A novel approach to improve detection of glucocorticoid doping in sport with new guidance for physicians prescribing for athletes.

The systemic effect of glucocorticoids (GCs) following injectable routes of administration presents a potential risk to both improving performance and causing harm to health in athletes. This review evaluates the current GC antidoping regulations defined by the World Anti-Doping Agency and presents a novel approach for defining permitted and prohibited use of glucocorticoids in sport based on the pharmacological potential for performance enhancement (PE) and risk of adverse effects on health. Known performance-enhancing doses of glucocorticoids are expressed in terms of cortisol-equivalent doses and thereby the dose associated with a high potential for PE for any GC and route of administration can be derived. Consequently, revised and substance-specific laboratory reporting values are presented to better distinguish between prohibited and permitted use in sport. In addition, washout periods are presented to enable clinicians to prescribe glucocorticoids safely and to avoid the risk of athletes testing positive for a doping test.

Detection of Stimulated Erythropoiesis by the RNA-Based 5'-Aminolevulinate Synthase 2 Biomarker in Dried Blood Spot Samples.

BACKGROUND: Despite implementation of the Athlete Biological Passport 10 years ago, blood doping remains difficult to detect. Thus, there is a need for new biomarkers to increase the sensitivity of the adaptive model. Transcriptomic biomarkers originating from immature reticulocytes may be reliable indicators of blood manipulations. Furthermore, the use of dried blood spots (DBSs) for antidoping purposes constitutes a complementary approach to venous blood collection. Here, we developed a method of quantifying the RNA-based 5'-aminolevulinate synthase 2 (ALAS2) biomarker in DBS. MATERIALS: The technical, interindividual, and intraindividual variabilities of the method, and the effects of storage conditions on the production levels of ALAS2 RNA were assessed. The method was used to monitor erythropoiesis stimulated endogenously (blood withdrawal) or exogenously (injection of recombinant human erythropoietin). RESULTS: When measured over a 7-week period, the intra- and interindividual variabilities of ALAS2 expression in DBS were 12.5%-42.4% and 49%, respectively. Following withdrawal of 1 unit of blood, the ALAS2 RNA in DBS increased significantly for up to 15 days. Variations in the expression level of this biomarker in DBS samples were more marked than those of the conventional hematological parameters, reticulocyte percentage and immature reticulocyte fraction. After exogenous stimulation of erythropoiesis via recombinant human erythropoietin injection, ALAS2 expression in DBS increased by a mean 8-fold. CONCLUSIONS: Monitoring of transcriptomic biomarkers in DBS could complement the measurement of hematological parameters in the Athlete Biological Passport and aid the detection of blood manipulations.

[The necessary dialogue between medicine and anti-doping for the integrity of sport and the athlete]. / Le dialogue nécessaire entre médecine et antidopage pour l'intégrité du sport et de l'athlète.

In the light of recurring anti-doping news, this article discusses some special cases and extends to the future prospects of developing effective anti-doping strategies. After recalling the current principles of the fight against doping, the use of substances tole-rated at a certain threshold, and the therapeutic use exemptions (TUE) are discussed. Authorized substances with a health risk for athletes are discussed before concluding on the development of the athlete's biological passport as a future common ground for anti-doping and medical follow-up of athletes. In conclusion, this approach emphasizes the imperative dialogue between anti-doping organizations and sports medicine in order to defend good practices preserving the intrinsic value of sport.

En réponse aux actualités antidopage, cet article traite quelques cas particuliers et s'étend aux perspectives futures de développement de stratégies antidopage efficaces. Après avoir rappelé les principes actuels de la lutte antidopage, il aborde l'utilisation de substances tolérées à un certain seuil et évoque les autorisations à usage thérapeutique (AUT). Les substances autorisées mais pouvant présenter un risque sanitaire pour les athlètes sont discutées avant de conclure sur le développement du passeport biologique de l'athlète comme futur terrain commun pour la lutte antidopage et le suivi médical des sportifs. En conclusion, cette approche souligne le dialogue impératif entre organisations antidopage et médecine du sport afin de défendre de bonnes pratiques à même de préserver la valeur intrinsèque du sport.

Urinary di-(2-ethylhexyl) phthalate metabolites for detecting transfusion of autologous blood stored in plasticizer-free bags.

