androstane‐3α,17β‐diol and of the androsterone/etiocholanolone, androsterone/testosterone, and 5α‐androstane‐3α,17β‐diol/epitestosterone ratios. Limited effects were instead measured after dermal intake. Indeed, the levels of miconazole after systemic administration were in the range of 0.1–12.5 μg/ml, whereas after dermal administration were below the limit of quantification (50 ng/ml). Significant alteration started to be registered at concentrations of miconazole higher than 0.5 μg/ml. These findings were primarily explained by the ability of miconazole in altering the kinetic/efficacy of deglucuronidation of the endogenous steroids by the enzyme β‐glucuronidase during the sample preparation process. The increase of both incubation time and amount of β‐glucuronidase was demonstrated to be effective countermeasures in the presence of miconazole to reduce the risk of uncorrected interpretation of the results., The results obtained from the investigation of the influence of miconazole administration on the parameters included in the “steroidal module” of the Athlete Biological Passport were presented. After oral and buccal administration of miconazole, the levels of the markers of the steroid profile were significantly altered, whereas no effects were registered after topical administration. These findings were primarily explained by the effect of miconazole on the kinetics/efficacy of the enzymatic hydrolysis during the sample preparation process. more...

In order to detect the misuse of testosterone (T) or boldenone (Bo) in doping control analysis, the confirmation of atypical findings employing the determination of carbon isotope ratios (CIR) is mandatory for issuing adverse analytical findings. Elevated concentrations of T (or elevated T/epitestosterone ratios) may result from confounding factors such as ethanol intake, and the presence of low urinary concentrations of Bo can originate from endogenous or urinary in situ production of small amounts of the steroid. As pharmaceutical preparations of Bo and T are generally depleted in 13 C, their CIR differ significantly from the 13 C-enriched endogenous steroids. Some rare cases have been reported on pharmaceutical preparations showing 13 C-enriched isotope ratios that complicate the current application of CIR in sports drug testing. Therefore, the CIR of a subset of n = 157 T preparations and n = 39 Bo preparations seized in Switzerland and Germany between 2013 and 2018 was analyzed in order to estimate the possible impact of steroid preparations showing 13 C-enriched isotope ratios on the current approach to detect their misuse. All investigated Bo preparations showed CIR in the expected range between - 26.7 and -30.3‰. Within the T samples, 95% showed the expected values below -26‰ while six samples fall between -25 and -26‰ and one sample was indistinguishable from endogenously produced T with a CIR of -23.3‰. more...

In order to detect the misuse of testosterone (T), urinary steroid concentrations and concentration ratios are quantified and monitored in a longitudinal manner to enable the identification of samples exhibiting atypical test results. These suspicious samples are then forwarded to isotope ratio mass spectrometry (IRMS)–based methods for confirmation. Especially concentration ratios like T over epitestosterone (E) or 5α-androstanediol over E proved to be valuable markers. Unfortunately, depending on the UGT2B17 genotype and/or the gender of the athlete, these markers may fail to provide evidence for T administrations when focusing exclusively on urine samples. In recent years, the potential of plasma steroids has been investigated and were found to be suitable to detect T administrations especially in female volunteers. A current drawback of this approach is the missing possibility to confirm that elevated steroid concentrations are solely derived from an administration of T and cannot be attributed to confounding factors. Therefore, an IRMS method for plasma steroids was developed and validated taking into account the comparably limited sample volume. As endogenous reference compounds, unconjugated cholesterol and dehydroepiandrosterone sulfate were found suitable, while androsterone and epiandrosterone (both sulfo-conjugated) were chosen as target analytes. The developed method is based on multi-dimensional gas chromatography coupled to IRMS in order to optimize the overall assay sensitivity. The approach was validated, and a reference population encompassing n = 65 males and females was investigated to calculate population-based thresholds. As proof-of-concept, samples from volunteers receiving T replacement therapies and excretion study samples were investigated. Graphical abstract more...

