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The first step in the detection of testosterone (T) doping is to measure the urinary steroid profile for the athlete biological passport (ABP). To harmonise the analysis between anti-doping laboratories, urinary steroid profiling is parametrised in deep detail and shall be performed by gas chromatography hyphenated to mass spectrometry (GC-MS). However, due to its requirement for extensive sample preparation, alternatives to GC-MS are being actively pursued. The aim of this study was the evaluation of Ultra-High-Performance Supercritical Fluid Chromatography hyphenated to tandem Mass Spectrometry (UHPSFC-MS/MS) as an alternative for the quantification of endogenous urinary steroids. In this context, we developed a high throughput sample extraction method, followed by a novel UHPSFC-MS/MS method for the analysis of 10 endogenous urinary steroids which are relevant for doping control analysis. Depending on the steroid, the herein presented method is capable of quantification from 0.5 ng/mL up to 10 µg/mL. After validation, the applicability of the method was evaluated by analysing 132 authentic urine samples, which demonstrated results similar to classical GC-MS analysis. Steroid concentrations determined by UHPSFC-MS/MS were slightly overestimated in comparison with GC-MS, but the ratios had <10 % difference between the two methods. As the ABP considers the steroid ratios for passport evaluation, the herein presented method could be used for steroid profiling without reducing the sensitivity of the ABP. Thus, we would propose to consider UHPSFC-MS/MS as an alternative to GC-MS after more tests would have been performed to support our findings. Furthermore, we have also investigated the potential of this technology for sample purification prior to Isotope Ratio Mass Spectrometry (IRMS) for the differentiation between exogenous and endogenous origin of T and its metabolites. While the achieved separation was sufficient to purify urine samples for IRMS analysis in our proof-of-concept study, the instrumental parameters should be further refined for future use., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

For antidoping laboratories, the determination of an illicit testosterone (T) administration in urine samples remains a difficult process as it requires the determination of the exogenous origin by carbon isotope ratios (CIRs) of testosterone and its metabolites. As a complement to the urinary analysis, targeting testosterone esters (e.g. testosterone undecanoate [TU]) in serum samples by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) could represent a simpler approach compared with isotope ratio mass spectrometry (IRMS). These two approaches both lead to the direct detection of the administration of exogenous T but with a difference in effort and complexity of the analysis. To compare the detection window obtained with the two strategies, serum and the corresponding urine samples collected from an administration study with oral TU were analysed. Results showed that, at all timepoints where the intact TU was detected in serum, the CIRs of urinary steroids were also not in agreement with an endogenous origin. IRMS analysis required more effort but resulted in slightly longer detection windows than the ester analysis. Finally, this comparison study showed that, in the presence of a suspicious urinary steroid profile, the LC-MS/MS steroid esters analysis in the corresponding serum samples can be very helpful. If steroid esters are not detected, the IRMS analysis can then be conducted on the urine sample afterwards. Overall, the combination of matrices might facilitate the detection of prohibited T administration in sports, especially for athletes with naturally low T/E ratios., (© 2024 John Wiley & Sons Ltd.)

Sulfate metabolites of endogenous anabolic androgenic steroids (EAAS) have been shown to prolong the detection times compared with the conventional urinary markers of the steroid profile for oral and intramuscular administrations of testosterone (T). In this work, the sensitivity of sulfate EAAS markers for the detection of T gel administration has been evaluated in six Caucasian and six Asian male volunteers. Fourteen sulfate metabolites were measured in basal and post-administration samples after multiple doses of T gel (100 mg/day, three consecutive days), and the detection times based on individual thresholds for each volunteer were evaluated. Sulfate concentrations did not show adequate sensitivity, but the results of sulfate ratios were much more promising. Androsterone sulfate/testosterone sulfate (A-S/T-S), epiandrosterone sulfate/epitestosterone sulfate (epiA-S/E-S), epiA-S/T-S, and etiocholanolone sulfate/epitestosterone sulfate (Etio-S/E-S) provided the most consistent detectability for all volunteers and populations, with detection times ranging from 60 to 96 h since the first dose. Additional ratios improved detectability to up to 7 days, but only in particular volunteers. In general, sensitivity was similar to or better than the conventional testosterone/epitestosterone ratio (T/E) of the steroid profile, which further reinforces the conclusion that sulfate EAAS metabolites can be a good complement for the current steroid profile., (© 2023 John Wiley & Sons, Ltd.)

Aims: Arsenic exposure is a significant global public health concern and has been implicated in endocrine disruption and increased oxidative stress, both of which are crucial pathogenic mechanisms of periodontitis. This study aimed to investigate the association of urinary total arsenic and arsenic species with periodontitis and to further explore the potential mediating roles of sex hormones and oxidative stress indicators., Methods: Data used in this study were derived from the 2013-2014 National Health and Nutrition Examination Survey (NHANES) in the US population. In all, 1063 participants with complete data were included in this study. Weighted logistic regression analyses were used to evaluate the relationship between urinary arsenic and periodontitis. Mediation analyses were used to explore the effects of potential mediators on these associations., Results: High concentrations of urinary dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), 2 types of toxic urinary arsenic (TUA2), and 4 types of toxic urinary arsenic (TUA4) were positively related to periodontitis (P < .05). After adjusting for potential confounders, the positive association remained significant (odds ratio, 1.32; 95% confidence interval, 1.01-1.71). Testosterone may partially mediate the relationship between MMA and periodontitis, with mediating effects of 21.78% and 39.73% of the total effect. No significant mediation effect of oxidative stress indicators was found for this relationship., Conclusions: This study reports a positive association between urinary MMA and periodontitis, and testosterone may mediate this relationship. Our findings serve as a call for action to avoid the deployment of arsenic-containing therapeutic agents as treatment modalities for oral afflictions., Competing Interests: Conflict of interest None disclosed., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Selective androgen receptor modulators (SARMs) are prohibited by the World Anti-Doping Agency (WADA) since 2008. Similarly, to anabolic androgenic steroids (AAS), SARMs are detrimental to health not only in athletes but also in the general population. However, studies of the occurrence of SARMs outside of sport are scarce. Swedish healthcare samples from the Drugs of Abuse Laboratory at Karolinska were analyzed using WADA-accredited screening methods at the Doping Control Laboratory in Stockholm to estimate the frequency of SARM use outside of the WADA laboratories. Twenty (4%) of the male urine samples (n = 542) were positive for SARMs, whereas none of the analyzed female samples (n = 100) contained any SARMs. The top three SARMs found were LGD-4033 followed by RAD140 and ostarine. Two or more SARMs were found in >50% of the SARM-positive samples. AASs were identified in 40% of samples containing SARMs. A difference between genders was observed where 34% male and 7% female samples contained AAS. Many samples displayed testosterone/epitestosterone values indicative of testosterone intake, without presence of other AAS, and hence, there is a risk that these samples are being falsely reported as negative. Our results indicate that SARM use might be a concern outside of sport. Subsequently, in addition to AAS, the healthcare system should also be informed about SARM abuse and the associated adverse side effects., (© 2023 John Wiley & Sons Ltd.)