BACKGROUND: Autologous blood transfusion (ABT) efficiently increases sport performance and is the most challenging doping method to detect. Current methods for detecting this practice center on the plasticizer di(2-ethlyhexyl) phthalate (DEHP), which enters the stored blood from blood bags. Quantification of this plasticizer and its metabolites in urine can detect the transfusion of autologous blood stored in these bags. However, DEHP-free blood bags are available on the market, including n-butyryl-tri-(n-hexyl)-citrate (BTHC) blood bags. Athletes may shift to using such bags to avoid the detection of urinary DEHP metabolites. STUDY DESIGN AND METHODS: A clinical randomized double-blinded two-phase study was conducted of healthy male volunteers who underwent ABT using DEHP-containing or BTHC blood bags. All subjects received a saline injection for the control phase and a blood donation followed by ABT 36 days later. Kinetic excretion of five urinary DEHP metabolites was quantified with liquid chromatography coupled with tandem mass spectrometry. RESULTS: Surprisingly, considerable levels of urinary DEHP metabolites were observed up to 1 day after blood transfusion with BTHC blood bags. The long-term metabolites mono-(2-ethyl-5-carboxypentyl) phthalate and mono-(2-carboxymethylhexyl) phthalate were the most sensitive biomarkers to detect ABT with BTHC blood bags. Levels of DEHP were high in BTHC bags (6.6%), the tubing in the transfusion kit (25.2%), and the white blood cell filter (22.3%). CONCLUSIONS: The BTHC bag contained DEHP, despite being labeled DEHP-free. Urinary DEHP metabolite measurement is a cost-effective way to detect ABT in the antidoping field even when BTHC bags are used for blood storage.

Hepcidin as a new biomarker for detecting autologous blood transfusion.

Autologous blood transfusion (ABT) is an efficient way to increase sport performance. It is also the most challenging doping method to detect. At present, individual follow-up of haematological variables via the athlete biological passport (ABP) is used to detect it. Quantification of a novel hepatic peptide called hepcidin may be a new alternative to detect ABT. In this prospective clinical trial, healthy subjects received a saline injection for the control phase, after which they donated blood that was stored and then transfused 36 days later. The impact of ABT on hepcidin as well as haematological parameters, iron metabolism, and inflammation markers was investigated. Blood transfusion had a particularly marked effect on hepcidin concentrations compared to the other biomarkers, which included haematological variables. Hepcidin concentrations increased significantly: 12 hr and 1 day after blood reinfusion, these concentrations rose by seven- and fourfold, respectively. No significant change was observed in the control phase. Hepcidin quantification is a cost-effective strategy that could be used in an "ironomics" strategy to improve the detection of ABT.

Longitudinal monitoring of endogenous steroids in human serum by UHPLC-MS/MS as a tool to detect testosterone abuse in sports.

The detection of testosterone abuse in sports is routinely achieved through the 'steroidal module' of the Athlete Biological Passport by GC-MS(/MS) quantification of selected endogenous anabolic androgenic steroids (EAAS) from athletes' urines. To overcome some limitations of the "urinary steroid profile" such as the presence of confounding factors (ethnicity, enzyme polymorphism, bacterial contamination, and ethanol), ultrahigh performance liquid chromatography (UHPLC) measurements of blood concentrations of testosterone, its major metabolites, and precursors could represent an interesting and complementary strategy. In this work, two UHPLC-MS/MS methods were developed for the quantification of testosterone and related compounds in human serum, including major progestogens, corticoids, and estrogens. The validated methods were then used for the analyses of serum samples collected from 19 healthy male volunteers after oral and transdermal testosterone administration. Results from unsupervised multiway analysis allowed variations of target analytes to be assessed simultaneously over a 96-h time period. Except for alteration of concentration values due to the circadian rhythm, which concerns mainly corticosteroids, DHEA, and progesterone, significant variations linked to the oral and transdermal testosterone administration were observed for testosterone, DHT, and androstenedione. As a second step of analysis, the longitudinal monitoring of these biomarkers using intra-individual thresholds showed, in comparison to urine, significant improvements in the detection of testosterone administration, especially for volunteers with del/del genotype for phase II UGT2B17 enzyme, not sensitive to the main urinary marker, T/E ratio. A substantial extension of the detection window after transdermal testosterone administration was also observed in serum matrix. The longitudinal follow-up proposed in this study represents a first example of 'blood steroid profile' in doping control analysis, which can be proposed in the future as a complement to the 'urinary module' for improving steroid abuse detection capabilities.

