Studies on the in vivo metabolism of methylstenbolone and detection of novel long term metabolites for doping control analysis

The anabolic-androgenic steroid methylstenbolone (MSTEN; 2 α ,17 α -dimethyl-17 β -hydroxy-5 α -androst-1-en-3-one) is available as a so-called designer steroid or nutritional supplement. It is occasionally detected in doping control samples, predominantly tested and confirmed as the glucuronic acid conjugate of methylstenbolone. The absence of other meaningful metabolites reported as target analytes for sports drug testing purposes can be explained by the advertised metabolic stability of methylstenbolone. In 2013, a first investigation into the human metabolism of methylstenbolone was published, and two hydroxylated metabolites were identified as potential targets for initial testing procedures in doping controls. These metabolites were not observed in recent doping control samples that yielded adverse analytical findings for methylstenbolone, and in the light of additional data originating from a recent publication on the in vivo metabolism of methylstenbolone in the horse, revisiting the metabolic reactions in humans appeared warranted. Therefore, deuterated methylstenbolone together with hydrogen isotope ratio mass spectrometry

These metabolites were not observed in recent doping control samples that yielded adverse analytical findings for methylstenbolone, and in the light of additional data originating from a recent publication on the in vivo metabolism of methylstenbolone in the horse, revisiting the metabolic reactions in humans appeared warranted. Therefore, deuterated methylstenbolone together with hydrogen isotope ratio mass spectrometry (IRMS) in combination with high accuracy/high resolution mass spectrometry were employed. After oral administration of a single dose of 10 mg of doubly labeled methylstenbolone, urine samples were collected for 29 days. Up to 40 different deuterated methylstenbolone metabolites were detected in post-administration samples, predominantly as glucuronic acid conjugates, and all were investigated regarding their potential to prolong the detection window for doping controls. Besides methylstenbolone excreted glucuronidated, three additional metabolites were still detectable at the end of the study on day 29. The most promising candidates for inclusion into routine sports drug testing methods (2α,17α-dimethyl-5α-androst-1-ene-3β,17β-diol and 2α,17α-dimethyl-5α-androst-1-ene-3α,17β-diol) were synthesized and characterized by NMR.

| INTRODUCTION
Anabolic-androgenic steroids such as methylstenbolone (MSTEN; 2α,17α-dimethyl-17β-hydroxy-5α-androst-1-en-3-one) are prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). 1 Despite an increasing number of antidoping legislations in numerous countries, a decrease in the trafficking of steroidal drugs (as well as other doping agents) has been barely noticeable and they are readily available via the world wide web. For many years, anabolic agents have been among the most frequently detected banned substances in sport. 2 In doping control analysis, the targeting of urinary metabolites of administered compounds is frequently the superior strategy over detecting the intact and unmodified compound itself. Therefore, comprehensive investigations of phase-I-and phase-II-metabolic reactions of doping agents have contributed to improved detectability in numerous cases.
Regarding methylstenbolone, so far only one study has reported on human in vivo metabolism, and two hydroxylated urinary metabolites have been described. 3 Both metabolites were detectable in urine for a longer time period (up to 7 days) than methylstenbolone itself. These results, however, did not reflect recent findings in doping control samples in which MSTEN glucuronide but not the reported hydroxylated metabolites were detected.
Also, additional hydroxylated metabolites identified in in vitro and humanized mouse metabolism studies on methylstenbolone are not directly applicable to sports drug testing. 4 In a recent investigation focusing on the metabolism of methylstenbolone in the horse, several metabolites were described that were detectable for a longer time period than methylstenbolone itself. 5 Based on these results, revisiting the metabolism of methylstenbolone in humans seemed advisable in order to elucidate the discrepancies found between the described and observed urinary metabolites and to facilitate the improved detection of this banned steroid in doping control analysis.
In order to generate a comprehensive profile of urinary methylstenbolone metabolites, a recently established approach utilizing hydrogen isotope ratio (HIR) determinations was applied. [6][7][8][9] Therefore, two-fold deuterated MSTEN was administered to one healthy male volunteer, and the collected urine samples were processed and analyzed using gas chromatography/thermal conversion/isotope ratio mass spectrometry (GC/TC/IRMS) to detect urinary steroids comprising at least one deuterium atom. All identified metabolites were further analyzed by gas chromatography/high resolution-high accuracy/mass spectrometry (GC-HRMS) to validate their deuterium content and to elucidate their structural characteristics.
Promising metabolite candidates were investigated regarding their potential to improve and prolong the traceability of methylstenbolone administration. In order to corroborate the utility of newly identified metabolites for doping control, an excretion study was conducted using unlabeled methylstenbolone, and urine samples were analyzed under authentic routine sports drug testing conditions, complemented by the herein discovered, synthesized, and additionally NMR-characterized metabolites.