The steroid module of the Athlete Biological Passport (ABP) is based on the analysis of six endogenous steroids in urine samples and a Bayesian statistical approach. However, the urinary steroid concentrations may be affected by confounders like microbial degradation, possible co-administration of diuretics as masking agents, insufficient conjugate hydrolysis or UGT2B17 gene polymorphisms affecting glucuronidation. Therefore, it can be helpful to use other matrices (ABP blood and serum samples) to quantify steroids and thereby support noticeable deviations in the Athlete Biological Passport, for example, abnormally increased urinary testosterone/epitestosterone (T/E) ratios. Aim of the study was to investigate the feasibility to re-use plasma obtained from athlete ABP blood samples for measuring a steroid profile. Therefore, testosterone, androstenedione, cortisol and cortisone were quantified in 36 intra-individual matching ABP blood and serum samples. The steroid levels measured in both matrices showed a high agreement indicating a good stability uninfluenced by storage temperature and duration. Our results pointed out the possibility to expand the athlete ABP blood analysis for steroid profiling., (© 2022 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

This study aims to investigate the effect of arsenic exposure on urinary levels of arsenic metabolites, semen parameters, and testosterone concentrations. A systematic comprehensive literature search was conducted up till 31st January 2024 using Embase, MEDLINE/Pubmed, and Scopus. This study adopted the Population Exposure Comparator Outcome and Study Design (PECOS) framework. Four studies with a total of 380 control subjects and 347 exposed men were included. Arsenic exposure significantly increased urinary levels of total arsenic (Mean Difference (MD) - 53.35 [95 % Confidence Interval (CI): - 100.14, - 6.55] P= 0.03), and reduced primary arsenic methylation index (PMI) (MD 0.22 [95 % CI: 0.14, 0.31] P< 0.00001), semen volume (MD 0.30 [95 % CI: 0.05, 0.54] P= 0.02) and total testosterone (MD 0.48 [95 % CI: 0.23, 0.73] P= 0.0002). In addition, arsenic exposure marginally reduced sperm concentration (MD 25.04 [95 % CI: - 45.42, 95.50] P= 0.49) and total sperm motility (MD 22.89 [95 % CI: - 14.15, 59.94] P= 0.23). The present meta-analysis demonstrates that arsenic exposure lowers semen quality and testosterone levels. Since the general human population is exposed to arsenic occupationally or domestically, adequate strategic measures should be put in place to limit arsenic exposure in an attempt to preserve semen quality. In addition, studies investigating interventions that may inhibit the bioaccumulation of arsenic in men who are exposed are recommended., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Steroids are cholesterol-derived biomolecules that play an essential role in biological processes. These substances used as growth promoters in animals are strictly regulated worldwide. Targeted assays are the conventional methods of monitoring steroid abuse, with limitations: only detect known metabolites. Metabolism leads to many potential compounds (isomers), which complicates the analysis. Thus, to overcome these limitations, non-targeted analysis offers new opportunities for a deeper understanding of metabolites related to steroid metabolism. Molecular networking (MN) appears to be an innovative strategy combining high-resolution mass spectrometry and specific data processing to study metabolic pathways. In the present study, two databases and networks of steroids were constructed to lay the foundations for the implementation of the GNPS-MN approach. Steroids of the same family were grouped together, nandrolone and testosterone were linked to other analogues. This network and associated database were then applied to a few urine samples in order to demonstrate the annotation capacity in steroidome study. The results show that MN strategy could be used to study steroid metabolism and highlight biomarkers., (© 2024. The Author(s).)

Objective: Human choriogonadotrophin (hCG) treatment of gonadotrophin-deficient infertile men uses hCG of urinary (uhCG) or recombinant (rhCG) origin, but these treatments have not been compared nor are there studies defining rhCG dosing in men., Design: hCG products were studied in randomized cross-over single-dose studies of standard (Study 1, 1500 IU and 62.5 µg, respectively) or high (Study 2, 5000 IU and 250 µg) dose and a multi-dose population pharmacology study of hCG use., Participants: Eight (Study 1) and seven (Study 2) volunteers in cross-over and 52 gonadotrophin-deficient men in the multi-dose study MEASUREMENTS: In cross-over studies, serum testosterone (T), dihydrotestosterone (DHT) and estradiol by liquid chromatography-mass spectrometry (LCMS) and serum hCG, LH, FSH, SHBG and T (observational study) by immunoassays., Results: After standard and high-dose injection, serum hCG and testosterone responses had similar timing and peak concentrations except for a mildly lower early (<48 h) serum testosterone with uhCG. In the multi-dosing study, both hCGs had similar pharmacokinetics (pooled half-life 5.8 days, p < .001), while serum testosterone concentrations were stable after injection and did not differ between hCG products. Bench testing verified that 20% of pens from 4/10 individuals were used inappropriately., Conclusions: Although hCG pharmacokinetics are not formally bioequivalent, the similar pharmacodynamic effects on serum testosterone indicate that at the doses tested both hCGs provide comparable clinical effects. The starting dose of rhCG for treating gonadotrophin-deficient men should be 62.5 µg (6 clicks) of the rhCG pen., (© 2024 Commonwealth of Australia. Clinical Endocrinology published by John Wiley & Sons Ltd.)

Sex and thyroid hormones are critical for male reproductive health. However, the associations between haloacetic acid (HAA) exposure - a known endocrine disruptor - and sex and thyroid hormones in humans remains unclear. We thus recruited 502 male participants seeking fertility evaluation from a reproductive center. We measured concentrations of sex and thyroid hormones in a single blood sample and dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA) in repeated urine samples. Multivariable linear regression models were constructed to evaluate the associations between HAA concentrations and hormone measurements. After adjusting for potential confounders and urinary creatinine concentrations, urinary concentrations of TCAA were inversely associated with serum levels of sex hormone-binding globulin (SHBG), testosterone (T), T/luteinizing hormone ratio (T/LH), and thyroid stimulating hormone (TSH) (all P for trend < 0.10). Compared with participants in the lowest quartile of TCAA concentrations, those in the highest quartile had reduced serum levels of SHGB by 14.2 % (95% CI: -26.7, -3.0 %), T by 11.1 % (95% CI: -21.7, -1.3 %), T/LH by 21.0 % (95% CI: -36.7, -7.1 %), and TSH by 19.1 % (95% CI: -39.7, -1.5 %). Additionally, we observed inverse associations between continuous measurements of urinary HAAs and serum levels of free T, bioactive T, and estradiol. Our findings suggest that male HAA exposure may be associated with disrupted sex and thyroid function., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yixin Wang reports financial support was provided by Foundation for Innovative Research Groups of the National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Organophosphate, pyrethroid, and neonicotinoid insecticides have resulted in adrenal and gonadal hormone disruption in animal and in vitro studies; limited epidemiologic evidence exists in humans. We assessed relationships of urinary insecticide metabolite concentrations with adrenal and gonadal hormones in adolescents living in Ecuadorean agricultural communities., Methods: In 2016, we examined 522 Ecuadorian adolescents (11-17y, 50.7% female, 22% Indigenous; ESPINA study). We measured urinary insecticide metabolites, blood acetylcholinesterase activity (AChE), and salivary testosterone, dehydroepiandrosterone (DHEA), 17β-estradiol, and cortisol. We used general linear models to assess linear (β = % hormone difference per 50% increase of metabolite concentration) and curvilinear relationships (β 2  = hormone difference per unit increase in squared ln-metabolite) between ln-metabolite or AChE and ln-hormone concentrations, stratified by sex, adjusting for anthropometric, demographic, and awakening response variables. Bayesian Kernel Machine Regression was used to assess non-linear associations and interactions., Results: The organophosphate metabolite malathion dicarboxylic acid (MDA) had positive associations with testosterone (β boys  = 5.88% [1.21%, 10.78%], β girls  = 4.10% [-0.02%, 8.39%]), and cortisol (β boys  = 6.06 [-0.23%, 12.75%]. Para-nitrophenol (organophosphate) had negatively-trending curvilinear associations, with testosterone (β 2 boys  = -0.17 (-0.33, -0.003), p = 0.04) and DHEA (β 2 boys  = -0.49 (-0.80, -0.19), p = 0.001) in boys. The neonicotinoid summary score (β boys  = 5.60% [0.14%, 11.36%]) and the neonicotinoid acetamiprid-N-desmethyl (β boys  = 3.90% [1.28%, 6.58%]) were positively associated with 17β-estradiol, measured in boys only. No associations between the pyrethroid 3-phenoxybenzoic acid and hormones were observed. In girls, bivariate response associations identified interactions of MDA, Para-nitrophenol, and 3,5,6-trichloro-2-pyridinol (organophosphates) with testosterone and DHEA concentrations. In boys, we observed an interaction of MDA and Para-nitrophenol with DHEA. No associations were identified for AChE., Conclusions: We observed evidence of endocrine disruption for specific organophosphate and neonicotinoid metabolite exposures in adolescents. Urinary organophosphate metabolites were associated with testosterone and DHEA concentrations, with stronger associations in boys than girls. Urinary neonicotinoids were positively associated with 17β-estradiol. Longitudinal repeat-measures analyses would be beneficial for causal inference., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The World Anti-Doping Agency (WADA) has introduced an Athlete Biological Passport (ABP) with a steroidal module, which is intended for the monitoring of longitudinal profiles of an athlete's steroid variables in urine to identify endogenous anabolic androgenic steroids that are administered exogenously. It has been in use since 2014. The prevalence of UGT2B17 gene deletion with relatively low levels of testosterone (T) glucuronide in urine is high in the Asian region. There are cases in which urinary T is below the detection limit in specific urine samples, for example, diluted urine, urine collected from females, or urine collected from UGT2B17 deletion individuals. Additional steroid markers T, 4-androstenedione (A4), and the T/A4 ratio in serum were newly added to the ABP steroidal module by WADA in 2023 to compensate for the urine steroid profile. In this study, populations of blood steroid markers in Asians (n = 510) were investigated and classified according to UGT2B17 polymorphism to confirm the effectiveness of blood steroid markers in monitoring Asian athletes. No significant difference in the T/A4 ratio was observed between the genotypes. Furthermore, an administration study of T enanthate in females (n = 10) who were classified according to UGT2B17 genotypes was performed. The concentration of T and the T/A4 ratio were found to be significantly increased in all post-administration samples until 15 days after administration (p < 0.01). The overall results supported the high effectiveness of subject-based monitoring for serum T and T/A4 ratio for recently identified shortcomings in the detection of T abuse in Asians., (© 2023 John Wiley & Sons Ltd.)