A 59-year-old male was found dead at home, with two empty vials of an oily preparation obtained from a manufacturer from East Europe. There was no label on the vial. The subject was a former weightlifter, also known as an anabolic steroids abuser. The local prosecutor ordered a body examination, which was unremarkable, and allowed collecting femoral blood, urine and scalp hair (6 cm, brown). He was treated for cardiac insufficiency with quinidine. Biological specimens were submitted not only to standard toxicological analyses including a screening with liquid chromatography (LC)–quadrupole time of flight, but also to a specific LC–tandem mass spectrometry method for anabolic steroids testing. Ethanol was not found in both blood and urine. Quinidine blood concentration (791 ng/mL) was therapeutic. No drug of abuse was identified. In blood, testosterone was less that 1 ng/mL and no other steroid was identified. In urine, testosterone/epitestosterone was 1.56 and boldenone was present at a concentration of 9 ng/mL. The hair test results, performed on the whole length, demonstrated repetitive steroids abuse, including not only testosterone (140 pg/mg), testosterone propionate (605 pg/mg) and testosterone decanoate (249 pg/mg), but also boldenone (160 pg/mg), trenbolone (143 pg/mg) and metandienone (60 pg/mg). Since forensic laboratories have limited access to steroid urinary metabolite reference material due to specific regulations (to avoid testing athletes before anti-doping verifications), hair analyses seem to be the best approach to document anabolic agents abuse. Indeed, in hair, the target drug is the parent compound; in addition, when compared to blood or urine, this matrix has a much larger window of detection. The pathologist concluded cardiac insufficiency in a context involving repetitive abuse of anabolic drugs. This case indicates that more attention should be paid to anabolic steroids, in a context of sudden cardiac death. more...

In women, hormonal fluctuations related to the menstrual cycle may impose a great source of variability for some biomarkers of testosterone (T) administration, which can ultimately disrupt the sensitivity of their longitudinal monitoring. In this study, the sensitivity of the current urinary and haematological markers of the Athlete Biological Passport (ABP), as well as serum steroid biomarkers, was investigated for the monitoring of a 28-day T gel treatment combined with endogenous fluctuation of the menstrual cycle in 14 healthy female subjects. Additionally, the analysis of urinary target compounds was performed on a subset of samples for endogenous/exogenous origin via isotope ratio mass spectrometry (IRMS). In serum, concentrations of T and dihydrotestosterone (DHT) increased significantly during the treatment, whereas in urine matrix the most affected biomarkers were found to be the ratios of testosterone/epitestosterone (T/E) and 5α-androstane-3α,17β-diol/epitestosterone (5αAdiol/E). The detection capability of both urinary biomarkers was heavily influenced by [E], which fluctuated depending on the menstrual cycle, and resulted in low sensitivity of the urinary steroidal ABP module. On the contrary, an alternative approach by the longitudinal monitoring of serum T and DHT concentrations with the newly proposed T/androstenedione ratio showed higher sensitivity. The confirmatory IRMS results demonstrated that less than one third of the tested urine samples fulfilled the criteria for positivity. Results from this study demonstrated that the 'blood steroid profile' represents a powerful complementary approach to the 'urinary module' and underlines the importance of gathering bundle of evidence to support the scenario of an endogenous prohibited substance administration. more...

Context Little is known about how exogenous testosterone (T) affects the steroid profile in women. More knowledge would give the antidoping community keys as to how to interpret tests and detect doping. Objective This work aimed to investigate the steroid profile in serum and urine in young healthy women after T administration. Methods In a randomized, double-blind, placebo-controlled study, 48 healthy young women were assigned to daily treatment with T cream (10 mg) or placebo (1:1) for 10 weeks. Urine and blood were collected before and at the end of treatment. Serum steroids were analyzed with liquid chromatography–tandem mass spectrometry, and urine levels of T, epitestosterone (E), and metabolites included in the Athlete Biological Passport (ABP) were analyzed with gas chromatography–tandem mass spectrometry. Results In serum, T and dihydrotestosterone levels increased, whereas sex hormone–binding globulin and 17-hydroxyprogesterone decreased after T treatment as compared to placebo. In urine, T and 5α-androstanediol increased in the T group. The median T increase in serum was 5.0-fold (range, 1.2-18.2) and correlated to a 2.2-fold (range, 0.4-14.4) median increase in T/E in urine (rs = 0.76). Only 2 of the 24 women receiving T reached the T/E cutoff ratio of 4, whereas when the results were added to the ABP, 6 of 15 participants showed atypically high T/E (40%). In comparison, 22/24 women in the T group increased serum T more than 99.9% of the upper confidence interval of nontreated values. Conclusion It seems that the T/E ratio is not sufficient to detect exogenous T in women. Serum total T concentrations could serve as a complementary marker of doping. more...