| Sample preparation for HIR determinations
The different steps in the sample preparation for all specimens collected during the elimination study with deuterated MSTEN are described in detail elsewhere. [12][13][14][15] In brief, 20 mL of urine was applied onto a preconditioned solid-phase extraction (SPE) cartridge (Chromabond ® C18, Macherey-Nagel, Düren, Germany), washed with water, and eluted with methanol. After evaporation, the dried residue was reconstituted in 2 mL of aqueous phosphate buffer (pH 7) and extracted with tert-butyl methyl ether to yield the unconjugated steroid fraction. To the aqueous residue, β-glucuronidase was added, and after 60 min at 50 C, an aqueous potassium carbonate buffer (pH 10) was added to allow for partitioning of the formerly glucuronidated steroids into tert-butyl methyl ether. The aqueous layer is subsequently acidified and applied onto another SPE cartridge followed by acidic solvolysis, employing sulfuric acid in ethyl acetate. Finally, the formerly sulfoconjugated steroids were extracted.
The fraction of unconjugated steroids was immediately forwarded to hydrogen isotope ratio determinations while the formerly glucuronidated and sulfated steroids were subjected to high performance liquid chromatographic clean-up. This was accomplished using an Agilent 1100 HPLC system (Waldbronn, Germany) equipped with a XBridge™ Shield RP18 5 mm (4.6 × 250 mm) column, protected by a XBridge™ Shield RP18 5 mm (4.6 × 20 mm) guard column from Waters (Eschborn, Germany). Solvents used for gradient elution were A: water and B: acetonitrile. The gradient started from 20% acetonitrile, increased to 100% acetonitrile within 25 min and was held for 5 min followed by re-equilibration for 5 min at 20% acetonitrile. The flow was set to 1 mL/min and the injection volume was 100 μL. Fractions were collected using a FOXY R1 automatic fraction collector (Axel Semrau, Sprockhövel, Germany) from 4.00 to 10.00, from 10.01 to 13.00, from 13.01 to 16.00, from 16.01 to 18.00, from 18.01 to 20.00, from 20.01 to 24.00, and from 24.01 to 30.00 min.
All fractions were evaporated and trimethylsilylated using Nmethyl-N-trimethylsilyltrifluoroacetamide in ethyl acetate (20/80, v/v) at 70 C for 45 min. 16 After evaporation, the samples were transferred into auto-sampler vials using 200 μL of ethyl acetate, dried in a desiccator, and reconstituted as necessary for IRMS or HRMS determinations.

| Gas chromatography/isotope ratio mass spectrometry setup
The instrumentation used for all HIR determinations was a Delta V Plus IRMS (Thermo Fisher Scientific, Bremen, Germany) coupled to a Trace GC 1310 equipped with a TriPlusRSH autosampler via the GC IsoLink CNH and the ConFlow IV (all Thermo). The thermal conversion ceramic tubing (320 mm length, 0.5 mm inner diameter, 1.5 mm outer diameter from IVA Analysentechnik, Meerbusch, Germany) was operated at 1450 C for pyrolysis. The IRMS was hyphenated to an ISQ mass spectrometer (Thermo) operated in positive electron ionization mode recording total ion chromatograms from m/z 50 to 800.

