Received: 14 July 2017 Revised: 10 August 2017 Accepted: 17 August 2017 Published online in Wiley Online Library: 27 October 2017

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1 Research article Received: 14 July 2017 Revised: 10 August 2017 Accepted: 17 August 2017 Published online in Wiley Online Library: 27 October 2017 ( DOI /dta.2291 Epiandrosterone sulfate prolongs the detectability of testosterone, 4-androstenedione, and dihydrotestosterone misuse by means of carbon isotope ratio mass spectrometry Thomas Piper, a * Marlen Putz, a Wilhelm Schänzer, a Valentin Pop, b Malcolm D. McLeod, c Dimanthi R. Uduwela, c Bradley J. Stevenson c and Mario Thevis a,d In the course of investigations into the metabolism of testosterone (T) by means of deuterated T and hydrogen isotope ratio mass spectrometry, a pronounced influence of the oral administration of T on sulfoconjugated steroid metabolites was observed. Especially in case of epiandrosterone sulfate (EPIA_S), the contribution of exogenous T to the urinary metabolite was traceable up to 8 days after a single oral dose of 40 mg of T. These findings initiated follow-up studies on the capability of EPIA_S to extend the detection of T and T analogue misuse by carbon isotope ratio (CIR) mass spectrometry in sports drug testing. Excretion study urine samples obtained after transdermal application of T and after oral administration of 4-androstenedione, dihydrotestosterone, and EPIA were investigated regarding urinary concentrations and CIR. With each administered steroid, EPIA_S was significantly depleted and prolonged the detectability when compared to routinely used steroidal target compounds by a factor of 2 to 5. In order to simplify the sample preparation procedure for sulfoconjugated compounds, enzymatic cleavage by Pseudomonas aeruginosa arylsulfatase was tested and implemented into CIR measurements for the first time. Further simplification was achieved by employing multidimensional gas chromatography to ensure the required peak purity for CIR determinations, instead of sample purification strategies using liquid chromatographic fractionation. Taking into account these results that demonstrate the unique and broad applicability of EPIA_S for the detection of illicit administrations of T or T-related steroids, careful consideration of how this steroid can be implemented into routine doping control analysis appears warranted. Copyright 2017 John Wiley & Sons, Ltd. Keywords: testosterone; carbon isotope ratio; epiandrosterone; doping; long-term detectability; sulfoconjugated steroids Introduction Detecting the misuse of testosterone (17β-hydroxyandrost-4-en-3- one, T) is still of major concern for doping control analysis and remains challenging due to the ubiquitous presence of T in human urine. Urinary concentration thresholds or diagnostic ratios such as T / epitestosterone (17α-hydroxyandrost-4-en-3-one, E) may suffice to identify suspicious samples in initial testing procedures but it cannot unambiguously prove the administration of an illicit compound. Confounding factors like ethanol intake or microbial degradation together with intra- and inter-individual variation diminish the probative force of these markers. [1] Carbon isotope ratios (CIR) allow for discrimination between an endogenous or exogenous source of excreted urinary T and T- metabolites. [2,3] Endogenous steroids reflect the isotopic composition of the individual s diet, whereas exogenous steroids commonly exhibit more depleted CIR, i.e. pharmaceutical preparations contain a lower 13 C content. CIR are expressed as δ 13 C-values against the Drug Test. Analysis 2017, 9, international standard Vienna Pee Dee Belemnite (VPDB) following equation (1) and given in or mur: [4] * Correspondence to: Thomas Piper, German Sport University Cologne, Center for Preventive Doping Research, Am Sportpark Müngersdorf 6, Cologne, Germany. t.piper@biochem.dshs-koeln.de a German Sport University Cologne, Center for Preventive Doping Research, Am Sportpark Müngersdorf 6, Cologne, Germany b Romanian Doping Control Laboratory, National Anti-Doping Agency, Bvd. Basarabia, nr , Bucharest, Romania c Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia d European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/ Bonn, Germany Copyright 2017 John Wiley & Sons, Ltd. 1695

2 1696 δ 13 C VPDB ¼. 13 C 12 C 13 C 12 C SAMPLE VPDB 1 (1) To compensate for the inter-individual variation in diet and, consequently, different δ 13 C-values, differences between so called endogenous reference compounds (ERC) like pregnanediol (5βpregnane-3α,20α-diol, PD) and the different target compounds (TC) are calculated based on equation (2): Δ ½ Š ¼ δ 13 C ERC δ 13 C TC (2) Following the administration of T or T-related steroids, metabolites found downstream in the metabolic pathway present with depleted CIR and, accordingly, Δ-values will increase as the ERC remains unaffected. Depending on the endogenous production and excretion rate of the different metabolites, the influence of the exogenous administration will be more or less pronounced over time as the depleted CIR will undergo dilution by the endogenous signal. The slower the turnover of the pool of a steroid is, the longer this pool will conserve the exogenous signal and, consequently, the longer this will be detectable in doping control specimens. In a recent T elimination study, epiandrosterone (3β-hydroxy-5αandrostan-17-one, EPIA) demonstrated the desired prolonged detectability of an exogenous CIR, most probably due to the slow turnover of its pool described above. [5] In an earlier study, EPIA also showed a prolonged