Detection of methandienone and its metabolites in equine urine, plasma and hair following a multidose oral administration.

Methandienone is an anabolic-androgenic steroid that is prohibited in equine sports due to its potential performance enhancing properties. Metabolism and detection of methandienone in equine urine have been investigated comprehensively in literature; however, there is a limited knowledge about its metabolites in equine plasma and no information about its detection in equine hair. Following a multi-dose oral administration of methandienone to two Thoroughbred horses, 17-epimethandienone, methyltestosterone, two mono-hydroxylated, two di-hydroxylated and three 17α-methylandrostanetriol metabolites were detected in plasma. The majority of these were present as free analytes, whilst the mono-hydroxylated metabolites and one isomer of 17α-methylandrostanetriol were partially conjugated. Estimated peak concentrations of methandienone were 6,000 and 11,100 pg/ml; meanwhile, they were 25.4 and 40.5 pg/ml for methyltestosterone. The most abundant analyte in the post-administration plasma samples of both horses was the mono-hydroxylated metabolite; however, the parent compound provided the longest detection (up to 96 h). Screening analysis of hair enabled the detection of methandienone in mane hair samples only, for up to 3 months. Its mono- and di-hydroxylated metabolites were detected with greater peak responses for up to 6 months post-administration in both mane and tail samples, showing that these metabolites could be better analytical targets for hair analysis when administered orally. A follow-up methodology with an extensive wash procedure confirmed the presence of methandienone and its metabolites in a number of post-administration hair samples. Final wash samples were also analysed to assess the degree of internal incorporation (via bloodstream) against possible external deposition (via sweat/sebum).

Detection of FG-4592 and metabolites in equine plasma, urine and hair following oral administration.

FG-4592 is a hypoxia-inducible factor inhibitor that has been approved for therapeutic use in some countries. This class of compounds can increase the oxygen carrying capacity of the blood and thus have the potential to be used as performance enhancing agents in sports. The purpose of this study was to investigate the detection of FG-4592 and metabolites in equine plasma and mane hair following a multiple dose oral administration to two Thoroughbred racehorses, to identify the best analytical targets for doping control laboratories. Urine samples were also analysed, and the results compared to previously published urine data. Liquid chromatography-high resolution mass spectrometry was used for metabolite identification in urine and plasma. Liquid chromatography-tandem mass spectrometry was used for full sample analysis of urine, plasma and hair samples and generation of urine and plasma profiles. FG-4592 and a mono-hydroxylated metabolite were detected in plasma. FG-4592 was detected with the greatest abundance and gave the longest duration of detection, up to 312 h post-administration, and would be the recommended target in routine doping samples. FG-4592 was detected in all mane hair samples collected post-administration, up to 166 days following the final dose, showing extended detection can be achieved with this matrix. To the best of the authors' knowledge, this is the first report of FG-4592 and metabolites in equine plasma and hair samples. Urine results were consistent with the previously published data, with FG-4592 offering the best target for detection and longest detection periods.

Detection of boldenone in the urine of female horses-ex vivo formation versus administration.

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.

Detection of adeno-associated viral DNA in equine post-administration frozen blood and plasma samples after long-term storage.

Gene doping in horses is a threat to the fairness in sport and has serious implications for animal welfare. To investigate the effect of long-term storage on the detection of AAV in plasma and whole blood, samples from an administration study using an adeno-associated virus serotype 6 expressing green fluorescence protein (AAV6-GFP) were stored at -20°C for 8 months before analysis. The AAV vector was detected in stored plasma samples, following the same detection profile as the fresh plasma samples. The stored blood showed lower overall DNA detection but followed the same detection profile as the plasma samples. This study provides confidence that re-analysing plasma samples and/or analysing a frozen 'B' sample with different matrix such as whole blood after prolonged storage will still result in the detection of gene doping material.

Long-read sequencing assays designed to detect potential gene editing events in the myostatin gene revealed distinct haplotype signatures in the Thoroughbred horse population.