Circulating microRNAs: The Future of Biomarkers in Anti-doping Field.

microRNAs (miRNAs) are small non-coding RNAs that regulate various biological processes. Cell-free miRNAs have been proposed as biomarkers of disease, including diagnosis, prognosis, and monitoring of treatment responses. These circulating miRNAs are highly stable in several body fluids, including plasma and serum; hence, in view of their potential use as novel, non-invasive biomarkers, the profiles of circulating miRNAs have been explored in the field of anti-doping. This chapter describes the enormous potential of circulating miRNAs as a new class of biomarkers for the detection of doping substances, and highlights the advantages of measuring these stable species over other methods that have already been implemented in anti-doping regimes. Incorporating longitudinal measurements of circulating miRNAs into the Athlete Biological Passport is proposed as an efficient strategy for the implementation of these new biomarkers. Furthermore, potential challenges related to the transition of measurements of circulating miRNAs from research settings to practical anti-doping applications are presented.

Antidoping programme and biological monitoring before and during the 2014 FIFA World Cup Brazil.

BACKGROUND: The FIFA has implemented an important antidoping programme for the 2014 FIFA World Cup. AIM: To perform the analyses before and during the World Cup with biological monitoring of blood and urine samples. METHODS: All qualified players from the 32 teams participating in the World Cup were tested out-of-competition. During the World Cup, 2-8 players per match were tested. Over 1000 samples were collected in total and analysed in the WADA accredited Laboratory of Lausanne. RESULTS: The quality of the analyses was at the required level as described in the WADA technical documents. The urinary steroid profiles of the players were stable and consistent with previously published papers on football players. During the competition, amphetamine was detected in a sample collected on a player who had a therapeutic use exemption for attention deficit hyperactivity disorder. The blood passport data showed no significant difference in haemoglobin values between out-of-competition and postmatch samples. CONCLUSIONS: Logistical issues linked to biological samples collection, and the overseas shipment during the World Cup did not impair the quality of the analyses, especially when used as the biological passport of football players.

Determination of ¹³C/¹² C ratios of endogenous urinary 5-amino-imidazole-4-carboxamide 1ß-D-ribofuranoside (AICAR).

RATIONALE: AICAR (5-aminoimidazole-4-carboxamide 1ß-D-ribofuranoside) is prohibited in sport according to rules established by the World Anti-Doping Agency. Doping control laboratories identify samples where AICAR abuse is suspected by measuring its urinary concentration and comparing the observed level with naturally occurring concentrations. As the inter-individual variance of urinary AICAR concentrations is large, this approach requires a complementary method to unambiguously prove the exogenous origin of AICAR. Therefore, a method for the determination of carbon isotope ratios (CIRs) of urinary AICAR has been developed and validated. METHODS: Concentrated urine samples were fractionated by means of liquid chromatography for analyte cleanup. Derivatization of AICAR yielding the trimethylsilylated analog was necessary to enable CIR determinations by gas chromatography/combustion/isotope ratio mass spectrometry. The method was tested for its repeatability and stability over time and a linear mixing model was applied to test for possible isotopic discrimination. A reference population of n = 63 males and females was investigated to calculate appropriate reference limits to differentiate endogenous from exogenous urinary AICAR. These limits were tested by an AICAR elimination study. RESULTS: The developed method fulfills all the requirements for adequate sports drug testing and was found to be fit for purpose. The investigated reference population showed a larger variability in the CIR of AICAR than of the endogenous steroids. Nevertheless, the calculated thresholds for differences between AICAR and endogenous steroids can be applied straightforwardly to evaluate suspicious doping control samples with the same statistical confidence as established e.g. for testosterone misuse. These thresholds enabled the detection of a single oral AICAR administration for more than 40 h. CONCLUSIONS: Determination of thee CIRs is the method of choice to distinguish between an endogenous and an exogenous source of urinary AICAR. The developed method will enable investigations into doping control samples with elevated urinary concentrations of AICAR and clearly differentiate between naturally produced/elevated and illicitly administered AICAR.