Boldenone is an anabolic-androgenic steroid (AAS) that is prohibited in equine sports. However, in certain situations, it is endogenous, potentially formed by the microbes in urine. An approach to the differentiation based on the detection of the biomarkers Δ1-progesterone, 20(S)-hydroxy-Δ1-progesterone and 20(S)-hydroxyprogesterone was assessed, and their concentrations were monitored in the urine of untreated female horses (n = 291) alongside boldenone, boldienone, testosterone and androstenedione. Using an ultra-sensitive analytical method, boldenone (256 ± 236 pg/mL, n = 290) and the biomarkers (Δ1-progesterone up to 57.6 pg/mL, n = 8; 20(S)-hydroxy-Δ1-progesterone 85.3 ± 181 pg/mL, n = 130; 20(S)-hydroxyprogesterone 43.5 ± 92.1 pg/mL, n = 158) were detected at low concentrations. The ex vivo production of Δ1-steroids was artificially induced following the storage of urine samples at room temperature for 7 days in order to assess the concentrations and ratios of the monitored steroids. The administration of inappropriately stored feed source also resulted in an increase in 20(S)-hydroxy-Δ1-progesterone concentrations and the biomarker ratios. Using the results from different datasets, an approach to differentiation was developed. In situations where the presence of boldenone exceeds a proposed action limit of 5 ng/mL, the presence of the biomarkers would be investigated. If Δ1-progesterone is above 50 pg/mL or if 20(S)-hydroxy-Δ1-progesterone is above 100 pg/mL with the ratio of 20(S)-hydroxy-Δ1-progesterone:20(S)-hydroxyprogesterone greater than 5:1, then this would indicate ex vivo transformation or consumption of altered feed rather than steroid administration. There remains a (small) possibility of a false negative result, but the model increases confidence that adverse analytical findings reported in female horses are caused by AAS administrations., (© 2023 John Wiley & Sons, Ltd.)

This work focused on the possible alterations of the markers of the steroidal module of the athlete biological passport, considering samples of athletes declaring and not-declaring the supplementation of thyroid hormones (TH) in the Doping Control Form (DCF). Concentrations of 5α-androstane-3α,17β-diol (5α-Adiol), 5β-androstane-3α,17β-diol (5β-Adiol), testosterone (T), androsterone (A), etiocholanolone (Etio), epitestosterone (E), pregnanediol (PD), dehydroepiandrosterone (DHEA), and 11β-hydroxy-androsterone (OHA) were calculated using internal standards and external calibration by gas chromatography-tandem mass spectrometry. Also, ratios between the above biomarkers were also estimated. The data set was composed of samples from females and males declaring and not-declaring TH supplementation in the DCF. To corroborate these observations, a controlled urinary excretion study was carried out with multiple doses of sodium liothyronine (T3). Female data showed significant differences for the concentrations of 5α-Adiol, A, DHEA, E, OHA, and T and the ratio A/Etio between FD and FND groups, whereas the male groups only showed significant differences in OHA concentration. In both cases, males and females declaring the consumption of levothyroxine showed narrower data distribution and diminished percentiles from 17% to 67% with respect to the not-declaring corresponding groups (p < 0.05). Concentrations of 5α-metabolites showed a higher depression for the FND, and both FD and MD groups showed a peculiar behavior for the PD concentrations. The controlled study agreed with the observations, mainly for the female group with significant differences for concentrations of E, Etio, 5α-Adiol, and 5β-Adiol after TH administration. The interpretation of the steroid markers of the ABP should consider TH administrations., (© 2023 John Wiley & Sons Ltd.)

The Athlete Biological Passport (ABP) is a longitudinal tool used in anti-doping to monitor biological parameters known to change with performance-enhancing drug use. The ABP consists of multiple modules, including two aimed at detecting the use of endogenous anabolic androgenic steroids: the urinary and serum steroid modules. Human chorionic gonadotropin (hCG) is a protein hormone potentially abused by male athletes to increase the production of endogenous testosterone. To date, no studies have investigated the impact of extended hCG administration on the urinary and serum steroid modules of the ABP. The goal of this study was to identify the impact of multiple hCG administrations on the parameters tracked as part of the urinary and serum steroid modules of the ABP. Ten recreationally active, healthy male individuals self-administered seven 250 μg hCG injections over 3 weeks. Serum and urine samples were collected before, during, and 2 weeks following the final injection. All ABP parameters were quantified in the respective matrix, and steroid profiles were created with Anti-Doping Administration and Management System adaptive model upper and lower limits for both matrices. In both serum and urine profiles, testosterone increased; however, the testosterone/epitestosterone ratio in urine and the testosterone/androstenedione ratio in serum showed minimal changes. Additionally, serum luteinizing hormone (LH) was quantified using an immunoassay, and a serum testosterone/LH ratio was generated. Serum LH values decreased during administration causing large increases in the serum T/LH ratio, indicating this ratio may be a more sensitive parameter for detecting hCG abuse than urinary testosterone/epitestosterone or serum testosterone/androstenedione., (© 2023 John Wiley & Sons Ltd.)