We present here the use of targeted, long-read sequencing of the myostatin (MSTN) gene as a model to detect potential gene editing events in Thoroughbred horses. MSTN is a negative regulator of muscle development, making the gene a prime candidate target for gene doping. By sequencing the complete gene in one PCR product, we can catalogue all mutations without the need to produce short-fragment libraries. A panel of reference material fragments with defined mutations was constructed and successfully sequenced by both Oxford Nanopore and Illumina-based methods, showing that gene doping editing events can be detected using this technology. To ascertain the normal variation within the population, we sequenced the MSTN gene in 119 UK Thoroughbred horses. Variants from the reference genome were assigned to haplotypes and eight distinct patterns, designated Hap1 (reference genome) to Hap8, were determined with haplotypes Hap2 and Hap3 (which includes the 'speed gene' variant) being far the most prevalent. Hap3 was most abundant in flat-racing horses, whereas Hap2 was most abundant in jump-racing. Within this data set, results for 105 racehorses from out-of-competition sampling were compared between matrices of extracted DNA and direct PCR of whole blood from lithium heparin gel tubes, and strong agreement was found between the two methods. The direct-blood PCR was achieved without compromising the sample prior to plasma separation for analytical chemistry, and could thus be used as part of a routine screening workflow for gene editing detection.

Novel holistic pharmacokinetic model applied to plasma and urine concentrations of 2,5-dihydroxybenzene sulphonate following administrations of calcium dobesilate and etamsylate to exercised horses.

Calcium dobesilate (CD) is a synthetic venoactive drug used in veterinary medicine to treat equine navicular disease. Etamsylate is a haemostatic agent used in horses for the treatment of exercise-induced pulmonary haemorrhage. Both etamsylate and CD dissociate in the circulatory system with 2,5-HBSA as the active drug. The aim of the research was to be able to provide detection time (DT) advice from pharmacokinetic (PK) studies in Thoroughbred horses to better inform trainers, and their veterinary surgeons, prescribing these substances for treatment of Thoroughbred racehorses. Two (pilot study) and six (final study) horses were given 28 and 9 repeated dose of CD (3 mg/kg BID) respectively. Two horses were each given a single intravenous (IV) dose of etamsylate (10 mg/kg). Plasma and urine 2,5-HBSA concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The CD pilot study revealed that steady state could be reached with a few days and that 2,5-HBSA in plasma and urine shows instability during storage at -20°C but appears stable at -80°C. A novel holistic non-linear mixed-effects three-compartmental PK model was developed that described both plasma and urine concentrations of 2,5-HBSA, from either CD or etamsylate administration. Typical values for 2,5-HBSA clearance and bioavailability were 2.0 mL/min/kg and 28% respectively. Using the parameters obtained from this PK model, in conjunction with methodology developed by Toutain, afforded a possible screening limit (SL) that can regulate for a DT of 3 days in urine; however, a corresponding SL in plasma would be below current levels of detection. However, it is the responsibility of the individual racing authorities to apply their own risk management with regard to SLs and DTs.

Use of mitochondrial sequencing to detect gene doping in horses via gene editing and somatic cell nuclear transfer.

Gene editing and subsequent cloning techniques offer great potential not only in genetic disease correction in domestic animals but also in livestock production by enhancement of desirable traits. The existence of the technology, however, leaves it open to potential misuse in performance-led sports such as horseracing and other equestrian events. Recent advances in equine gene editing, regarding the generation of gene-edited embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer, have highlighted the need to develop tools to detect potential prohibited use of the technology. One possible method involves the characterisation of the mitochondrial genome (which is not routinely preserved during cloning) and comparing it with the sequence of the registered dam. We present here our approach to whole-mitochondrial sequencing using tiled long-range PCR and next-generation sequencing. To determine whether the background mutation rate in the mitochondrial genome could potentially confound results, we sequenced 10 sets of dam and foal duos. We found variation between duos but none within duos, indicating that this method is feasible for future screening systems. Analysis of WGS data from over 100 Thoroughbred horses revealed wide variation in the mitochondria sequence within the breed, further displaying the utility of this approach.

Direct sequence confirmation of qPCR products for gene doping assay validation in horses.

The misuse of gene therapy by the introduction of transgenes via plasmid or viral vectors as a doping agent is an increasing concern in human and animal sports, not only in consideration to fair competition but also in potential detrimental effects to welfare. Doping events can be detected by polymerase chain reaction (PCR) amplification of a transgene-specific region of DNA. Quantitative real-time PCR (qPCR) is particularly suited to confirmatory investigations where precise limits of detection can be calculated. To fully validate a qPCR experiment, it is highly desirable to confirm the identity of the amplicon. Although post-PCR techniques such as melt curve and fragment size analysis can provide strong evidence that the amplicon is as expected, sequence identity confirmation may be beneficial as part of regulatory proceedings. We present here our investigation into two alternative processes for the direct assessment of qPCR products for five genes using next-generation sequencing: ligation of sequence-ready adapters to qPCR products and qPCR assays performed with primers tailed with Illumina flow cell binding sites. To fully test the robustness of the techniques at concentrations required for gene doping detection, we also calculated a putative limit of detection for the assays. Both ligated adapters and tailed primers were successful in producing sequence data for the qPCR products without further amplification. Ligated adapters are preferred, however, as they do not require re-optimisation of existing qPCR assays.