The interpretation of the steroidal module of the Athlete Biological Passport (ABP) in female athletes is complex due to the large variation of the endogenous urinary steroids. The menstrual cycle seems to be one of the largest confounders of the steroid profile. The duration of the different phases in the menstrual cycle differs between women and is difficult to predict only by counting days after menstruation. Here, we have determined the follicle, ovulation, and luteal phases, by assessing the menstrual hormones in serum samples collected from 17 healthy women with regular menses. Urine samples were collected three times per week during two consecutive cycles to measure the urinary steroid concentrations used in the ABP. The metabolite that was mostly affected by the menstrual phases was epitestosterone (E), where the median concentration was 133% higher in the ovulation phase compared to the follicle phase (p < 0.0001). The women with a large coefficient of variation (CV) in their first cycle also had a large CV in their second cycle and vice versa. The inter‐individual difference was extensive with a range of 11%–230% difference between the lowest and the highest T/E ratio during a cycle. In conclusion, E and ratios with E as denominator are problematic biomarkers for doping in female athletes. The timing of the sample collection in the menstrual cycle will have a large influence on the steroid profile. The results of this study highlight the need to find additional biomarkers for T doping in females., The menstrual cycle is one of the largest confounders of the steroid profile in female athletes. We have determined the follicle, ovulation, and luteal phases, by assessing the menstrual hormones in serum samples in 17 women as well as determined the urinary ABP steroids for two consecutive cycles. The impact of the different phases of the menstrual cycle on the steroid profile and the correlation between menstrual hormones and urinary steroid are investigated. more...

The Paternò-Büchi (PB) reaction is a common organic reaction in which a carbonyl radical formed by exposure to UV radiation reacts with an alkene to form an oxetane ring. Recent analytical applications of this reaction have included the determination of C═C bond position in lipid fatty acyl tails using tandem mass spectrometry. Our group has recently investigated methods for structurally modifying steroid isomers to improve their identification and resolution using ion mobility spectrometry. Herein, we report the first application of the Paternò-Büchi reaction to form steroid oxetanes using a simple, low-cost, and high efficiency method with a low pressure mercury lamp. This methodology is performed on several endogenous steroid isomers, resulting in unique ion mobility spectra that provide a unique fingerprint for each. These fingerprint spectra can add confidence in identification of those compounds, especially in complex biological matrixes. Testosterone and epitestosterone, an epimer pair commonly interrogated in a number of applications such as for their use as performance enhancing drugs, displayed one and three unique ion mobility peaks, respectively. These spectra and their measured collision cross sections (CCS) allow for unambiguous differentiation of these and several other steroid isomer groups analyzed in this work. Finally, multiple anabolic androgenic steroids prohibited by the World Anti-Doping Agency were tested with this method and resulted in unique CCS for their PB reaction products. This approach can offer improved confidence in their identification as well as for many other banned substances. more...

Herein we demonstrate the first application of ozone-induced cleavage of endocyclic C═C double bonds for improved steroid isomer separation using ion mobility-mass spectrometry. Steroids represent a challenging biomolecular class for ion mobility (IM) separations due to their structural rigidity and subtle stereochemical differences. In this work, we compare the effects of ozonolysis on the relative mobilities of a model stereoisomer pair, testosterone and epitestosterone. A solution-phase ozonolysis approach is used due to its simplicity, relatively low cost, and potential for rapid, online analysis. Despite the presence of solvent-based addition products, we observe that these steroids undergo an ozone-based cleavage resulting in unique, stable gas-phase conformations. The resulting resolution between testosterone and epitestosterone, with collision cross section values of 176.6 and 193.3 A2, respectively, demonstrates a significant improvement in comparison with previous IM-based approaches. The significantly smaller conformation observed for epitestosterone is stabilized by a three-point interaction between the oxygen-containing functional groups and a sodium ion; this same conformation cannot be sterically achieved by testosterone. Identification of this specific structural difference is strengthened by experimental results showing the disappearance of this conformation following in-source water loss, which eliminates the potential for that three-point interaction. Computational modeling of the lowest energy gas-phase structures for these ozone products corroborates the experimental results. In conclusion, this approach provides tremendous potential as a rapid IM separation method for steroid isomers and other endocyclic C═C double bond containing molecules. more...