| Gas chromatography/high resolution mass spectrometry
In order to presumably identify the structure and the deuterium content of the detected metabolites, all HIR samples were diluted by a factor of 5 to 20 and re-injected on an Agilent 7200 Accurate-Mass Q-TOF system coupled to an Agilent 7890A gas chromatograph (Santa Clara, CA). The GC column and parameters were equivalent to those described above, the injection volume was reduced to 2 μL and the pulsed splitless option was chosen with an initial pressure of 40 psi. Data covering a scan range from m/z 50 to 800 with a scan speed of 333 ms/spectrum were acquired and evaluated using MassHunter software (version B.06, Agilent). The instrument was mass calibrated prior to each sequence in order to achieve mass accuracy in the range of ±5 ppm.
Additional studies to improve the identification and detectability of those metabolites important for doping control analysis were conducted on a Q Exactive GC Orbitrap GC-MS/MS system (Thermo) under routine doping control conditions. In a first step, selected samples were re-prepared as described above employing the HPLC cleanup but subsequently derivatized using MSTFA/NH 4 I/ethanethiol 1000:2:3 (v:w:v). 17 Finally, sample preparation was conducted according to routine analytical conditions to demonstrate the feasibility of directly implementing the most promising target analytes. 18,19 Also, the GC was equipped with a HP-Ultra 1 analytical column, but the temperature program was slightly different. The initial temperature of 180 C was increased with 3 C/min to 240 C and then with 40 C/min to 320 C and held for 2 min. Injections were performed in split mode at 300 C with a split and purge flow of 5 mL/min each. A constant flow was set to 1 mL/min and transfer line temperatures were at 280 and 300 C. Data were acquired in full MS mode covering a scan range of m/z 50 to 700 or in parallel reaction monitoring using an isolation window of m/z 1.3. The resolution was set to 60.000 and data were evaluated using Xcalibur (Version 4.0, Thermo). Daily mass calibration of the instrument yielded mass accuracy in the range of ±2 ppm.

| Synthesis of the new long-term metabolite
Ten milligrams of methylstenbolone was dissolved in 2 mL of tetrahydrofuran (THF). Then, 200 μL of a solution of lithium aluminum hydride (1 M in THF) was added dropwise. The mixture was vortexed and after 10 min at room temperature, 5 mL of water was added and the reaction products were liquid-liquid-extracted twice using 5 mL of TBME. After evaporation, the dried residue was reconstituted in 2 mL of acetonitrile/water (50/50) and forwarded to HPLC clean-up on the above mentioned system. The employed gradient was modified to start with 60% acetonitrile, increasing to 80% acetonitrile in 10 min, then to 100% acetonitrile in 0.1 min, where it was held for 5 min prior to re-equilibration for 5 min at starting conditions. The injection volume was reduced to 50 μL to prevent column overload. In addition to the fraction containing methylstenbolone (collection window from 8.21 to 9.00 min), two fractions containing metabolites were collected (from 7.10 min to 7.70 min and from 7.71 min to 8.20 min) and forwarded to nuclear magnetic resonance (NMR) spectroscopy.

| Nuclear magnetic resonance spectroscopy
Prior to NMR analysis, the reduced methylstenbolone products were subjected to another LC fractionation step using an Agilent T A B L E 1 Confirmed deuterated metabolites found in urine after administration of 10 mg of deuterated methylstenbolone. G refers to metabolites excreted glucuronidated or unconjugated and S to sulfated metabolites. Metabolites marked with an asterisk were found in both fractions. The retention times are given for the routine set up employed on the GC-Orbitrap