Background: Most current state-of-the-art strategies to generate individual adaptive reference ranges are designed to monitor one clinical parameter at a time. An innovative methodology is proposed for the simultaneous longitudinal monitoring of multiple biomarkers. The estimation of individual thresholds is performed by applying a Bayesian modeling strategy to a multivariate score integrating several biomarkers (compound concentration and/or ratio). This multimodal monitoring was applied to data from a clinical study involving 14 female volunteers with normal menstrual cycles receiving testosterone via transdermal route, as to test its ability to detect testosterone administration. The study samples consisted of urine and blood collected during 4 weeks of a control phase and 4 weeks with a daily testosterone gel application., Results: Integrating multiple biomarkers improved the detection of testosterone gel administration with substantially higher sensitivity compared with the distinct follow-up of each biomarker, when applied to selected urine and serum steroid biomarkers, as well as the combination of both. Among the 175 known positive samples, 38% were identified by the multimodal approach using urine biomarkers, 79% using serum biomarkers and 83% by combining biomarkers from both biological matrices, whereas 10%, 67% and 64% were respectively detected using standard unimodal monitoring., Significance and Novelty: The detection of abnormal patterns can be improved using multimodal approaches. The combination of urine and serum biomarkers reduced the overall number of false-negatives, thus evidencing promising complementarity between urine and blood sampling for doping control, as highlighted in the case of the use of transdermal testosterone preparations. The generation in a multimodal setting of adaptive and personalized reference ranges opens up new opportunities in clinical and anti-doping profiling. The integration of multiple parameters in a longitudinal monitoring is expected to provide a more complete evaluation of individual profiles generating actionable intelligence to further guide sample collection, analysis protocols and decision-making in clinics and anti-doping., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The steroid module of the athlete biological passport (ABP) aims to detect doping with endogenous steroids by longitudinally monitoring epitestosterone (E), testosterone (T), and four metabolically related steroids and their ratios. There are large variations in the urinary levels of the androgen metabolites due to genetic polymorphisms, drug use, menstrual cycle, and other factors. In this study, we aimed to increase our understanding of the natural, within-individual variations of the established ABP markers in males and females over time, looking at samples collected both in and out-of-competition (IC/OOC). Urinary steroid profiles from 323 Swedish athletes, with at least five samples per athlete, were extracted from ADAMS together with information on type of sport, IC/OOC, and time of day. Data were analyzed using coefficient of variation (CV%) to examine within-subject variability and linear mixed effects models to estimate within-subject change in the metabolites over time. The metabolites and ratios expressed higher individual CV% in females (23-56) than in males (18-39). Samples taken OOC showed larger intra-individual variations than samples collected IC for most of the ABP metabolites in both sexes. The median concentrations were higher IC for some metabolites, particularly testosterone being 52% higher among females. Time of day influenced the intra-individual variation of the urinary steroid profile with decreases in androgen metabolites over time, if measured in evening versus daytime. These findings can aid in the testing strategies and interpretation of the steroidal module of ABP., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

When testing for anabolic androgenic steroids (AAS) outside sports communities, for example, in healthcare and forensic medicine, urine is the matrix of choice. However, there are drawbacks with urinary sampling, and serum might be useful as a complementary matrix. The aim was to develop an LC-MS/MS method for serum measuring AAS frequently used outside of sport, including testosterone (T), steroid esters, and eight other synthetic AAS. The sample pretreatment included sample precipitation and evaporation. Limit of quantification for the AAS was 0.05-0.5 ng/mL, and linearity was 0.05-20 ng/mL for most of the substances. Generally, the within- and between-day CV results, matrix effect, and process efficiency were <15%. The AAS were stable for at least 6 months at -20°C. Serum samples were obtained from previous studies. A novel finding from an administration study was that T enanthate was present in serum even after 5 years of storage at -20°C. Serum samples from self-reporting AAS individuals, where T esters were detected, were positive for testosterone using the urinary testosterone/epitestosterone criterion >10. Of those identified as positive in traditional urinary doping tests (n = 15), AAS in serum were found in 80% of the subjects. Our results show that serum may be a valid complementary matrix to urine samples for AAS testing., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The steroidal module of the athlete biological passport (ABP) targets the use of pseudo-endogenous androgenous anabolic steroids in elite sport by monitoring urinary steroid profiles. Urine and blood samples were collected weekly during two consecutive oral contraceptive pill (OCP) cycles in 15 physically active women to investigate the low urinary steroid concentrations and putative confounding effect of OCP. In urine, testosterone (T) and epitestosterone (E) were below the limit of quantification of 1 ng/ml in 62% of the samples. Biomarkers' variability ranged between 31% and 41%, with a significantly lesser variability for ratios (except for T/E [41%]): 20% for androsterone/etiocholanolone (p < 0.001) and 25% for 5α-androstane-3α,17β-diol/5ß-androstane-3α,17β-diol (p < 0.001). In serum, markers' variability (testosterone: 24%, androstenedione: 23%, dihydrotestosterone: 19%, and T/A4: 16%) was significantly lower than in urine (p < 0.001). Urinary A/Etio increased by >18% after the first 2 weeks (p < 0.05) following withdrawal blood loss. In contrast, serum T (0.98 nmol/l during the first week) and T/A4 (0.34 the first week) decreased significantly by more than 25% and 17% (p < 0.05), respectively, in the following weeks. Our results outline steroidal variations during the OCP cycle, highlighting exogenous hormonal preparations as confounder for steroid concentrations in blood. Low steroid levels in urine samples have a clear negative impact on the subsequent interpretation of steroid profile of the ABP. With a greater analytical sensitivity and lesser variability for steroids in healthy active women, serum represents a complementary matrix to urine in the ABP steroidal module., (© 2022 John Wiley & Sons Ltd.)

To analyze doping control samples from female athletes demands understanding of non-doping factors that affect the steroid profile. These could be physiological factors such as exercise, alcohol consumption, hormonal changes during the menstrual cycle, or the effect of commonly used approved drugs like combined oral contraceptives. Urine samples have been the main way of doping testing, but serum samples are proposed as a complement. Testosterone, dihydrotestosterone, or the ratio of testosterone and androstenedione has been proposed as a biomarker for testosterone doping because it increases after transdermal testosterone administration. In this double-blind, randomized, placebo-controlled study of 340 healthy females, we analyzed the serum steroid levels, including glucuronide metabolites, before and after 3 months of combined oral contraceptives or placebo. At follow up, sample collection in the placebo group was randomly distributed between different menstrual cycle phases. This enabled to analyze changes in concentrations between the follicular, ovulation, and luteal phases. Combined oral contraceptives decreased all serum steroids including the glucuronide metabolites. As expected, serum testosterone levels increased during the ovulation phase, and also androstenedione and androstenediol, whereas the glucuronide metabolites remained unaffected. Neither combined oral contraceptives nor menstrual cycle phases did affect the ratio of testosterone and androstenedione in serum, and consequently this ratio seems promising as a marker of doping with endogenous anabolic androgenic steroids in women., (© 2022 John Wiley & Sons Ltd.)