influence after the administration of dehydroepiandrosterone (3β-hydroxyandrost-5-en-17-one, DHEA), a so called T-prohormone, and so further studies were considered warranted. [6] In order to evaluate if EPIA offers a similar advantageous behavior not only after oral T or DHEA administrations but also after different routes of administration or different T-related steroids, the following administration studies were considered: transdermal application of T (T-GEL), oral application of 4- androstenedione (androst-4-ene-3,17-dione, ADION), dihydrotestosterone (17β-hydroxy-5α-androstan-3-one, DHT), and EPIA itself, which is marketed as a DHT booster on the black market. As EPIA is exclusively converted to its sulfoconjugate in humans, the corresponding sample preparation is complicated and commonly necessitates an acidic solvolysis carried out after the enzymatic hydrolysis of all glucuronides. [6,7] The possibility to simplify this step in sample preparation by using the recently described Pseudomonas aeruginosa arylsulfatase (PaS) was tested for CIR determination here for the first time. [8] Further simplification of the analytical procedure was accomplished by using multidimensional gas chromatography (MDGC) prior to isotope ratio mass spectrometry (IRMS) determinations, instead of purifying analytes by high performance liquid chromatography (HPLC). [9] Experimental Reagents and chemicals Pyridine, glacial acetic acid, sodium hydroxide (NaOH), methanol (MeOH), sulfuric acid (H 2 SO 4 ), acetonitrile (ACN), tert-butyl methyl ether (TBME) and cyclohexane were purchased from Merck (Darmstadt, Germany). Chromabond C18 cartridges (500 mg, 6 ml) were obtained from Macherey & Nagel (Düren, Germany). Acetic anhydride was from Fluka/Sigma-Aldrich (Steinheim, Germany). All solvents and reagents were of analytical grade. β- glucuronidase from Escherichia coli was from Roche Diagnostics GmbH (Mannheim, Germany) and two strains of PaS (wild type PaS [8] and a mutant PVFV PaS) were expressed in E. coli as previously described. The details of PaS mutant generation will be described in a forthcoming publication. Steroid reference materials 3β-hydroxy-5α-androstane (RSTD), 5α-androst-16-en-3α-ol (16EN), 5α-androstane-3α,17β-diol (5a), 5βandrostane-3α,17β-diol (5b), and androst-5-ene-3β,17β-diol (5EN) were supplied by Steraloids (Newport, RI, USA), while 3α-hydroxy- 5α-androstan-17-one (A), 3α-hydroxy-5β-androstan-17-one (ETIO), E, T, DHEA, DHT, EPIA and PD were supplied by Sigma-Aldrich, and ADION was from Fluka/Sigma-Aldrich. The CO 2 tank gas (Linde, Pullach, Germany) was calibrated against a secondary reference material USADA 33-1 provided by Cornell University (Ithaca, NY, USA). [10] Elimination studies Testosterone gel For 7 consecutive days, two healthy male volunteers (both 38 years old) applied 2 bags of TESTOGEL (25 mg of T in 2.5 g of gel, Jenapharm, Jena, Germany) once daily in the evening. T extracted from the gel showed a δ 13 C VPDB -value of 29.1 ± 0.3 (n = 3). Urine samples were collected each morning during the application period and for 10 days before and after the study. All samples were stored frozen at 20 C until analysis. Within this study, 2 samples collected prior to the administration were prepared and used as blank urines together with all samples collected during the administration and the first 3 sample after cessation. Epiandrosterone One healthy male volunteer aged 42 years administered one capsule of ANDROVAR (Hardrock Supplements, USA) ordered via the internet. Each capsule was declared to contain 100 mg of EPIA. The product was tested prior to the study by HPLC coupled to time of flight mass spectrometry, and a purity > 99 % and a content of approx. 100 mg per capsule was confirmed. The CIR was δ 13 C VPDB = 30.2 ±0.1 (n = 6). Prior to the administration, 3 urine samples were collected as blank specimens. For the first 2 days post-administration, each urine was collected followed by 13 days of sampling the morning urine only, which resulted in a total of n = 25 specimens. All urine samples were stored frozen at 20 C until analysis. 4-Androstenedione One healthy male volunteer aged 65 years administered 80 mg of ADION dissolved in 20 ml of a mixture containing ethanol (EtOH)/water (25/75, v/v). The ADION was pure with a measured δ 13 C VPDB -value of 31.8 ± 0.3 (n = 3). Directly before the application, one blank urine was collected. Then, each urine was sampled for the following 24 h, 3 urine samples were collected during the second day, and for the next 6 days one urine each resulting in a total of n = 16 specimens that were stored at +4 C until analysis, which was conducted within 2 weeks after sampling. Dihydrotestosterone T. Piper et al. Fifty milligrams of DHT were dissolved in 20 ml of EtOH/water as described before and administered orally by a healthy male volunteer aged 42 years. The purity was > 97% and the CIR was δ 13 C VPDB = 28.9 ± 0.1 (n = 3). The administration took place after sampling of a blank urine. Then, selected specimens were collected for 3 days followed by collection of morning urines for the wileyonlinelibrary.com/journal/dta Copyright 2017 John Wiley & Sons, Ltd. Drug Test. Analysis 2017, 9,