Confounding factors and genetic polymorphism in the evaluation of individual steroid profiling.

In the fight against doping, steroid profiling is a powerful tool to detect drug misuse with endogenous anabolic androgenic steroids. To establish sensitive and reliable models, the factors influencing profiling should be recognised. We performed an extensive literature review of the multiple factors that could influence the quantitative levels and ratios of endogenous steroids in urine matrix. For a comprehensive and scientific evaluation of the urinary steroid profile, it is necessary to define the target analytes as well as testosterone metabolism. The two main confounding factors, that is, endogenous and exogenous factors, are detailed to show the complex process of quantifying the steroid profile within WADA-accredited laboratories. Technical aspects are also discussed as they could have a significant impact on the steroid profile, and thus the steroid module of the athlete biological passport (ABP). The different factors impacting the major components of the steroid profile must be understood to ensure scientifically sound interpretation through the Bayesian model of the ABP. Not only should the statistical data be considered but also the experts in the field must be consulted for successful implementation of the steroidal module.

Monitoring of biological markers indicative of doping: the athlete biological passport.

The athlete biological passport (ABP) was recently implemented in anti-doping work and is based on the individual and longitudinal monitoring of haematological or urine markers. These may be influenced by illicit procedures performed by some athletes with the intent to improve exercise performance. Hence the ABP is a valuable tool in the fight against doping. Actually, the passport has been defined as an individual and longitudinal observation of markers. These markers need to belong to the biological cascade influenced by the application of forbidden hormones or more generally, affected by biological manipulations which can improve the performance of the athlete. So far, the haematological and steroid profile modules of the ABP have been implemented in major sport organisations, and a further module is under development. The individual and longitudinal monitoring of some blood and urine markers are of interest, because the intraindividual variability is lower than the corresponding interindividual variability. Among the key prerequisites for the implementation of the ABP is its prospect to resist to the legal and scientific challenges. The ABP should be implemented in the most transparent way and with the necessary independence between planning, interpretation and result management of the passport. To ensure this, the Athlete Passport Management Unit (APMU) was developed and the WADA implemented different technical documents associated to the passport. This was carried out to ensure the correct implementation of a profile which can also stand the challenge of any scientific or legal criticism. This goal can be reached only by following strictly important steps in the chain of production of the results and in the management of the interpretation of the passport. Various technical documents have been then associated to the guidelines which correspond to the requirements for passport operation. The ABP has been completed very recently by the steroid profile module. As for the haematological module, individual and longitudinal monitoring have been applied and the interpretation cascade is also managed by a specific APMU in a similar way as applied in the haematological module. Thus, after exclusion of any possible pathology, specific variation from the individual norms will be then considered as a potential misuse of hormones or other modulators to enhance performance.

Challenges and threats to implementing the fight against doping in sport.

Prominent doping cases in certain sports have recently raised public awareness of doping and reinforced the perception that doping is widespread. Efforts to deal with doping in sport have intensified in recent years, yet the general public believes that the 'cheaters' are ahead of the testers. Therefore, there is an urgent need to change the antidoping strategy. For example, the increase in the number of individual drug tests conducted between 2005 and 2012 was approximately 90 000 and equivalent to an increase of about 50%, yet the number of adverse analytical findings remained broadly the same. There is also a strikingly different prevalence of doping substances and methods in sports such as a 0.03% prevalence of anabolic steroids in football compared to 0.4% in the overall WADA statistics. Future efforts in the fight against doping should therefore be more heavily based on preventative strategies such as education and on the analysis of data and forensic intelligence and also on the experiences of relevant stakeholders such as the national antidoping organisations, the laboratories, athletes or team physicians and related biomedical support staff. This strategy is essential to instigate the change needed to more effectively fight doping in sport.

Time for change: a roadmap to guide the implementation of the World Anti-Doping Code 2015.