To detect doping with endogenous steroids, six urinary steroids are longitudinally monitored in the athlete biological passport (ABP). These steroids include testosterone, etiocholanolone, androsterone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol, and the testosterone isomer epitestosterone. It is known that the intake of hormonal contraceptives may interfere with the ABP biomarkers. A previous study showed that athletes using hormonal contraceptives (HCs) display lower urinary epitestosterone concentrations than non-using athletes. In this study, we analyzed the urinary steroid profile prior to and three months after administration of an oral HC including levonorgestrel and ethinylestradiol (n = 55). The urinary concentrations of all the ABP metabolites decreased after three months, with epitestosterone showing the largest decline (median 6.78 to 3.04 ng/mL, p˂0.0001) followed by 5α-androstane-3α,17β-diol (median 23.5 to 12.83 ng/mL, p˂0.0001), and testosterone (median 5.32 to 3.66, p˂0.0001). Epitestosterone is included in two of the five ratios in the ABP (T/E and 5αAdiol/E), and consequently these ratios increased 1.7-fold (range 0.27 to 8.50) and 1.26-fold (range 0.14 to 5.91), respectively. Some of these changes may mimic the changes seen after administration of endogenous steroids leading to atypical findings. Notably, even though participants used the same contraceptive treatment schedule, the HC-mediated epitestosterone change varied to a large extent (median 0.43-fold, range 0.06 to 6.5) and were associated with a functional T˃C promoter polymorphism in CYP17A1. Moreover, the epitestosterone changes correlated with HC-induced testosterone and gonadotropins changes in serum, indicating that urinary epitestosterone reflects the androgen load in HC-using women. more...

In order to detect the abuse of substances in sports, the knowledge of their metabolism is of undisputable importance. As in vivo administration of compounds faces ethical problems and might even not be applicable for nonapproved compounds, cell-based models might be a versatile tool for biotransformation studies. We coincubated HepG2 cells with metandienone and D3 -epitestosterone for 14 days. Phase I and II metabolites were analyzed by high-performance liquid chromatography (HPLC)-tandem mass spectrometry and confirmed by gas chromatography-mass spectrometry (GC-MS). The metandienone metabolites formed by HepG2 cells were comparable with those renally excreted by humans. HepG2 cells also generated the two long-term metabolites 17β-hydroxymethyl-17α-methyl-18-nor-androst-1,4,13-trien-3-one and 17α-hydroxymethyl-17β-methyl-18-nor-androst-1,4,13-trien-3-one used in doping analyses, though in an inverse ratio compared with that observed in human urine. In conclusion, we showed that HepG2 cells are suitable as model for the investigation of biotransformation of androgens, especially for the anabolic androgenic steroid metandienone. They further proved to cover phase I and II metabolic pathways, which combined with a prolonged incubation time with metandienone resulted in the generation of its respective long-term metabolites known from in vivo metabolism. Moreover, we showed the usability of D3 -epitestosterone as internal standard for the incubation. The method used herein appears to be suitable and advantageous compared with other models for the investigation of doping-relevant compounds, probably enabling the discovery of candidate metabolites for doping analyses. more...