3 Epiandrosterone sulfate prolongs the detectability of testosterone, 4-androstenedione and dihydrotestosterone misuse next 7 days. All samples were stored at +4 C until analysis conducted within 2 weeks after sampling. For all studies, participants provided written informed consent and study approval was obtained from the Ethical Committee of the National Institute of Sports of Romania (Bucharest, Romania). Sample preparation for CIR determinations All samples collected during the elimination studies for T-GEL and for EPIA were prepared following established protocols. [7,11] The samples derived from the administration studies conducted with DHT and ADION were prepared following a new protocol encompassing enzymatic cleavage of the sulfoconjugated steroids, while for the glucuronylated steroids an already published method was adopted. [12] The complete procedure and its validation will be subject of an independent publication and therefore is only described briefly herein: To a conditioned C18 cartridge, 5 to 20 ml of urine was applied, washed and eluted with MeOH. After evaporation, the dry residue was reconstituted with 2 ml of sodium phosphate buffer (0.2 M, ph 7) and 5 ml of TBME was added. After shaking and centrifugation, the organic layer was discarded and 100 μl of β-glucuronidase was added to the aqueous phase and the samples were incubated for 1 h at 50 C. After glucuronide hydrolysis, the ph was adjusted to 10 by adding 1 ml of an aqueous carbonate buffer (K 2 CO 3 /KHCO 3 1:1, w/w, 200 g/l) and 5 ml of TBME was added for another liquid-liquid-extraction. The aqueous layer was kept for the sulfoconjugated steroids and the organic layer now containing the formerly glucuronylated steroids was evaporated to dryness and subjected to acetylation using 75 μl of pyridine and 75 μl of acetic anhydride for 1 h at 70 C. After evaporation to dryness, the samples were reconstituted in 3 ml of ACN/water (50/50, v/v) and applied to a conditioned C18 cartridge. After washing with 2 ml of the same solvent, the first fraction containing ETIO, A, and T was eluted with 5 ml of ACN/water (75/25, v/v). Then, the second fraction containing 16EN, 5a, 5b and PD was eluted with 5 ml ACN. The eluates were dried and transferred into auto sampler vials with 250 μl of cyclohexane. Fraction 1 was split and 25 μl (ca. 1/10 th ) of the volume was used for diluted injections, accounting for the higher concentrations of ETIO and A compared to T. Samples were dried and reconstituted in cyclohexane as necessary for the MDGC-IRMS determinations to fall into the linear range of the IRMS instrument. To the aqueous residue containing the sulfoconjugated steroids, 3 ml of water and 300 μl of glacial acetic acid were added to adjust to a ph < 5. Then the samples were applied to a conditioned C18 cartridge, washed with water and eluted with MeOH. After drying 2 ml of Tris-HCL buffer (0.05 M) and 60 μl of PaS (19 mg/ml) were added. The samples were incubated either 4 h at 50 C or overnight at 37 C. Then 1 ml of the above mentioned aqueous carbonate buffer was added together with 5 ml of TBME. After shaking and centrifugation the organic layer containing formerly sulfoconjugated steroids was transferred and evaporated to dryness. After acetylation and sample transfer as described above, the samples were reconstituted in an appropriate amount of cyclohexane for MDGC-IRMS measurements to fall into the linear range of the IRMS instrument. (length 30 m, i.d mm and film thickness 1 μm) was installed while in the second dimension an Agilent J&W Scientific DB-17MS (length 30 m, i.d mm, film thickness 0.25 μm) was used. Injections were performed with up to 4 μl in splitless mode at 300 C, and each measurement was monitored by using the internal standard double method adding 1 μl of a solution containing 40 μg/ml of RSTD in cyclohexane. GC oven temperature programs were adjusted as needed, exemplarily those for DHEA and EPIA will be described in detail. The first GC started at 100 C (held for 1.5 min), then increased at 30 C/min to 260 C, then with 10 C to 300 C, held for 37 min under constant pressure at 4 bar. Carrier gas was He (purity grade 5.0). Sample transfer from the first to the second dimension took place from 20.8 to 21.8 min for RSTD and from 26.5 to 28.6 min for DHEA and EPIA by changing the auxiliary pressure from 3.15 to 3.20 bar. In case of analytes with significantly different concentrations this collection window was modified to either collect DHEA (26.7 to 27.5 min) or EPIA (27.3 to 28.6 min). The second GC was held at an initial temperature of 100 C for 31 min, then heated at 40 C/min to 273 C, then at 1 C/min to 281 C followed by 40 C/min to 320 C held for 3 min. This resulted in a total run time of 47.5 min. Analytes were combusted to CO 2 at 950 C and Isodat 3.0 (ThermoFisher, Bremen, Germany) was used for data acquisition and evaluation. To monitor peak purity and specificity, a Thermo ISQ single quadrupole mass spectrometer was appended to the end of the second GC column. The MS was operated in electron ionization mode and data was collected in full scan mode from m/z 50 to 500 using Thermo Xcalibur version 2.2. Gas chromatography/high resolution mass spectrometry (GC/Q-TOF) In order to semi-quantify the sulfoconjugated steroids and to monitor relative yields during method development employing enzymatic cleavage of the sulfoconjugated analytes, hydrolysed samples were injected into an Agilent 7200 Accurate-Mass Q-TOF system coupled to an Agilent 7890A gas chromatograph (Santa Clara, CA, USA). Injections were performed in pulsed splitless mode at 280 C with an injection volume of 2 μl and an injection pulsed pressure of 25 psi. The GC column was a HP5-MS (length 30 m, i.d mm, film thickness 0.25 μm) from Agilent. The temperature program started at 100 C held for 2 min, then with 40 C/min to 250 C, then with 3 C/min to 276 C and finally with 40 C/min to 310 C held for 2 min. During analysis a constant flow of 1.2 ml/min as applied, carrier gas was He (purity 4.6). The EI source was operated at 40 ev at a