A medical and scientific multidisciplinary consensus meeting was held from 29 to 30 November 2013 on Anti-Doping in Sport at the Home of FIFA in Zurich, Switzerland, to create a roadmap for the implementation of the 2015 World Anti-Doping Code. The consensus statement and accompanying papers set out the priorities for the antidoping community in research, science and medicine. The participants achieved consensus on a strategy for the implementation of the 2015 World Anti-Doping Code. Key components of this strategy include: (1) sport-specific risk assessment, (2) prevalence measurement, (3) sport-specific test distribution plans, (4) storage and reanalysis, (5) analytical challenges, (6) forensic intelligence, (7) psychological approach to optimise the most deterrent effect, (8) the Athlete Biological Passport (ABP) and confounding factors, (9) data management system (Anti-Doping Administration & Management System (ADAMS), (10) education, (11) research needs and necessary advances, (12) inadvertent doping and (13) management and ethics: biological data. True implementation of the 2015 World Anti-Doping Code will depend largely on the ability to align thinking around these core concepts and strategies. FIFA, jointly with all other engaged International Federations of sports (Ifs), the International Olympic Committee (IOC) and World Anti-Doping Agency (WADA), are ideally placed to lead transformational change with the unwavering support of the wider antidoping community. The outcome of the consensus meeting was the creation of the ad hoc Working Group charged with the responsibility of moving this agenda forward.

Circulating miRNAs: a new generation of anti-doping biomarkers.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. Cell-free miRNAs detected in blood plasma are used as specific and sensitive markers of physiological processes and some diseases. Circulating miRNAs are highly stable in body fluids, for example plasma. Therefore, profiles of circulating miRNAs have been investigated for potential use as novel, non-invasive anti-doping biomarkers. This review describes the biological mechanisms underlying the variation of circulating miRNAs, revealing that they have great potential as a new class of biomarker for detection of doping substances. The latest developments in extraction and profiling technology, and the technical design of experiments useful for anti-doping, are also discussed. Longitudinal measurements of circulating miRNAs in the context of the athlete biological passport are proposed as an efficient strategy for the use of these new markers. The review also emphasizes potential challenges for the translation of circulating miRNAs from research into practical anti-doping applications.

Effect of changes in the deuterium content of drinking water on the hydrogen isotope ratio of urinary steroids in the context of sports drug testing.

The hydrogen isotope ratio (HIR) of body water and, therefore, of all endogenously synthesized compounds in humans, is mainly affected by the HIR of ingested drinking water. As a consequence, the entire organism and all of its synthesized substrates will reflect alterations in the isotope ratio of drinking water, which depends on the duration of exposure. To investigate the effect of this change on endogenous urinary steroids relevant to doping-control analysis the hydrogen isotope composition of potable water was suddenly enriched from -50 to 200 ‰ and maintained at this level for two weeks for two individuals. The steroids under investigation were 5ß-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5ß-androstan-17-one (ETIO), 5α-androstane-3α,17ß-diol, and 5ß-androstane-3α,17ß-diol (excreted as glucuronides) and ETIO, ANDRO and 3ß-hydroxyandrost-5-en-17-one (excreted as sulfates). The HIR of body water was estimated by determination of the HIR of total native urine, to trace the induced changes. The hydrogen in steroids is partly derived from the total amount of body water and cholesterol-enrichment could be calculated by use of these data. Although the sum of changes in the isotopic composition of body water was 150 ‰, shifts of approximately 30 ‰ were observed for urinary steroids. Parallel enrichment in their HIR was observed for most of the steroids, and none of the differences between the HIR of individual steroids was elevated beyond recently established thresholds. This finding is important to sports drug testing because it supports the intended use of this novel and complementary methodology even in cases where athletes have drunk water of different HIR, a plausible and, presumably, inevitable scenario while traveling.

Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing.