Plasma and urine pharmacokinetics of hydroxyzine and cetirizine following repeated oral administrations to exercised horses.

Hydroxyzine and cetirizine are first- and second-generation oral antihistamine drugs, respectively, used to treat allergic reactions in horses. Cetirizine is also a metabolite of hydroxyzine, which may lead to complexities in regulating their use in equine sporting events. The aim of the research was to be able to provide detection times (DT) from pharmacokinetic studies in thoroughbred horses to better inform trainers, and their veterinary surgeons, prescribing these substances for treatment of Thoroughbred racehorses. Six and two horses were given 9 repeated administrations of hydroxyzine HCl (500 mg BID) or cetirizine HCl (190 mg BID), respectively. Plasma and urine hydroxyzine and cetirizine concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A holistic non-linear mixed effects PK model was developed that described both plasma and urine concentrations of hydroxyzine and cetirizine, either from administration of each individually or cetirizine as a metabolite of hydroxyzine. Using the parameters obtained from this PK model in conjunction with methodology developed by Toutain afforded possible screening limits (SL) that can regulate for a DT of 4 days in either plasma or urine. Hydroxyzine and cetirizine concentration prediction intervals for the 80th , 95th and 99th percentiles of a virtual horse population were performed in order to assess the statistical protection of the DT. However, it is down to the individual racing authorities to apply their own risk management.

Identification of equine in vitro metabolites of seven non-steroidal selective androgen receptor modulators for doping control purposes.

Selective androgen receptor modulators, SARMs, are a large class of compounds developed to provide therapeutic anabolic effects with minimal androgenic side effects. A wide range of these compounds are available to purchase online and thus provide the potential for abuse in sports. Knowledge of the metabolism of these compounds is essential to aid their detection in doping control samples. In vitro models allow a quick, cost-effective response where administration studies are yet to be carried out. In this study, the equine phase I metabolism of the non-steroidal SARMs GSK2881078, LGD-2226, LGD-3303, PF-06260414, ACP-105, RAD-140 and S-23 was investigated using equine liver microsomes. Liquid chromatography coupled to a QExactive Orbitrap mass spectrometer allowed identification of metabolites with high resolution and mass accuracy. Three metabolites were identified for both GSK2881078 and LGD-2226, four for LGD-3303 and RAD-140, five for PF-06260414, twelve for ACP-105 and ten for S-23. The equine metabolism of GSK-2881078, LGD-2226, LGD-3303 and PF-06260414 is reported for the first time. Although the equine metabolism of ACP-105, RAD-140 and S-23 has previously been reported, the results obtained in this study have been compared with published data.

Pharmacokinetics of paracetamol in the Thoroughbred horse following an oral multi-dose administration.

Paracetamol is a widely used, non-opioid analgesic and antipyretic drug. Scientific evidence suggests that it is an effective pain treatment in equine medicine. However, there is very little published information about the pharmacokinetics of the drug in the horse. The aim of the research was to determine the pharmacokinetics of paracetamol in equine plasma and urine to inform treatment of Thoroughbred racehorses. In this multi-dose study, paracetamol was administered orally at 20 mg/kg to six Thoroughbred horses. Pre- and post-administration urine and plasma samples were collected and analysed using a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Pharmacokinetic analysis of urine and plasma paracetamol clearance profiles was carried out, which enabled the calculation of possible screening limits (SL) that can regulate for a detection time of 120 h. Additionally, an estimation of orthocetamol concentration levels in urine was carried out to investigate any underlying relationship between the para- and ortho-isomers as both were suspected to contribute to basal levels, possibly due to environmental feed sources.

The intravenous pharmacokinetics of butorphanol and detomidine dosed in combination compared with individual dose administrations to exercised horses.