temperature of 250 C. The scan range of m/z was covered at a scan speed of 200 ms/spectrum. For data acquisition and evaluation MassHunter software (version B.06, Agilent) was used. Steroid profile determinations All urinary steroid concentrations were determined following established routine protocols. [1] Gas chromatography/isotope ratio mass spectrometry setup The CIR determinations for the T-GEL and the EPIA excretion study were carried out using conditions described elsewhere. [5,13] The MDGC-IRMS setup followed a published configuration with some modifications. [9] In the first dimension, an OPTIMA 1 column Method development regarding enzymatic cleavage of sulfoconjugated steroids As no data was available on the suitability of PaS with regards to CIR determinations, this aspect was carefully investigated. A comparison to the currently applied acidic solvolysis was carried out and 1697 Drug Test. Analysis 2017, 9, Copyright 2017 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/dta

4 T. Piper et al. the new method was validated in accordance to established protocols including linear mixing models. [6,11,14] Several reaction conditions (time and temperature) were investigated regarding the overall yield and the resulting CIR. The full validation for the glucuronylated steroids and MDGC-IRMS is ongoing and will be published in the near future. Results and discussion Method validation for sulfoconjugated steroids Method development In contrast to the general ability of the acidic solvolysis to cleave every sulfoconjugated steroid, PaS exhibited a very specific enzymatic activity. As demonstrated in Figure 1, only defined steroids are liberated encompassing DHEA, EPIA and 5EN resulting in favorably clean chromatograms. This improvement in selectivity was in part achieved at the expense of analytes recovery as the employed protocol with PaS resulted in lower yields, arguably due to incomplete deconjugation. Depending on the applied reaction conditions, this yield ranged from 40 to 80 % compared to the acidic solvolysis. Comparing both strains of PaS, both worked similar well with PVFV showing slightly elevated yields compared to the wild type PaS. Therefor the PVFV was used throughout this study. Incomplete reaction yields can be accompanied by isotopic fractionation and changes in CIR have been demonstrated for steroids undergoing spontaneous and incomplete deconjugation. [15] While the reaction yields could probably be optimized further by varying the amounts of enzyme or incubation times, this step was not of utmost priority as no significant (t-test, p <0.05) influence on the measured CIR was detected (Table 1). Table 1. Comparison of CIR measured in different aliquots of the same urine sample after acidic solvolysis or employing PaS. All values given in δ 13 C VPDB [ ]. Acidic solvolysis PaS n RSTD DHEA EPIA RSTD DHEA EPIA Mean SD of 4, a combined analyte transfer results in the same CIR as under separate transfer conditions. Especially after administration of exogenous steroids, the concentration of EPIA can be significantly elevated. In such cases, separation of analytes was a mandatory step to ensure valid results. Validation results In accordance to published protocols for validation of CIR methods, the repeatability of the new approach was tested by means of a six- Method performance The ability of the developed MDGC-IRMS approach to determine DHEA and EPIA is demonstrated in Figures 2 and 3. Interfering matrix components still present can easily be separated on the first GC dimension. From here, only areas of interest in the chromatogram are transferred to the second dimension, containing the RSTD or the analytes of interest. The transfer is accomplished as reported earlier by changing pressures applied to the Deans switch device. [9] Both analytes of interest can either be transferred by employing a single window or separately, which then necessitates 2 injections of the same sample. As long as the concentrations of DHEA and EPIA are comparable, i.e. they do not differ by more than a factor Figure 2. FID chromatogram obtained after injection of one urine sample without sample transfer to the second dimension to fix retention times. Collection windows were programmed as depicted and described in the Experimental section Figure 1. Total ion chromatograms of the same urine sample treated with acidic solvolysis (upper part) or PaS (lower part) to liberate the sulfoconjugated steroids. Some identified peaks are highlighted exemplarily. wileyonlinelibrary.com/journal/dta Copyright 2017 John Wiley & Sons, Ltd. Drug Test. Analysis 2017, 9,

5 Epiandrosterone sulfate prolongs the detectability of testosterone, 4-androstenedione and dihydrotestosterone misuse Figure 3. MDGC-IRMS chromatogram of one urine sample injected three times using different sample transfer options: a) combined transfer of DHEA and EPIA, b) small transfer window for DHEA only, c) the same for EPIA. Further information in the text. fold preparation of one blank urine sample and by a standard addition technique referred to as linear mixing models. [14] The results obtained on repeatability are listed in Table 2 and showed comparable or improved values to already published data on steroids. [11] Linear mixing models allow for determination of both inter- and intra-day precision and can demonstrate the absence of isotopic fractionation during sample preparation and measurement. [16] The parameters obtained for the linear regression are listed in Table 3 and demonstrate that the developed method is fit-forpurpose. In general, standard deviations were lower compared to published