IntroductionIn female athletes, the interpretation of doping tests is complex due to hormonal variations during the menstrual cycle and hormonal contraceptive use, both influencing the urinary steroid profile. Exercise is suggested to affect circulating steroid hormone levels, and in women, the urinary steroid profile differs between in competition testing and out of competition testing. No previous study has investigated the relationship between amount of exercise and the urinary steroid profile in female elite athletes.PurposeTo compare the urinary steroid profile between female Olympic athletes and age- and BMI-matched untrained controls, and to study the urinary steroid profile in relation to serum hormones and amount of exercise.MethodsIn this cross-sectional study conducted at the Women’s Health Research Unit, Karolinska University Hospital, Stockholm, 94 female elite athletes and 86 untrained controls were included. Serum estrogens and testosterone and the urinary steroid profile were analyzed by liquid chromatography–tandem mass spectrometry and gas chromatography-tandem mass spectrometry, respectively. Exercise hours/week were evaluated by questionnaire.ResultsAlthough serum steroid hormones were comparable between groups, the athletes demonstrated approximately 30% lower urinary steroid metabolites of testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstan-3α, 17β-diol, and 5β-androstan-3α, 17β-diol compared to the controls. The urinary steroid metabolites correlated positively with serum steroid hormones. In the athletes, urinary steroid metabolites: androsterone (rs = −0.28, p = 0.007), epitestosterone (rs = −0.22, p = 0.034), 5αAdiol (rs = −0.31, p = 0.002) and testosterone (rs = −0.24, p = 0.026), were negatively correlated with amount of training (hours per week).ConclusionThe urinary concentrations of steroid metabolites were lower in elite athletes than in sedentary controls, although serum steroids were comparable between groups. Moreover, exercise time was negatively associated with the urinary concentrations. Our findings suggest alternative excretion routes of androgens in the athletes related to training. more...

At the Swedish national forensic toxicology laboratory, a measured testosterone/epitestosterone (T/E) ratio ≥ 12 together with testosterone/luteinizing hormone (T/LH) in urine > 400 nmol/IU is considered as a proof of exogenous testosterone administration. However, according to the rules of the World Anti-Doping Agency (WADA), samples with T/E ratio > 4 are considered suspicious and shall be further analysed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to confirm the origin of testosterone and its metabolites. The aim of this study was to investigate the possibility of false negative results and to estimate the frequency of negative results using the current criteria for detection of abuse of testosterone in forensic investigations. Urine and serum samples were collected by the police at suspected infringement of the doping law in Sweden. Fifty-eight male subjects were included in the study. Urinary testosterone was determined by gas chromatography-mass spectrometry (GC-MS), serum testosterone and LH-by immunoassay. The origin of testosterone and its metabolites was confirmed by means of GC-C-IRMS. Twenty-six of the 57 analysed subjects tested positive for exogenous testosterone using the criteria T/E ≥ 12 combined with T/LH > 400 nmol/IU. The IRMS analyses confirmed 47 positives; thus, 21 were considered false negatives. Negative predictive value was 32% (95% confidence interval [CI]: 16%-50%) and sensitivity 55%. No false positive subjects were found. The number of false negative cases using the current criteria for the detection of testosterone abuse and hence the low sensitivity indicates a need to discuss introduction of new strategies in forensic doping investigations. more...

In this work we have investigated the influence of the intake of two synthetic isoflavones, methoxyisoflavone and ipriflavone, on the urinary concentration of endogenous steroids, and on their relative ratios, of doping relevance. Specifically, the concentrations of testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio), 5α-androstan-3α,17α-diol (5αAdiol), 5β-androstan-3α,17α-diol (5βAdiol), and the ratios T/E, A/T, A/Etio, 5αAdiol/5βAdiol, 5αAdiol/E, were considered, in the framework of the Steroidal Module of the Athlete Biological Passport (ABP). The above set of parameters were complemented by the urinary levels of luteinizing hormone (total LH) and the ratio between T and LH (T/total LH), to assess the possible effects on the biosynthesis of the mentioned steroids. Five healthy Caucasian male volunteers were selected for the study. Urine samples were collected before and during the administration of (i) methoxyisoflavone (Methoxyisoflavone, MyProtein) and (ii) ipriflavone (Osteofix ®, Chiesi Farmaceutici). For the analysis of the urinary steroid profile, after enzymatic hydrolysis with β-glucuronidase from Escherichia Coli (E. Coli) and liquid–liquid extraction with tert-buthylmethyl ether, all samples were analyzed by gas chromatography coupled to tandem mass spectrometry (GC–MS/MS), while for the determination of total LH all urine samples were directly analyzed by a chemiluminescent immunometric assay technique (Siemens Immulite 2000 LH). Our results show that the administration of either methoxyisoflavone or ipriflavone causes an alteration of the urinary concentrations and concentration ratios of the investigated steroids, in the range 55–80% from the baseline values. Furthermore, an oversecretion of LH after the daily intake of methoxyisoflavone or ipriflavone was also recorded in all volunteers, corresponding to an increase in the biosynthesis and excretion of T and some of its metabolites. These changes trigger a disregulation in the pattern of urinary excretion of the steroids included in the Steroidal Module of the ABP, which makes more difficult the interpretation of the longitudinal steroid profile based on the definition of individual normality ranges for each athlete. Our data are also consistent with previous evidence regarding the in vitro effects of natural and synthetic isoflavones, suggesting that their monitoring in doping control routine analysis would be very beneficial for the result management activities. more...