The urinary steroid profile established for the monitoring of eventual testosterone or testosterone precursor application by athletes includes concentrations and ratios of various endogenously produced steroidal hormones and metabolites. Due to enzymatic activities in urine specimens, the concentrations of these endogenous steroids and consequently their ratios may alter, leading to potential misinterpretation of analytical results. Microbiological contamination in athletes' urine samples can occur due to urinary tract infections or due to contamination by the non-sterile sample collection conditions. Depending on the duration of transportation of urine samples, the transport and storage conditions may favour microorganisms' growth, and therefore, the enzymatic activity can be accelerated. Degradation effects on endogenous steroids caused by microorganisms have been observed, such as hydrolysis of steroid conjugates, increase of testosterone in the free fraction or modification of the steroid structure by oxidoreductive reactions. The World Anti-Doping Agency (WADA) implemented criteria to check for signs of microbial degradation in a technical document dealing with the detection, analysis and reporting of endogenous androgenic anabolic steroids (TD EAAS) in urine samples. During the endogenous steroid profile confirmation procedures (CPs) of the WADA accredited Seibersdorf Laboratory, significant differences in the concentrations of markers of the steroid profile were observed compared to the initial testing procedures (ITPs). The changes in concentrations of the urinary steroid profile were attributed to the reduction of the 17-keto group to a 17β-hydroxy group caused by increased enzymatic activity during the hydrolysis step. In order to monitor the 17-keto reduction activity in athletes' urine specimens, possible marker substances containing a 17-keto group were synthesised and added in the internal standards mixture (ISTD) of the ITP. The presence of the reduced 17β-hydroxy form of the marker substance indicated enzymatic activity leading to 17-keto reduction reactions. The substance 3β-ethoxy-5α-androstane-17-one was defined to be suitable to indicate 17-keto reduction reactions occurring during hydrolysis carried out at moderate temperatures., (© 2022 John Wiley & Sons, Ltd.)

Boldenone is an anabolic-androgenic steroid that is prohibited in equine sports. However, in certain situations, it is endogenous or is believed to be formed by microbes in urine, and therefore, an approach for the differentiation is required. Following the identification of Δ1-progesterone and 20(S)-hydroxy-Δ1-progesterone as potential biomarkers of microbial activity, the presence of six steroids was investigated in the postrace urine of castrated male horses (geldings, n = 158). In line with endogenous findings from several other species when ultrasensitive methods are employed, boldenone was detected at low concentrations in all urine samples (27.0-1330 pg/ml). Furthermore, testosterone and androstenedione were detected in 157 samples (≤12,400 and 944 pg/ml, respectively), boldienone in two samples (≤22.0 pg/ml) and 20(S)-hydroxy-Δ1-progesterone in 20 samples (≤66.0 pg/ml). Δ1-Progesterone was not detected in any population samples analysed on arrival at the laboratory. The ex vivo transformation of boldienone, boldenone, androstenedione, Δ1-progesterone and 20(S)-hydroxy-Δ1-progesterone was induced following the storage of urine samples at room temperature for 7 days but not after refrigeration. Because the administration of inappropriately stored feed sources also resulted in an increase in 20(S)-hydroxy-Δ1-progesterone concentrations, a biomarker approach to distinguish steroid administrations was proposed. In situations where the presence of boldenone would exceed a proposed action limit, the presence of Δ1-progesterone and 20(S)-hydroxy-Δ1-progesterone would be investigated. If either Δ1-progesterone or 20(S)-hydroxy-Δ1-progesterone would exceed 50 and 100 pg/ml, respectively, for instance, then this would indicate ex vivo transformation or consumption of altered feed rather than steroid administration., (© 2022 John Wiley & Sons, Ltd.)

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., (© 2021 John Wiley & Sons, Ltd.)

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., (© 2021 State of New South Wales. Drug Testing and Analysis © 2021 John Wiley & Sons Ltd.)

Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol®. It is prohibited in sports at all times; nevertheless, there are several findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail. Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4α-chloro-17β-hydroxymethyl-17α-methyl-18-nor-5α-androstan-13-en-3α-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite. The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17α-hydroxymethyl-17β-methyl-18-nor-androsta-4,13-dien-3β-ol. It may serve as additional confirmatory metabolite. It is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated, to improve identification of cheating athletes. This study also offers some deeper insights into the metabolism of DHCMT and of 17α-methyl steroids in general., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The testing strategy for the detection of testosterone (T) or T-prohormones is based on the longitudinal evaluation of urinary steroid concentrations accompanied by subsequent isotope ratio mass spectrometry (IRMS)-based confirmation of samples showing atypical concentrations or concentration ratios. In recent years, the IRMS methodology focussed more and more on T itself and on the metabolites of T, 5α- and 5β-androstanediol. These target analytes showed the best sensitivity and retrospectivity, but their use has occasionally been challenging due to their comparably low urinary concentrations. Conversely, the carbon isotope ratios (CIR) of the main urinary metabolites of T, androsterone (A) and etiocholanolone (EITO), can readily be measured even from low urine volumes; those however, commonly offer a lower sensitivity and shorter retrospectivity in uncovering T misuse. Within this study, the CIRs of A and ETIO were combined with their urinary concentrations, resulting in a single parameter referred to as 'difference from weighted mean' (DWM). Both glucuronidated and sulfated steroids were investigated, encompassing a reference population (n = 110), longitudinal studies on three individuals, influence of ethanol in two individuals, and re-analysis of several administration studies including T, dihydrotestosterone, androstenedione, epiandrosterone, dehydroepiandrosterone, and T-gel. Especially DWM calculated for the sulfoconjugated steroids significantly prolonged the detection time of steroid hormone administrations when individual reference ranges were applied. Administration studies employing T encompassing CIR common for Europe (-23.8‰ and -24.4‰) were investigated and, even though for a significantly shorter time period and less pronounced, DWM could demonstrate the exogenous source of T metabolites., (© 2021 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

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., (© 2021 John Wiley & Sons, Ltd.)

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., (© 2021 John Wiley & Sons, Ltd.)