results, indicating that either the omitted HPLC clean up step contributed to the assay s uncertainty or the use of PaS allows for more reproducible results than the acidic solvolysis. In addition, the linear range of the MDGC-IRMS was tested by repeated injections of mixed pure standards covering a range from 10 to 200 ng of steroid on column. Resulting peak heights fell between 120 to 2800 mv and within this range the instrument operates linearly. Peak heights above 4000 mv showed constantly enriched values for EPIA due to the fact that both peaks (Figure 3) are eluting in close proximity and steroid concentrations exceeded the separation power of the column at this level. In such cases, dilution of the sample or separation of both analytes in two analytical runs is necessary. The use of 5EN as an additional ERC especially for those urine samples showing a high content of DHEA will be investigated in the future as first tests showed promising results (data not shown). Additionally, reference population investigations are scheduled and will be published at a later stage. Table 2. Method repeatability obtained by a six-fold preparation of one blank urine. All values given in δ 13 C VPDB [ ]. sample RSTD DHEA EPIA BW BW BW BW BW BW Mean SD Elimination studies Albeit comparable, the results of each administration study will be presented separately to enhance readability. Afterwards, the results will be summarized and final conclusions presented. Testosterone gel Almost 20 years ago, the pharmacokinetic properties of T-GEL were investigated and a preferred metabolism to steroids with a 5αconfiguration (DHT and 5a) was detected and attributed to increased 5α-reductase activity in the skin. [17] In accordance with this finding, the urinary metabolites T and 5a excreted in their 1699 Drug Test. Analysis 2017, 9, Copyright 2017 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/dta

6 T. Piper et al. Table 3. Results obtained for the Gaussian least squares fit on the linear mixing model for EPIA and DHEA. Listed are the slope of the line of best fit and its intercept with the y-axis together with the associated standard deviations. The combined measurement uncertainty is given for k = 1. Steroid Slope (a) SD (a) Intercept (b) SD(b) Std added Combined m u EPIA DHEA glucuronylated form were the most promising candidates to detect the misuse of T-GEL using IRMS. [14] Both volunteers of this study showed T/E ratios of 1.3 and 1.6 prior to the first T-GEL application, and this ratio was only slightly elevated over the course of the study reaching maximum values of 2.8 and 3.3, respectively. Compared to the presumed endogenous production of T in adult males of ca. 7 mg/day, the amount added via exogenous T is considered relatively small. [18] This is not only reflected by the found urinary concentrations but also by the determined CIR as shown in Figure 4. In agreement with earlier reported cases, the response in CIR grows slowly over time. [14] Presumably this is due to the fact that the endogenous T pool is only partially substituted by the exogenous T, and as the endogenous production is downregulated the portion of exogenous T continuously becomes larger. Whether the endogenous T production or the absorption of T-GEL varies inter-individually remains unclear (Figure 4). Only in volunteer 2, the urinary T reaches approximately the CIR of the administered T while the values in volunteer 1 do not fall below 25. Especially for volunteer 1, the response of EPIA is of particular interest as it becomes even more depleted than T and 5a over the studied course of time and the Δ-values for PD-EPIA fall above the established threshold of 3 clearly indicating the administration of a steroid. [5] This is not the case in volunteer 2, but even here, at the end of the study, Δ-values for PD-EPIA exceed the threshold. In both cases the already described tendency of EPIA to retain the exogenous signature over a longer time period than the glucuronylated steroids is visible. [5] Taking the preliminary results of only two volunteers into consideration, EPIA seems to be an interesting complement for the detection of T-GEL administrations. Depending on the individual it might be less or more sensitive than the established TCs T and 5a but should definitely indicate the misuse of steroids if applied as TC. Epiandrosterone Literature data on EPIA and doping control analysis is scarce. Ueki et.al. investigated the potential of EPIA excreted glucuronylated to detect misuse of steroids. [19] And EPIA excreted sulfoconjugated was investigated regarding its potential to detect the misuse of DHEA using its urinary concentration or CIR. [6,20] Only when applying CIR, EPIA provided a prolonged detection window for DHEA (mis)use in one volunteer. [6] Triggered by this observation and the recent finding of an extended signature in EPIA after T administration, an excretion study with EPIA was conducted to investigate further aspects of this compound s metabolic fate and elimination. Additionally, EPIA is marketed as a doping agent itself and is easily available via the internet. As the advertisements suggest enhanced DHT levels, it appears relevant to identify possible markers of EPIA administration and to identify steroidal target analytes offering the best retrospectivity regarding CIR determinations. As expected, EPIA itself was the most promising marker for EPIA administrations (Figure 5). Its CIR values were depleted over the entire time period of sample collection and were found exceeding the established 1700 Figure 4. CIR after administration of T-GEL daily during days 3 to 9 in 2 different male volunteers. Open circles represent PD, open diamonds T, open triangles 5a (all excreted glucuronylated) and black squares EPIA excreted sulfoconjugated. All values given in δ 13 C VPDB [ ]. Figure 5. CIR after administration of 100 mg EPIA orally. Grey triangles represent DHEA excreted sulfoconjugated as ERC, grey diamonds A excreted sulfoconjugated, open diamonds A excreted glucuronylated, open circles 5a excreted glucuronylated, and black squares EPIA excreted sulfoconjugated. All values given in δ 13 C VPDB [ ]. wileyonlinelibrary.com/journal/dta Copyright 2017 John Wiley & Sons, Ltd. Drug Test. Analysis 2017, 9,