To detect doping with pseudo-endogenous anabolic steroids in sports, a urinary steroid profile with glucuronidated plus unconjugated androgens is used. In addition to analyze androgen glucuronide metabolites, it can be of interest to also include sulfate metabolites in the urinary steroid profile. The combined ratios of epitestosterone sulfate/epitestosterone glucuronide to the ratios of testosterone sulfate/testosterone glucuronide ((ES/EG)/(TS/TG)) have previously been investigated as a complementary biomarker for testosterone doping. In this restudy, the aim was to evaluate this biomarker in a larger study sample population. A single dose of 500-mg testosterone enanthate was administered to 54 healthy male volunteers. Urine was collected prior to (Day 0) administration and throughout 15 days and analyzed for the sulfate and glucuronide conjugates of testosterone and epitestosterone. The results show that the combined ratio increased to a larger extent than the traditional T/E ratio in all subjects. This increase was independent on UGT2B17 gene polymorphism. Moreover, a delayed peak of the combined ratio was observed in ~60% of the participants. The results confirm that complementary analyses of the sulfate metabolites may be a useful approach to detect testosterone doping in men. more...

Serum analysis has received much attention in regulatory analysis of food-producing animals, especially for anabolic steroids. The possibility of confirming the parent drugs with minimum metabolization enables the detection of intact steroid esters, whose identification represents unequivocal proof of drug administration. This work involved the development and validation of a quantitative LC-MS/MS method to determine 30 steroids and steroid esters in bovine serum. Sensitivity was improved using microwave-assisted chemical derivatization with methoxyamine hydrochloride. The validation was successfully conducted in accordance with the Decision 657/2002/EC guidelines. An in vivo experiment was performed on 12 crossbred steers in which two commercial formulations containing boldenone undecylenate and testosterone propionate were administrated via intramuscular injections. The samples were collected over a period of 120 days, in which both intact esters were identified within 11 days postadministration. 17β-Boldenone was observed after 92 days for 2 steers and 56 days for the other animals. The applicability of a cut-off level to the ratio between 17β-testosterone and epitestosterone was evaluated in an attempt to differentiate testosterone abuse from endogenous production. It could be observed that a calculated ratio above this level is strong evidence of drug administration, although a high false-negative rate was obtained. more...

The steroidal module of the Athlete Biological Passport (ABP) has been used since 2014 for the longitudinal monitoring of urinary testosterone and its metabolites in order to identify samples suspicious for the use of synthetic forms of endogenous anabolic androgenic steroids (EAAS). Samples identified by the module may then be confirmed by isotope ratio mass spectrometry (IRMS) to establish clearly the exogenous origin of testosterone and/or metabolites in the sample. To examine the detection capability of the steroidal ABP model, testosterone administration studies were performed with various doses and three routes of administration - transdermal, intramuscular, and subcutaneous with 15 subjects for each route of administration. Urine samples were collected before, during, and after administration and steroid profiles were analyzed using the steroidal ABP module in ADAMS. A subset of samples from each mode of administration was also analyzed by IRMS. The steroidal ABP module was more sensitive to testosterone use than population-based thresholds and with high dose administrations there was very good agreement between the IRMS results and samples flagged by the module. However, with low dose administration the ABP module was unable to identify samples where testosterone use was still detectable by IRMS analysis. The testosterone/epitestosterone (T/E) ratio was the most diagnostic parameter for longitudinal monitoring with the exception of low testosterone excretors for whom the 5 alpha-androstane-3 alpha, 17 beta-diol/epitestosterone (5 alpha Adiol/E) ratio may provide more sensitivity. more...