Background: Higher endogenous testosterone levels are associated with reduced chronic disease risk and mortality. Since the mid-20th century, there have been significant changes in dietary patterns, and men's testosterone levels have declined in western countries. Cross-sectional studies show inconsistent associations between fat intake and testosterone in men., Methods: Studies eligible for inclusion were intervention studies, with minimal confounding variables, comparing the effect of low-fat vs high-fat diets on men's sex hormones. 9 databases were searched from their inception to October 2020, yielding 6 eligible studies, with a total of 206 participants. Random effects meta-analyses were performed using Cochrane's Review Manager software. Cochrane's risk of bias tool was used for quality assessment., Results: There were significant decreases in sex hormones on low-fat vs high-fat diets. Standardised mean differences with 95 % confidence intervals (CI) for outcomes were: total testosterone [-0.38 (95 % CI -0.75 to -0.01) P = 0.04]; free testosterone [-0.37 (95 % CI -0.63 to -0.11) P = 0.005]; urinary testosterone [-0.38 (CI 95 % -0.66 to -0.09) P = 0.009]; and dihydrotestosterone [-0.3 (CI 95 % -0.56 to -0.03) P = 0.03]. There were no significant differences for luteinising hormone or sex hormone binding globulin. Subgroup analysis for total testosterone, European and North American men, showed a stronger effect [-0.52 (95 % CI -0.75 to -0.3) P < 0.001]., Conclusions: Low-fat diets appear to decrease testosterone levels in men, but further randomised controlled trials are needed to confirm this effect. Men with European ancestry may experience a greater decrease in testosterone, in response to a low-fat diet., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Many studies investigating pubertal development use Tanner staging to assess maturation. Endocrine markers in urine and saliva may provide an objective, sensitive, and non-invasive method for assessing development., Objective: Our objective was to examine whether changes in endocrine levels can indicate the onset of pubertal development prior to changes in self-rated Tanner stage., Methods: Thirty-five girls and 42 boys aged 7 to 15 years were enrolled in the Growth and Puberty (GAP) study, a longitudinal pilot study conducted from 2007-2009 involving children of women enrolled in the Agricultural Health Study (AHS) in Iowa. We collected saliva and urine samples and assessed pubertal development by self-rated Tanner staging (pubic hair, breast development (girls), genital development (boys)) at three visits over six months. We measured dehydroepiandrosterone (DHEA) in saliva and creatinine-adjusted luteinizing hormone (LH), testosterone, follicle stimulating hormone (FSH), estrone 3-glucuronide (E13G) and pregnanediol 3-glucuronide (Pd3G) concentrations in first morning urine. We evaluated the relationships over time between Tanner stage and each biomarker using repeated measures analysis., Results: Among girls still reporting Tanner breast stage 1 at the final visit, FSH levels increased over the 6-month follow-up period and were no longer lower than higher stage girls at the end of follow-up. We observed a similar pattern for testosterone in boys. By visit 3, boys still reporting Tanner genital stage 1 or pubic hair stage 1 had attained DHEA levels that were comparable to those among boys reporting Tanner stages 2 or 3., Conclusions: Increasing concentrations of FSH in girls and DHEA and testosterone in boys over a 6-month period revealed the start of the pubertal process prior to changes in self-rated Tanner stage. Repeated, non-invasive endocrine measures may complement the more subjective assessment of physical markers in studies determining pubertal onset., Competing Interests: MG, LMK, JWM, JSK, MM, CM and DPS declare no competing interests exist. AJCJ, JAM, CB-C and DSC are employed by a commercial company, Social & Scientific Systems, Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Testosterone doping in sports is detected through the measurement of the carbon isotopic signature (δ 13 C) of testosterone and its metabolites in urine. A critical step in achieving accurate and precise δ 13 C values during compound-specific stable carbon isotope analysis (CSIA) is the removal of interfering matrix components. To this end, the World Anti-Doping Agency (WADA) recommends the use of high-performance liquid chromatography (HPLC) as a method of sample pretreatment. We provide a description of an automated two-dimensional HPLC (2D-HPLC) purification method for urine extracts that has made possible the CSIA of underivatized steroids, requiring only 36 min per sample. Eight urinary steroids including testosterone (T) and dehydroepiandrosterone (DHEA) and four of their metabolites as well as two endogenous reference compounds were collected during HPLC purification. Comparative GC chromatograms are used to contrast the efficiency of two-dimensional (2D) purification to a previously established 1D-HPLC method. The 2D purification leads to improved sample purity while simultaneously decreasing the analysis time, allowing for unprecedented sample throughput. Precision of δ 13 C for all analyzed compounds in negative and positive controls was 0.5‰ or better, which is comparable with the precision of pure reference materials at similar intensities., (© 2020 John Wiley & Sons, Ltd.)

Background: The serum prostate-specific antigen is the most widespread biomarker for prostate disease. Its low specificity for prostatic malignancies is a matter of concern and the reason why new biomarkers for screening purposes are needed. The correlation between altered production of the main steroids and prostate carcinoma (PCa) occurrence is historically known. The purpose of this study is to evaluate the modifications of a comprehensive urinary endogenous steroidal profile (USP) induced by PCa, by multivariate statistical methods., Methods: A total of 283 Italian subjects were included in the study, 139 controls and 144 PCa-affected patients. The USP, including 17 steroids and five urinary steroidal ratios, was quantitatively evaluated using gas chromatography coupled with single quadrupole mass spectrometry (GC-MS). The data were interpreted using a chemometric, multivariate approach (intrinsically more sensible to alterations with respect to traditional statistics) and a model for the discrimination of cancer-affected profiles was built., Results: Two multivariate classification models were calculated, the former including three steroids with the highest statistical significance (e.g. testosterone, etiocholanolone and 7β-OH-DHEA) and PSA values, the latter considering the three steroids' levels only. Both models yielded high sensitivity and specificity scores near to 70%, resulting significantly higher than PSA alone., Conclusions: Three USP steroids resulted significantly altered in our PCa population. These preliminary results, combined with the simplicity and low-cost of the analysis, open to further investigation of the potential role of this restricted USP in PCa diagnosis.

Conjugation with glucuronic acid is one of the major metabolic reactions in human steroid hormone catabolism. Recently, increasing interest has been raised concerning the biological roles of steroid glucuronides. We have therefore developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 15 urinary steroid hormone glucuronides in human urine: androsterone glucuronide (An-G), etiocholanolone glucuronide (Etio-G), epiandrosterone glucuronide (epiAn-G), dihydrotestosterone glucuronide (DHT-G), dehydroepiandrosterone glucuronide (DHEA-G), testosterone glucuronide (T-G), epitestosterone glucuronide (epiT-G), estrone glucuronide (E1-3 G), 17β-estradiol 17-glucuronide (E2-17 G), 17β-estradiol 3-glucuronide (E2-3 G), estriol 16-glucuronide (E3-16 G), pregnenolone glucuronide (Preg-G), tetrahydro-11-deoxycorticosterone 3-glucuronide (THDOC-3 G), cortisol 21-glucuronide (F-G) and pregnanediol glucuronide (PD-G). Sample workup included protein precipitation and solid phase extraction. Internal standards were used to correct for the loss of analytes during sample preparation and analysis. The method showed good linearity (R 2 ≥0.99) and recovery ranged from 89.6 % to 113.8 %. Limit of quantification ranged from 1.9 nmol/L for F-G to 21.4 nmol/L for An-G. Intra-day and inter-day accuracy and precision were below 15 % for all quality controls. The method was successfully applied to 67 urine samples from children and adolescents in whom total concentrations of free and conjugated steroids had been previously determined by GC-MS after enzymatic hydrolysis. Free and sulfated steroids were also measured by LC-MS/MS. In general, the sums of the respective glucuronidated, sulfated and free forms of an analyte corresponded well with its total amount determined after enzymatic hydrolysis by GC-MS. Regarding the most prominent steroid metabolites, the total mean levels of androsterone and etiocholanolone showed an increase up to 5820.0 nmol/L and 4017.8 nmol/L in the group of 15-20 year-old children, respectively. Glucuronide conjugates (4374.3 nmol/L and 3588.5 nmol/L, respectively) dominated. DHEA was excreted mostly as sulfate (0-1 month of age: 184.5 nmol/L; 15-20 years of age: 1618.4 nmol/L) in all age groups. Cortisol was present predominantly as sulfate (mean: 173.8 nmol/L) in newborns. Levels of sulfated cortisol decreased with age, its glucuronidated form increased. The levels of free cortisol were relatively constant throughout childhood. Sex hormones were preferably excreted as glucuronides. In general, steroid hormone metabolites were conjugated to various extents with glucuronic acid or sulfuric acid and their ratio changed over lifetime., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Energetic investment in human reproduction has long been recognized as costly, influencing developmental, physiological, and behavioral patterns in males and females. These effects are largely coordinated through the actions of reproductive hormones (eg, testosterone, estradiol, and progesterone). Here, the utility and limitations of minimally invasive sampling techniques are explored, providing a novel perspective on how reproductive hormone measurements can enhance reproductive endocrinology research. Salivary steroid measures are most commonly used, although several dried blood spot and urine assays are also available, and researchers continue to explore the efficacy of other sample types. These relatively simple measures have facilitated the collection of multiple samples from a single participant, allowing researchers to more accurately track the diurnal and cyclical variation exhibited by many reproductive hormones. Ultimately, the ability to collect fine-grained participant data allows biological anthropologists to better test questions central to human reproductive ecology, life history theory, and public health. For example, fieldwork using these techniques suggests that testosterone profile variation across populations is influenced by energetic constraints and reproductive status. Moreover, hormone concentrations shape the development of sex characteristics, with implications for evolutionary questions related to sexual selection. Hormone levels also can be used to identify a range of medical concerns (eg, suppressed hormone production levels linked with psychosocial stress). These findings highlight how minimally invasive collection techniques can be applied to test diverse evolutionary hypotheses and identify important health concerns. Still, more work is needed to standardize collection and laboratory analysis procedures, thereby enabling more direct data comparisons between researchers., (© 2020 Wiley Periodicals LLC.)