7 Epiandrosterone sulfate prolongs the detectability of testosterone, 4-androstenedione and dihydrotestosterone misuse threshold for DHEA-EPIA for over 10 days. Other significantly influenced urinary markers were A excreted both glucuronylated (A_G) or sulfoconjugated (A_S), and 5a. However, these returned to normal CIR values within 2 days after administration. Only A_S showed a slightly elevated turn-over time resulting in more depleted values between day 2 and 5 after administration compared to A_G. As no CIR threshold for A_S has been established to date, it is difficult to estimate how long the exogenous signal (still visible after 5 days) would indicate an illicit steroid administration. Of note, the urinary EPIA did not fully reflect the CIR of the administered steroid ( 30.2 ) but only showed depleted values around 29. In contrast, other metabolites such as A_G or 5a were found at 30 and above directly after administration. A possible explanation would be isotopic fractionation of the administered EPIA as described for DHEA applications and resulting CIR of ETIO. [21] Alternatively, a slow turnover of the endogenous EPIA pool is conceivable, which does not allow to entirely replace the natural EPIA by a single dose. Consequently, the excreted EPIA would remain a mixture of endogenous and exogenous EPIA explaining the observed values. Repeated administrations of EPIA could answer this question and might be addressed in future studies. By means of the steroid profile, the administration of EPIA was difficult to detect. Shortly after administration, the concentration of A_G increased to levels > ng/ml; however, as soon as 8 h post-administration the concentration dropped to < 4000 ng/ml. Due to an increase in 5a concentrations, elevated and atypical 5a/5b ratios up to 3.9 were detected, returning to normal levels after 22 h. As indicated in the literature, [19] EPIA emerged in the fraction of unconjugated and glucuronylated steroids directly after administration but also disappeared within the first 22 h. The excretion of sulfoconjugated EPIA was considerably prolonged and elevated after the administration as shown in Figure 6. As no validated method for the determination of urinary EPIA concentrations was available, a ratio of the area under the peak of EPIA divided by that of DHEA was chosen to monitor changes in EPIA concentrations after the application. This ratio correlates perfectly with the found Δ-values for EPIA-DHEA (r = 0.74, p < 0.001) and was the only urinary concentration or concentration ratio that prolonged the retrospective detection of steroid administration. As this ratio was also found elevated after DHT and ADION applications, further research on the individual stability of this marker and its possible use in sports drug testing seemed advisable. DHT was not significantly elevated after EPIA administrations in contrast to the aforementioned advertisement. Androstenedione Effects of ADION administration on male and female volunteers has been thoroughly investigated in the past, and especially in males, effects were found less pronounced compared to T administrations. [22 24] One interesting point in the context of sports drug testing may be the fact that ADION is not only converted to T but also to E and therefore administrations result in less elevated T/E ratios. [25] Therefore, ETIO and A concentrations were considered as promising candidates to detect ADION administrations together with different 6-hydroxylated metabolites. [26] Regarding CIR, formestane and ETIO or A have been suggested as possible markers. [27,28] Another confirmed metabolite of ADION (and T) EPIA was already described in 1964 and triggered these investigations in order to check its suitability in sports drug testing. [29] As shown in Figure 7, EPIA again significantly prolongs the retrospective traceability of administered pseudo-endogenous steroids. While ETIO and A return to unsuspicious CIR within 24 h after application, EPIA remains depleted beyond the established threshold around 130 h. Taking into account the other commonly investigated TCs T, 5a and 5b, the sample collected after 26 h would still be suspicious, but at 31 h after administration WADA thresholds are no longer exceeded. As observed above for EPIA administration, the urinary EPIA did not reflect the CIR of the administered ADION of 31.8 but maintained its depleted CIR for an outstandingly long period. The steroid profile based on glucuronylated steroid parameters (such as the absolute concentration of ETIO, A, and formestane plus the T/E ratio) is influenced for not more than 15 h after application. The ratio of EPIA/DHEA in the sulfate fraction was elevated for 37 h. Dihydrotestosterone Steroid profile parameters enabling the detection of DHT administrations were investigated in the 1990s and besides elevated urinary concentrations of DHT itself the inversion of the 5a/5b ratio was found to be diagnostic for administrations together with DHT/E or 5a/E. [30 32] These findings have been substantiated in all follow-up studies. [26] Regarding CIR after DHT administrations, Shackleton et. al. identified 5a as a promising marker together with its 17α-epimer (5α-androstane-3α,17α-diol) shortly after. [33] Within this study, both glucuronylated 5α-steroid metabolites of DHT, 5a and A, were found influenced (Figure 8). While A showed Figure 6. Ratio of EPIA/DHEA (both excreted sulfoconjugated) over time after administration of 100 mg EPIA at t = 0. Figure 7. CIR after administration of 80 mg ADION orally. Open circles represent PD as ERC, open diamonds A, grey diamonds ETIO (all excreted glucuronylated), and black squares EPIA excreted sulfoconjugated. All values given in δ 13 C VPDB [ ] Drug Test. Analysis 2017, 9, Copyright 2017 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/dta