In equine and racing practice, detomidine and butorphanol are commonly used in combination for their sedative properties. The aim of the study was to produce detection times to better inform European veterinary surgeons, so that both drugs can be used appropriately under regulatory rules. Three independent groups of 7, 8 and 6 horses, respectively, were given either a single intravenous administration of butorphanol (100 µg/kg), a single intravenous administration of detomidine (10 µg/kg) or a combination of both at 25 (butorphanol) and 10 (detomidine) µg/kg. Plasma and urine concentrations of butorphanol, detomidine and 3-hydroxydetomidine at predetermined time points were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The intravenous pharmacokinetics of butorphanol dosed individually compared with co-administration with detomidine had approximately a twofold larger clearance (646 ± 137 vs. 380 ± 86 ml hr-1  kg-1 ) but similar terminal half-life (5.21 ± 1.56 vs. 5.43 ± 0.44 hr). Pseudo-steady-state urine to plasma butorphanol concentration ratios were 730 and 560, respectively. The intravenous pharmacokinetics of detomidine dosed as a single administration compared with co-administration with butorphanol had similar clearance (3,278 ± 1,412 vs. 2,519 ± 630 ml hr-1  kg-1 ) but a slightly shorter terminal half-life (0.57 ± 0.06 vs. 0.70 ± 0.11 hr). Pseudo-steady-state urine to plasma detomidine concentration ratios are 4 and 8, respectively. The 3-hydroxy metabolite of detomidine was detected for at least 35 hr in urine from both the single and co-administrations. Detection times of 72 and 48 hr are recommended for the control of butorphanol and detomidine, respectively, in horseracing and equestrian competitions.

Bioformation of boldenone and related precursors/metabolites in equine feces and urine, with relevance to doping control.

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.

Re-evaluation of the pharmacokinetics of xylazine administered to Thoroughbred horses.

Xylazine is widely used worldwide as a short-acting sedative in general equine and racing practice. In the UK, although it has a legitimate use during training, equine anti-doping rules state it is a prohibited substance on race day. The aim of the study was to produce a detection time (DT) to better inform European veterinary surgeons so that xylazine can be used appropriately under regulatory rules. Previous publications have various limitations pertaining to analysis method, particularly for plasma and limited length of time of sample collection. In this study, pharmacokinetic data were produced for xylazine and 4-OH-xylazine in equine urine and plasma following a single intravenous xylazine dose of 0.4 mg/kg to six Thoroughbred horses. Pharmacokinetic parameters were generated from a 3-compartmental model with clearance = 15.8 ± 4.88 ml min-1  kg-1 , Vss = 1.44 ± 0.38 L/kg, terminal half-life = 29.8 ± 12.7 hr and a DT determined at 71 hr for the administration of xylazine (Chanazine® ) in plasma and urine. Urine screening should aim to detect the 4-OH-xylazine metabolite, which can act as an indicator for the xylazine plasma concentration. A DT of 72 hr has been agreed by the European Horserace Scientific Liaison Committee, to be implemented in June 2019.

Investigation of the metabolism of the selective androgen receptor modulator LGD-4033 in equine urine, plasma and hair following oral administration.

LGD-4033 is one of a number of selective androgen receptor modulators (SARMs) that are being developed by the pharmaceutical industry to provide the therapeutic benefits of anabolic androgenic steroids, without the less desirable side effects. Though not available therapeutically, SARMs are available for purchase online as supplement products. The potential for performance enhancing effects associated with these products makes them a significant concern with regards to doping control in sports. The purpose of this study was to investigate the metabolism of LGD-4033 in the horse following oral administration, in order to identify the most appropriate analytical targets for doping control laboratories. LGD-4033 was orally administered to two Thoroughbred horses and urine, plasma and hair samples were collected and analysed for parent drug and metabolites. LC-HRMS was used for metabolite identification in urine and plasma. Eight metabolites were detected in urine, five of which were excreted only as phase II conjugates, with the longest detection time being observed for di- and tri-hydroxylated metabolites. The parent compound could only be detected in urine in the conjugated fraction. Seven metabolites were detected in plasma along with the parent compound where mono-hydroxylated metabolites provided the longest duration of detection. Preliminary investigations with hair samples using LC-MS/MS analysis indicated the presence of trace amounts of the parent compound and one of the mono-hydroxylated metabolites. In vitro incubation of LGD-4033 with equine liver microsomes was also performed for comparison, yielding 11 phase I metabolites. All of the metabolites observed in vivo were also observed in vitro.

Monitoring dehydroepiandrosterone (DHEA) in the urine of Thoroughbred geldings for doping control purposes.