The second-to-fourth digit (2D:4D) ratio is a sexually-dimorphic biomarker for prenatal sex hormone exposure. We investigated whether titi monkeys (Plecturocebus cupreus) exhibit sexually-dimorphic 2D:4D ratio, and whether variation in 2D:4D ratio correlates with maternal testosterone and estrogen levels during early pregnancy. Subjects were 61 adult titi monkeys (32 males, 29 females). For 26 subjects, maternal urine samples were collected approximately 15-20 weeks before birth and assayed for testosterone and estrone conjugate (E 1 C). Titi monkeys exhibited a human-like pattern of sexual dimorphism in right-hand 2D:4D ratio, with females exhibiting higher 2D:4D ratio than males (β = -0.29, p = 0.023). For left-hand 2D:4D ratio, high levels of maternal E 1 C predicted low offspring 2D:4D ratio (β = -0.48, p = 0.009). For right-hand 2D:4D ratio, high levels of testosterone (β = -0.53, p = 0.005) and testosterone-to-E 1 C ratio (β = -0.41, p = 0.028) predicted low offspring 2D:4D ratio. For 2D:4D ratio asymmetry (right-hand - left-hand), high levels of testosterone (β = -0.43, p = 0.03) and testosterone-to-E 1 C ratio (β = -0.53, p = 0.003) predicted low (right-biased) asymmetry. This is the first report of sexually-dimorphic 2D:4D ratio in New World monkeys, and the results support a growing literature suggesting prenatal sex hormones may modulate offspring 2D:4D ratio., (© 2019 Wiley Periodicals, Inc.)

The detection of clostebol misuse in sports has been growing recently, especially in Italy, due to the ample availability of pharmaceutical formulations containing clostebol acetate (Trofodermin®) and the use of more sensitive instrumentation by the antidoping laboratories. Most of these cases have been claimed to be related to a nonconscious use of the drug or through contact with relatives or teammates using it. We have investigated, through the application of the well-known and currently used gas chromatographic mass spectrometric procedures, the likelihood of these allegations and have demonstrated that after a single transdermal administration of 5 mg of clostebol acetate and a transient contact with the application area, it is possible to generate adverse analytical findings in antidoping controls. We have reviewed the Phase I and Phase II clostebol metabolism in order to generate evidences that may help the sport authorities reviewing these cases. The main clostebol metabolite (4-chloro-androst-4-en-3α-ol-17-one, M1) generally used at the screening level as well as other three metabolites (M2-M4) are mainly excreted as glucuronides, whereas M5 (4ζ-chloro-5ζ-androstan-3β-ol-17-one) is predominantly excreted as sulfate. Neither the 5α-reductases activity (impaired by the presence of the chlorine in C4) nor specific sulfotransferases present in the skin allowed a clear distinction of the administration route. Studies with a larger number of volunteers and probably investigating another physiological fluid allowed in antidoping such as blood are needed for a deeper investigation. It is not unreasonable to establish a reporting level for M1, maybe creating some false negatives but excluding nonintentional doping scenarios., (© 2020 John Wiley & Sons, Ltd.)

Neuroendocrine research on the formation of social bonds has primarily focused on the role of nonapeptides. However, steroid hormones often act simultaneously to either inhibit or facilitate bonding. Testosterone is proposed to mediate a trade-off between male mating effort and nurturing behavior; therefore, low levels are predicted during periods of nurturing infant care and social bonding. In species where social bonding and support regulates hypothalamic-pituitary-adrenal axis activity, we also expect glucocorticoid levels to be low during bonding periods. We investigated how immunoreactive urinary testosterone (iuT) and cortisol (iuC) were related to triadic male-infant-male interactions - a ritualized male bonding behavior - as well as infant care in male Barbary macaques (Macaca sylvanus). We collected >3000 h of behavioral observation data during full-day focal animal follows from 14 adult males and quantified iuT and iuC from 650 urine samples. iuT was negatively correlated with rates of triadic interactions within subjects, but positively correlated between subjects. iuC was negatively correlated with triadic interactions both within and between subjects. Time spent caring for infants was positively correlated to both iuT and iuC within subjects, but not between subjects. The observed negative relationship between iuT and triadic interactions within subjects may be beneficial to lower competitive tendencies between adult males and to not inhibit bond formation. However, the positive correlation of iuT with triadic interactions between subjects was unexpected. We speculate that it could be due to a link between triadic interactions and coalition formation. A negative relationship between triadic interactions and iuC could reflect increased bonding and perceived social support as triadic interactions predict future coalition formation in this species, or reflect buffered tensions between males. The positive relationship of iuT and iuC with infant care suggests that the handling of infants may be less nurturing but rather protective or competitive in this species. Measuring steroid hormones in relation to bonding and nurturing can help us interpret behaviors within the ecological contexts that they occur., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 5α-androstane-3α, 17β-diol/epitestosterone (5αAdiol/E) ratio may provide more sensitivity., (© 2020 John Wiley & Sons, Ltd.)

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 regulates the male reproductive system and acts directly or indirectly on nearly all systems during fetal, pubertal and adult life. Testosterone homeostasis depends on its synthesis and degradation. The major biotransformation reactions are hydroxylation by different cytochrome P450 (CYP) isoforms. There are no described methods to determine the profile of testosterone-hydroxylated metabolites in human urine. The aim of this study was to develop an analytical method to determine testosterone-hydroxylated metabolites in human urine using UPLC-MS. Seven testosterone-hydroxylated metabolites, androstenedione, and testosterone, were identified by comparison of their t ret and positive electrospray ionization (ESI + ) data, with those of analytical standards. The method developed is sensitive, specific, repeatable, and precise. Limits of detection and quantitation for all compounds ranged from 1.360 to 13.054 ng/ml and 4.234-39.679 ng/ml, respectively. The percentages of recovery were between 81.2 and 128.8%. The applicability of the analytical method was confirmed by analysis of urine samples obtained from two groups of healthy men (25-30 and 50-75 years old). All analytes were identified with slightly different metabolites profiles in both groups. In conclusion, the UPLC-MS method developed here was validated for the analysis of testosterone-hydroxylated metabolites in human urine., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

UDP-glucuronosyltransferase 2B17 (UGT2B17) is a highly variable androgen-metabolizing and drug-metabolizing enzyme. UGT2B17 exhibits a unique ontogeny profile characterized by a dramatic increase in hepatic protein expression from prepubertal age to adulthood. Age, sex, copy number variation (CNV), and single nucleotide polymorphisms only explain 26% of variability in protein expression, highlighting the need for a phenotypic biomarker for predicting interindividual variability in glucuronidation of UGT2B17 substrates. Here, we propose testosterone glucuronide (TG) normalized by androsterone glucuronide (TG/AG) as a urinary UGT2B17 biomarker, and examine the associations among urinary TG/AG and age, sex, and CNV. We performed targeted metabolomics of 12 androgen conjugates with liquid-chromatography tandem mass spectrometry in 63 pediatric subjects ages 7-18 years followed over 7 visits in 3 years. Consistent with the reported developmental trajectory of UGT2B17 protein expression, urinary TG/AG is significantly associated with age, sex, and CNV. In conclusion, TG/AG shows promise as a phenotypic urinary UGT2B17 biomarker., (© 2020 The Authors Clinical Pharmacology & Therapeutics © 2020 American Society for Clinical Pharmacology and Therapeutics.)