8 T. Piper et al Figure 8. CIR after administration of 50 mg DHT orally. Grey triangles represent DHEA excreted sulfoconjugated and open circles PD excreted glucuronylated, both as ERCs. TCs are open triangles 5a and open diamonds A (both excreted glucuronylated), and black squares EPIA excreted sulfoconjugated. All values given in δ 13 C VPDB [ ]. significantly depleted CIR for less than 24 h, 5a was influenced for 35 h. Again, the sulfoconjugated EPIA proved to be the best marker for the application of an exogenous steroid with depleted CIR values exceeding the established thresholds for 70 h. Especially DHT administrations are difficult to confirm unambiguously as usually only 5a shows depleted values if urine specimens are investigated using current routine approaches. To avoid false positive testing in this instance, WADA established a unique threshold combination of an elevated Δ-value together with an absolute δ-value for the found 5a which minimized the chance for an adverse analytical finding. Here, EPIA will be an important complement enabling the substantiation of positive findings on the one hand while significantly lowering the chance for false positive testing by adding a second TC on the other. Within the steroid profile both diagnostic ratios (5a/5b and DHT/E) were found increased for approx. 20 h while 5a/E was influenced for more than 30 h. The ratio of EPIA/DHEA fell in-between and was influenced for 24h. Conclusion EPIA excreted sulfoconjugated was found to be significantly influenced for a longer period regarding its CIR after oral T administrations. This triggered several follow-up studies and after the administration of ADION and DHT, EPIA was depleted for significantly longer in CIR, and Δ-values calculated using either PD or DHEA exceeded established thresholds for significantly longer compared to currently applied routine protocols in sports drug testing. Compared to detection windows employing usual TCs like 5a, 5b and T, EPIA enabled the detection of exogenous steroids for a 2 to 3 times longer period. Even after low-dose T-GEL administrations EPIA was found depleted and can serve as a complementary marker. Additionally, EPIA is available as a prohormone on the dietary supplement market and not surprisingly, EPIA itself was found to be the best marker to proof EPIA administrations. CIR were found influenced approx. 5 times longer than the conventional TCs. In order to enable and simplify the implementation of EPIA into current drug testing methods focusing mainly on steroids excreted as glucuronides, a new arylsulfatase enzyme was tested regarding its potential in CIR determinations. The enzyme works well for both tested analytes (DHEA and EPIA) but might show limitations for other sulfoconjugated steroids. Implementing this enzyme in combination with a MDGC-IRMS approach allows for a fast and reliable detection of steroid administrations and will be an interesting and promising complement to current approaches that rely only on glucuronylated steroids. EPIA shows the possibility either to be used directly as a screening tool or to serve as a confirmation parameter in those cases giving suspicious but inconclusive results using the current appaoach. Acknowledgements This project was partly conducted with support of the World Anti- Doping Agency (#14D02MT, #16A11AC, #16AO6MM) and the Manfred-Donike Institute for Doping Analysis (Cologne, Germany). The support of Fausto Pigozzo from Thermo Fisher Scientific providing the MDGC system is also acknowledged. References [1] U. Mareck, H. Geyer, G. Opfermann, M. Thevis, W. Schänzer. Factors influencing the steroid profile in doping control analysis. J Mass Spectrom. 2008, 43, [2] T. Piper, C. Emery, M. Saugy. Recent developments in the use of isotope ratio mass spectrometry in sports drug testing. Anal Bioanal Chem. 2011, 401, [3] World Anti-Doping Agency. TD2016-IRMS. Available at: wada-ama.org/en/resources/science-medicine/td2016-irms. [23 June, 2017]. [4] T. B. Coplen. Guidelines and recommended terms of expression of stable-isotope-ratio and gas-ratio measurement results. Rapid Commun Mass Spectrom. 2011, 25, [5] T. Piper, W. Schänzer, M. Thevis. Genotype-dependent metabolism of exogenous testosterone new biomarkers result in prolonged detectability. Drug Test Analysis. 2016, 8, [6] T.Piper,G.Opfermann,M.Thevis,W.Schänzer.Determinationof 13 C/ 12 C ratios of endogenous urinary steroids excreted as sulphoconjugates. Rapid Commun Mass Spectrom. 2010, 24, [7] T. Piper, A. Thomas, M. Thevis, M. Saugy. Investigations on hydrogen isotope ratios of endogenous urinary steroids: Reference population based thresholds and proof-of-concept. Drug Test Analysis. 2012, 4, [8] B. J. Stevenson, C. C. Waller, P. Ma, et al. Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates. Drug Test Analysis. 