Carbon isotope ratio (CIR) analysis has been routinely and successfully applied to doping control analysis for many years to uncover the misuse of endogenous steroids such as testosterone. Over the years, several challenges and limitations of this approach became apparent, e.g., the influence of inadequate chromatographic separation on CIR values or the emergence of steroid preparations comprising identical CIRs as endogenous steroids. While the latter has been addressed recently by the implementation of hydrogen isotope ratios (HIR), an improved sample preparation for CIR avoiding co-eluting compounds is presented herein together with newly established reference values of those endogenous steroids being relevant for doping controls. From the fraction of glucuronidated steroids 5ß-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-Hydroxy-5ß-androstane-11,17-dione, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5ß-androstan-17-one (ETIO), 3ß-hydroxy-androst-5-en-17-one (DHEA), 5α- and 5ß-androstane-3α,17ß-diol (5aDIOL and 5bDIOL), 17ß-hydroxy-androst-4-en-3-one and 17α-hydroxy-androst-4-en-3-one were included. In addition, sulfate conjugates of ANDRO, ETIO, DHEA, 3ß-hydroxy-5α-androstan-17-one plus 17α- and androst-5-ene-3ß,17ß-diol were considered and analyzed after acidic solvolysis. The results obtained for the reference population encompassing n = 67 males and females confirmed earlier findings regarding factors influencing endogenous CIR. Variations in sample preparation influenced CIR measurements especially for 5aDIOL and 5bDIOL, the most valuable steroidal analytes for the detection of testosterone misuse. Earlier investigations on the HIR of the same reference population enabled the evaluation of combined measurements of CIR and HIR and its usefulness regarding both steroid metabolism studies and doping control analysis. The combination of both stable isotopes would allow for lower reference limits providing the same statistical power and certainty to distinguish between the endo- or exogenous origin of a urinary steroid.

To what extent does adding tobacco to cannabis expose young users to nicotine?

INTRODUCTION: To determine if mulling, the process of adding tobacco to cannabis for its consumption, exposes young cannabis users to significant levels of nicotine. METHODS: This observational study performed in 2009-2010 among Swiss youths aged 16-25 years involved the completion of a self-administrated questionnaire and the collection of a urine sample on the same day. Measures of urinary cotinine were blindly performed using liquid chromatography coupled-mass spectrometry. A total of 197 eligible participants were divided in 3 groups based on their consumption profile in the past 5 days: 70 abstainers (ABS) not having used cigarettes or cannabis, 57 cannabis users adding tobacco to the cannabis they smoke (MUL) but not having smoked cigarettes, and 70 cigarette smokers (CIG) not having smoked cannabis. RESULTS: Exposure to nicotine was at its lowest among ABS with a mean (SE) cotinine level of 3.2 (1.4) ng/ml compared, respectively, with 214.6 (43.8) and 397.9 (57.4) for MUL and CIG (p < .001). While consumption profile appeared as the only significant factor of influence when examining nicotine exposure from the ABS and MUL participants on multivariate analysis, it did not result in substantial differences among MUL and CIG groups. CONCLUSIONS: Urinary cotinine levels found among MUL are high enough to indicate a significant exposure to nicotine originating from the mulling process. In line with our results, health professionals should pay attention to mulling as it is likely to influence cannabis and cigarette use as well as the efficacy of cessation interventions.

Corrigendum: Hidden figures: Revisiting doping prevalence estimates previously reported for two major international sport events in the context of further empirical evidence and the extant literature.

[This corrects the article DOI: 10.3389/fspor.2022.1017329.].

Longitudinal Profiling of Endogenous Steroids in Blood Using the Athlete Biological Passport Approach.

CONTEXT: Detection of endogenous anabolic androgenic steroids (EAAS), like testosterone (T), as doping agents has been improved with the launch of the Steroidal Module of the Athlete Biological Passport (ABP) in urine samples. OBJECTIVE: To target doping practices with EAAS, particularly in individuals with low level of biomarkers excreted in urine, by including new target compounds measured in blood. DESIGN: T and T/androstenedione (T/A4) distributions were obtained from 4 years of anti-doping data and applied as priors to analyze individual profiles from 2 T administration studies in female and male subjects. SETTING: Anti-doping laboratory. Elite athletes (n = 823) and male and female clinical trials subjects (n = 19 and 14, respectively). INTERVENTION(S): Two open-label administration studies were carried out. One involved a control phase period followed by patch and then oral T administration in male volunteers and the other followed female volunteers during 3 menstrual cycles with 28 days of daily transdermal T application during the second month. MAIN OUTCOME MEASURE(S): Serum samples were analyzed for T and A4 and the performance of a longitudinal ABP-based approach was evaluated for T and T/A4. RESULTS: An ABP-based approach set at a 99% specificity flagged all female subjects during the transdermal T application period and 44% of subjects 3 days after the treatment. T showed the best sensitivity (74%) in response to transdermal T application in males. CONCLUSIONS: Inclusion of T and T/A4 as markers in the Steroidal Module can improve the performance of the ABP to identify T transdermal application, particularly in females.