The use of testosterone and its pro-drugs, such as dehydroepiandrosterone (DHEA), is currently regulated in horseracing by the application of international testosterone thresholds. However, additional steroidomic approaches, such as steroid ratios, to distinguish overall adrenal stimulation from drug administrations and an equine biological passport for longitudinal steroid profiling of individual animals could be advantageous in equine doping testing. Thus, DHEA concentrations and related ratios (testosterone [T] to DHEA and DHEA to epitestosterone [E]) were assessed in the reference population by quantitative analysis of 200 post-race gelding urine samples using liquid chromatography-tandem mass spectrometry. DHEA concentrations ranged between 0.9 and 136.6 ng/mL (mean 12.8 ng/mL), T:DHEA ratios between 0.06 and 1.85 (mean 0.43), and DHEA:E ratios between 0.21 and 13.56 (mean 2.20). Based on the reference population statistical upper limits of 5.4 for T:DHEA ratio and 48.1 for DHEA:E ratio are proposed with a risk of 1 in 10 000 for a normal outlier exceeding the value. Analysis of post-administration urine samples collected following administrations of DHEA, Equi-Bolic® (a mix of DHEA and pregnenolone) and testosterone propionate to geldings showed that the upper limit for T:DHEA ratio was exceeded following testosterone propionate administration and DHEA:E ratio following DHEA administrations and thus these ratios could be used as additional biomarkers when determining the cause of an atypical testosterone concentration. Additionally, DHEA concentrations and ratios can be used as a starting point to establish reference ranges for an equine biological passport.

Interlaboratory trial for the measurement of total cobalt in equine urine and plasma by ICP-MS.

Cobalt is an essential mineral micronutrient and is regularly present in equine nutritional and feed supplements. Therefore, cobalt is naturally present at low concentrations in biological samples. The administration of cobalt chloride is considered to be blood doping and is thus prohibited. To control the misuse of cobalt, it was mandatory to establish an international threshold for cobalt in plasma and/or in urine. To achieve this goal, an international collaboration, consisting of an interlaboratory comparison between 5 laboratories for the urine study and 8 laboratories for the plasma study, has been undertaken. Quantification of cobalt in the biological samples was performed by inductively coupled plasma-mass spectrometry (ICP-MS). Ring tests were based on the analysis of 5 urine samples supplemented at concentrations ranging from 5 up to 500 ng/mL and 5 plasma samples spiked at concentrations ranging from 0.5 up to 25 ng/mL. The results obtained from the different laboratories were collected, compiled, and compared to assess the reproducibility and robustness of cobalt quantification measurements. The statistical approach for the ring test for total cobalt in urine was based on the determination of percentage deviations from the calculated means, while robust statistics based on the calculated median were applied to the ring test for total cobalt in plasma. The inter-laboratory comparisons in urine and in plasma were successful so that 97.6% of the urine samples and 97.5% of the plasma samples gave satisfactory results. Threshold values for cobalt in plasma and urine were established from data only obtained by laboratories involved in the ring test. Copyright © 2017 John Wiley & Sons, Ltd.

Application of testosterone to epitestosterone ratio to horse urine - a complementary approach to detect the administrations of testosterone and its pro-drugs in Thoroughbred geldings.

Detection of testosterone and/or its pro-drugs in the gelding is currently regulated by the application of an international threshold for urinary testosterone of 20 ng/mL. The use of steroid ratios may provide a useful supplementary approach to aid in differentiating between the administration of these steroids and unusual physiological conditions that may result in atypically high testosterone concentrations. In the current study, an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify testosterone (T) and epitestosterone (E). The method was used to analyze 200 post-race urine samples from geldings in order to generate the ratios for the reference population. Following statistical analysis of the data, an upper limit of 5 for T:E ratio in geldings is proposed. Samples collected from 15 geldings with atypical urinary testosterone concentrations (>15 ng/mL) but otherwise normal steroid profile, had T:E ratios within those observed for the reference population. The applicability of an upper T:E ratio to detect an administration was demonstrated by the analysis of a selection of incurred samples from testosterone propionate, dehydroepiandrosterone (DHEA), and a mixture of DHEA and pregnenolone (Equi-Bolic®) administrations. These produced testosterone concentrations above the threshold of 20 ng/mL, but also T:E ratios above the proposed limit of 5. In conclusion, consideration of the T:E ratio appears to be a valuable complementary aid to evaluate whether an atypical testosterone concentration could be caused by a natural biological outlier as opposed to the administration of these steroids. Copyright © 2016 John Wiley & Sons, Ltd.