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 Δδ13C 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, its 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. more...

Five intratesticular sex steroids (testosterone, dihydrotestosterone, androstenedione, estradiol and epitestosterone) along with six serum hormones (LH, FSH, prolactin, SHBG, testosterone and estradiol) were determined in 84 non-obstructive azoospermic men, in order to evaluate to what extent serum and testicular tissue as well as individual hormones in the same material mutually correlate. With exception of androstenedione, tight correlations were found among tissue content of sex steroids, while only weak correlation was recorded between serum and testicular concentrations of major sex steroids testosterone and estradiol. It points to importance of measurement of intratesticular steroids in combination with examination of sperm parameters for assessment of testicular function and spermatogenesis. more...

The impact of dehydroepiandrosterone (DHEA) administration has been widely studied for anti-doping purposes in men, whereas only a few studies have been performed in women. In the present study, the impact of DHEA on the steroid profile parameters and their carbon isotopic ratios was explored. Eleven healthy young women and 10 healthy young men received two treatments: One with 100 mg/day of DHEA for 28 days and one with a placebo according to a double-blind crossover protocol. Urine and saliva (only in females) samples were collected before and for 72 hours after each short-term treatment. In all female subjects, concentrations of the urinary parameters of the steroid profile were highly impacted by short-term DHEA administration including epitestosterone (E). Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis was performed and positive results were observed for E in the four female subjects where E concentration was adequate for such analysis, whereas men results remained negative for E. Last, the ability of the Anti-Doping Administration and Management System (ADAMS) software used for the athlete biological passport to identify such doping was assessed. Of the 11 passports generated for female subjects, 10 were automatically classified as an atypical passport finding (ATPF). For the remaining passport with normal status in one woman, the variability of the concentrations prevented the ADAMS software from adjusting individual limits. The most impacted markers in women were T/E and 5αAdiol/E, with a detection window of 36 hours for 5αAdiol/E. In addition, good correlations were observed for DHEA and T concentrations in urine and saliva in females. more...

Objective To investigate the excretion and conjugation profile of testosterone (T), Epitestosterone (EpiT), and other androgen metabolites in different phases of pregnancy and postpregnancy as a reflection of the “androgenic exposure.” Design Consecutive recruitment of pregnant women. Setting Maternity outpatient low-risk pregnancy clinic. Patients Seventy-seven pregnant women. Interventions Collection of urine for analyses of sulfate (S) and glucuronide (G) conjugates and metabolic ratios of androgens and androgen metabolites using liquid chromatography-tandem mass spectrometry. Main Outcome Measures Excretion profiles and metabolic ratios of G and S conjugates of T, EpiT, dehydroepiandrosterone (DHEA), androsterone (A), etiocholanolone (Etio), and dihydrotestosterone in relation to trimester and postpartum, body mass index, fetal sex, and ethnicity. Results T-S excretion increased significantly between the second and third trimester, whereas excretion of T-G did not change. In contrast, both conjugates of EpiT increased markedly, more so for the S-(17-fold) than the G-conjugate (1.6-fold). The preference for S over G conjugation was conspicuous for EpiT and DHEA (S/G ratio 2.1 and 4.7, respectively, in the third trimester), whereas the reverse was true for T, A, and Etio (S/G 0.6, 0.13, and 0.11, respectively). Conclusions Pregnancy influences the androgen excretion profile, with the most profound change being an increase in EpiT excretion throughout the trimesters. EpiT may modulate the effect of T, but its exact role during pregnancy is not known. There were marked differences in the S/G conjugate ratios between androgens upstream and downstream from T in the metabolic network. These results are interesting to compare with the androgen disposition in women with endocrine disorders or abuse of steroids., The urinary excretion rate and pattern of conjugated androgens vary widely in pregnancy among women, androgens, and trimesters and may reflect the androgenic “exposure” to the feto-maternal unit. more...