The use of anabolic androgenic steroids (AAS) and other performance enhancing substances can change over time, so there is a need to constantly update what substances are used and can be detected. Six women and 30 men anabolic androgenic steroid users were recruited who filled out an anonymous questionnaire about their use of performance enhancing substances during the past year. Sampling took place on a single occasion and included blood and urine collection. Our aim was to identify which doping agents can be detected in men and women self-reporting AAS use. The first choice of substances differed between men (testosterone) and women (oxandrolone). The use of growth hormones was reported among men (10%) and women (50%). Growth hormone releasing factors/secretagogs were reported by about ~ 20% in both genders. Nandrolone was the most frequently detected anabolic androgenic steroid even in those who did not report use in the past year. Of the current male testosterone users, 82% exhibited testosterone/epitestosterone (T/E) ratios of > 4. Men with current testosterone use displayed 4-fold and 6-fold higher median T/E, respectively, when compared with recent and previous testosterone users (P = 0.0001). Dermal testosterone use in women (n = 2) was not associated with a T/E ratio of > 4, but with supra-physiological total serum testosterone concentrations. Changes in gonadotropins and hematological parameters were associated with the time of the last anabolic androgenic steroid intake in men, whereas in women these biomarkers were within the normal range. This highlights gender specific differences and indicates the need for additional biomarkers in female athletes., (© 2020 John Wiley & Sons, Ltd.)

In Europe, chemical castration has been adopted as a treatment for paraphilia since the 1930s. Among the various chemical castration agents, luteinizing hormone-releasing hormone (LHRH) agonists are now used widely because of their effectiveness and safety. In South Korea, a legislation of chemical castration to control the sexual impulses of sexual offenders was enforced in July 2011. Most of these subjects are treated with leuprorelin acetate, an LHRH agonist, for chemical castration. Despite this, there are few studies that address the long-term influence of LHRH agonists on testosterone (T) and epitestosterone (E) levels in chemical castration subjects. In order to analyze the urinary levels of T in chemical castration subjects, whose T levels are extremely low, we developed and validated an analytical method for the detection of both T and E in human urine using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The urine samples were hydrolyzed, extracted, and analyzed by LC-MS/MS with electrospray ionization in the positive-ion mode. The limits of detection were 0.02 ng/mL and the limits of quantitation were 0.05 ng/mL, which provided great sensitivity. The established method was applied to urine samples from chemical castration subjects and healthy male volunteers. The chemical castration subjects showed significantly lower urinary T levels than the control subjects. In addition, the urinary E levels were also lower in the chemical castration subjects; however, the T/E ratios were constant and did not show a notable decrease because of the simultaneous decrease in both urinary T and E. The urinary T levels and T/E ratio did not exceed the doping control criteria for exogenous T ingestion for any subject. This study shows the trend of urinary T and E levels in long-term treated chemical castration subjects by establishing a highly sensitive LC-MS/MS method, that provides useful information for monitoring chemical castration., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Boldenone (1-dehydrotestosterone) is an exogenous anabolic-androgenic steroid (AAS) but is also known to be endogenous in the entire male horse and potentially formed by microbes in voided urine, the gastrointestinal tract, or feed resulting in its detection in urine samples. In this study, equine fecal and urine samples were incubated in the presence of selected stable isotope labeled AAS precursors to investigate whether microbial activity could result in 1-dehydrogenation, in particular the formation of boldenone. Fecal matter was initially selected for investigation because of its high microbial activity, which could help to identify potential 1-dehydrogenated biomarkers that might also be present in low quantities in urine. Fecal incubations displayed Δ1-dehydrogenase activity, as evidenced by the use of isotope labeled precursors to show the formation of boldenone and boldione from testosterone and androstenedione, as well as the formation of Δ1-progesterone and boldione from progesterone. Unlabeled forms were also produced in unspiked fecal samples with Δ1-progesterone being identified for the first time. Subsequent incubation of urine samples with the labeled AAS precursors demonstrated that Δ1-dehydrogenase activity can also occur in this matrix. In all urine samples where labeled boldenone or boldione were detected, labeled Δ1-progesterone was also detected. Δ1-progesterone was not detected any non-incubated urine samples or following an administration of boldenone undecylenate to one mare/filly. Δ1-progesterone appears to be a candidate for further investigation as a suitable biomarker to help evaluate whether boldenone present in a urine sample may have arisen due to microbial activity rather than by its exogenous administration., (© 2019 John Wiley & Sons, Ltd.)

Research suggests that women's sexual psychology and behavior change across the ovulatory cycle, but very little is known about how fluctuations in estradiol and progesterone - two hormones that systematically vary across the ovulatory cycle - affect romantic relationship dynamics. We present the first dyadic study to assess daily hormonal fluctuations and personal and relationship well-being from both partners' perspectives. Specifically, we recruited women who were not using hormonal contraception and their partners for a 15-day diary study. Participants collected daily urine samples to assess estradiol, progesterone, and testosterone, and they responded to daily questions about their relationship. Results revealed that increases in estradiol negatively affected women's relationship evaluations. Men perceived these changes, which in turn, affected men's well-being. The present findings highlight the importance of women's hormonal fluctuations in shaping relationship dynamics and provide, for the first time, information about how such fluctuations affect male partners., (Copyright © 2019 Elsevier B.V. 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 work reports on a modularized electrochemical method for the determination of the hormones cortisol, progesterone, testosterone and 17β-estradiol in urine. These hormones were employed as templates when generating molecular imprints from aniline and metanilic acid by electropolymerization on the surface of screen-printed electrodes. The electrically conductive imprint was characterized by SEM, AFM and cyclic voltammetry. A four-channel system was then established to enable simultaneous determination of the hormones by cyclic voltammetry. The detection limits for cortisol, progesterone, testosterone and 17β-estradiol are as low as 2, 2.5, 10 and 9 ag·mL -1 (for S/N = 3). Graphical abstract A four-channel system was established to enable simultaneous determination of 4 steroid hormones by cyclic voltammetry and by using moleculalry imprinted polymers.

Testosterone doping remains a prevalent and potent form of drug cheating among elite athletes. In men, the urine testosterone (T) to epitestosterone (E) ratio (T/E ratio) can identify administration of exogenous T by its suppression of endogenous T production through strong negative feedback on endogenous T and E production as well as spill over into urine of extra testosterone. However, this mechanism may be partially inoperative in females whose much lower circulating T derives from three sources, none subject to powerful negative T feedback. Hence, additional methods to detect T doping in females are required. In this study we report two cases of elite female athletes who were sanctioned for T doping proven by measurement of serum T using liquid chromatography-mass spectrometry (LC-MS), when serial urine T and T/E ratio in one were not indicative of T doping, and in the other were nullified by incidental genetic inactivation of T glucuronidation through the uridine diphosphoglucuronosyl transferase 2B17 (UGT2B17) deletion genotype-phenotype. These findings indicate the potential for serum T measurement by LC-MS to detect T doping in female athletes, especially if implemented in the Bayesian format of an athlete biological passport., (© 2019 John Wiley & Sons, Ltd.)

A 34-year old male was found breathless and panting at home by his girlfriend three hours after a gym workout. Minutes later, he collapsed and died. Autopsy, histological and chemical analyses were conducted. The examination of the heart showed left ventricular hypertrophy, while the right coronary artery showed only a small vascular lumen (3 mm in diameter), due to its anatomical structure. In femoral blood concentrations of approx. 1 μg/L clenbuterol, approx. 56 μg/L stanozolol and approx. 8 μg/L metandienone, with trenbolone (