2015, 7, [9] A. Casilli, T. Piper, F. Azamor de Oliveira, et al. Optimization of an online heart-cutting multidimensional gas chromatography cleanup step for isotopic ratio mass spectrometry and simultaneous quadrupole mass spectrometry measurements of endogenous anabolic steroid in urine. Drug Test Analysis. 2016, 8, [10] Y. Zhang, H. J. Tobias, J. T. Brenna. Steroid isotopic standards for gas chromatography-combustion isotope ratio mass spectrometry (GCC- IRMS). Steroids. 2009, 74, [11] T. Piper, C. Emery, A. Thomas, M. Saugy, M. Thevis. Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sport drug testing. Anal Bioanal Chem. 2013, 405, [12] C. Saudan, C. Emery, F. Marclay, E. Strahm, P. Mangin, M. Saugy. Validation and performance comparison of two carbon isotope ratio methods to control the misuse of androgen in humans. J Chrom B. 2009, 877, [13] T. Piper, M. Putz, P. Delahaut, M. Thevis. Carbon isotope ratios of endogenous steroids in Belgian Blue and Holstein cattle: Method development, reference population studies and application to androgen misuse control. Rapid Commun Mass Spectrom. 2017, 31, [14] T. Piper, U. Mareck, H. Geyer, et al. Determination of 13 C/ 12 C ratios of endogenous urinary steroids: method validation, reference population and application to doping control purposes. Rapid Commun Mass Spectrom. 2008, 22, [15] T. Piper, H. Geyer, W. Schänzer. Degradation of urine samples and its influence on the 13 C/ 12 C ratios of excreted steroids. Drug Test Analysis. 2010, 2, wileyonlinelibrary.com/journal/dta Copyright 2017 John Wiley & Sons, Ltd. Drug Test. Analysis 2017, 9,

9 1703 Epiandrosterone sulfate prolongs the detectability of testosterone, 4-androstenedione and dihydrotestosterone misuse [16] T. Piper, M. Thevis. Applications of isotope ratio mass spectrometry in sports drug testing accounting for isotope fractionation in analysis of biological samples. Methods in Enzymology. 2017, 596, [17] R. S. Swerdloff, C. Wang, G. Cunningham, et al. Long-Term Pharmacokinetics of Transdermal Testosterone Gel in Hypogonadal Men. JClinEnd&Metab. 2000, 85, [18] S. G. Korenman, H. Wilson, M. B. Lipsett. Testosterone production rates in normal adults. J clin Invest. 1963, 42, [19] M. Ueki, M. Okano, A. Ikekita, T. Hiruma, M. Sato. Epiandrosterone Glucuronide as a Sign to Indicate Natural Hormone Doping, in Recent advances in doping analysis, (Eds: M. Donike, H. Geyer, A. Gotzmann, U. Mareck-Engelke, S. Rauth) Sport und Buch Strauß, Köln, 1997, pp [20] L. Dehennin, M. Ferry, P. Lafarge, G. Peres, J. P. Lafarge. Oral administration of dehydroepiandrosterone to healthy men: Alteration of the urinary androgen profile and consequences for the detection of abuse in sport by gas chromatography-mass spectrometry. Steroids. 1998, 63, [21] A. T. Cawley, R. Kazlauskas, G. J. Trout, J. H. Rogerson, A. V. George. Isotopic Fractionation of Endogenous Anabolic Androgenic Steroids and its Relationship to Doping Control in Sports. J Chrom Sci. 2005, 43, [22] D. S. King, R. L. Sharp, M. D. Vukovich, et al. Effect of oral androstenedione on serum testosterone and adaptions to resistance training in young men - a randomized controlled trial. JAMA. 1999, 281, [23] A. T. Kicman, T. Bassindale, D. A. Cowan, S. Dale, A. J. Hutt, A. R. Leeds. Effect of Androstenedione Ingestion on Plasma Testosterone in Young Women; a Dietary Supplement with Potential Health Risk. Clin Chem. 2003, 49, [24] T. Bassindale, D. A. Cowan, S. Dale, et al. Effects of Oral Administration of Androstenedione on Plasma Androgens in Young Women Using Hormonal Contraception. J Clin Endocrinol Metab. 2004, 89, [25] D. H. Catlin, B. Z. Leder, B. D. Ahrens, C. K. Hatton, J. S. Finkelstein. Effects of androstenedione administration on epitestosterone metabolism in men. Steroids. 2002, 67, [26] M. K. Shelby, D. J. Crouch, D. L. Black, T. A. Robert, R. Heltsley. Screening Indicators of Dehydroepiandrosterone, Androstenedione, and Dihydrotestosterone Use: A Literature Review. JAnalToxicol. 2011, 35, [27] A. T. Cawley, G. J. Trout, R. Kazlauskas, A. V. George. The detection of androstenedione abuse in sport: a mass spectrometry strategy to identify the 4-hydroxyandrostenedione metabolite. Rapid Commun Mass Spectrom. 2008, 22, [28] J. Wang, M. Wu, X. Liu, Y. Xu. Profiling of urinary steroids by gas chromatography-mass spectrometry detection and confirmation of androstenedione administration using isotope ratio mass spectrometry. Steroids. 2011, 76, [29] E. E. Baulieu, P. Mauvis-Jarvis. Studies on Testosterone Metabolism II. Metabolism of Testosterone-4-14 C and Androst-4-ene-3,17-dione- 1,2-3 H. J Biol Chem. 1964, 239, [30] G. J. Southan, R. V. Brooks, D. A. Cowan, A. T. Kicman, N. Unnadkat, C. J. Walker. Possible indices for the detection of the administration of dihydrotestosterone to athletes. J Steroid Biochem Molec Biol. 1992, 42, [31] M. Donike, M. Ueki, Y. Kuroda, et al. Detection of dihydrotestosterone (DHT) doping: alterations in the steroid profile and reference ranges for DHT and its 5 alpha-metabolites. J Sports Med Phys Fitness. 1995, 35, [32] A. T. Kicman, S. B. Coutts, C. J. Walker, D. A. Cowan. Proposed Confirmatory Procedure for Detecting 5a-Dihydrotestosterone Doping in Male Athletes. Clin Chem. 1995, 41, [33] C. H. L. Shackleton, E. Roitman, A. Phillips, T. Chang. Androstanediol and 5-androstenediol profiling for detecting exogenously administered dihydrotestosterone, epitestosterone, and dehydroepiandrosterone: Potential use in gas chromatography isotope ratio mass spectrometry. Steroids. 1997, 62, Drug Test. Analysis 2017, 9, Copyright 2017 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/dta