| Urinary MSTEN metabolites detected by HIR
The employed untargeted metabolite identification approach allows for the sensitive detection of those analytes that retain at least one of the drug's deuterium atoms during metabolic conversion, as long as the metabolite recovery from the urine is ensured by the applied sample preparation and derivatization technique. Furthermore, the physico-chemical properties of the analyte needs to be compatible with gas chromatographic analyses. In Figure 1 Especially at the beginning of the excretion study, glucuroconjugated methylstenbolone represents one of the most abundant metabolites together with several hydroxylated metabolites excreted into urine as unconjugated or glucuronidated metabolic products. After approximately 5 days, peaks are still visible in the HIR chromatograms close to the detection limit.
As shown in Figure 1 (lower part, zoom area), the isotope ratio of pregnanediol (PD) seems also to be influenced by the administration of methylstenbolone. This is, however, not attributed to the metabolic conversion of methylstenbolone to pregnanediol but to an artifact of the measurement. During the thermal conversion process, deuterium atoms can be absorbed by the heated aluminum tubing and released when the subsequently eluting compound exiting the GC column is pyrolyzed. Therefore, confirmation of the deuterium content of signals identified in the HIR chromatogram by GC-HRMS under comparable chromatographic conditions is particularly important in order to identify and exclude such analytical artifacts. In the present study, over 40 metabolites were confirmed to contain at least one deuterium atom.

| MSTEN metabolites suitable for doping control analysis
As the applied derivatization technique (MSTFA in ethyl acetate) during the first step of metabolite detection was not in accordance with F I G U R E 2 High resolution-parallel reaction monitoring mass spectra of metabolite 5. In the upper part, the deuterated metabolite using the precursor ion at m/z 549.3 is shown, in the lower part the non-deuterated metabolite with precursor ion at m/z 548.3, the deuterium label is present in the A-ring the usually applied routine doping control methods, the samples were re-prepared in agreement with routine protocols.
At first, besides changing the derivatization technique, all sample preparation steps were kept including the HPLC fractionation-based target analyte purification. The partitioning of unconjugated steroids was omitted as no long term metabolites were expected to exist in this fraction. All metabolites found at this stage of the study are summarized in Table 1.
In agreement with literature data on human and especially horse metabolism, the majority of the methylstenbolone metabolites was found to be hydroxylated (24 out of 40 metabolites. 3,5 Regarding phase-II-metabolism, 25 metabolites were excreted glucuronidated and only six metabolites were found simultaneously as both glucurono-and sulfo-conjugated analytes. Comparing the order of elution and mass spectra, the herein observed metabolites 14 and 18 were assigned to urinary metabolites of methylstenbolone reported by Calvacanti et al. 3 Unfortunately, applying a similar approach concerning the numerous metabolites identified in the horse proved considerably more complex as the gas chromatographic conditions differed substantially and the majority of the metabolites was discussed based on liquid chromatography-mass spectrometry-derived data. 5 With a distinct loss of 103 Da under EI-MS conditions, one metabolite (number 5 in Table 1)  or C-21 (i.e. the methyl group attached to C-2). Especially for 19-hydroxylated steroids, the loss of m/z 103 is well established. 22,23 With the data to hand, it is not possible to identify unequivocally the position of hydroxylation and further research will be required to clarify this aspect. The metabolite was visible up to 18 days post-administration, but three other metabolites outperformed this result.

| Methylstenbolone
The administered compound itself is noted to be particularly resistant concerning metabolic reactions, and taking into account the results of this study the introduction of an additional methyl group into the Aring of the steroid indeed appears to affect several phase-I-metabolic routes usually observed in steroid metabolism. 24 After preparing 10 mL of urine and HPLC purification, the deuterated glucurono-and F I G U R E 4 (A) Full scan high resolution mass spectra obtained for the epi-NEW_LTM, (B) magnification of the range m/z 320 to 480, the grey arrow indicates the characteristic loss of 44 Da. Given elemental compositions were calculated within a mass error of ±5 ppm. Further information in the text F I G U R E 5 NOESY experiment on the epi-NEW_LTM demonstrating the interaction of protons located on C-3, C-5, C-9, and C-14 as well as C-19, C-8, and C-18, respectively sulfoconjugated MSTEN (metabolites 22 and 39, respectively, Table 1) were visible until the end of the study, i.e. for 29 days after a single oral application. In Figure 3, the extracted ion chromatograms for m/z 461.3253 (± 10 ppm) are depicted. This finding is in good agreement with the results obtained for routine doping control samples yielding AAFs based on the sole presence of (glucurono-conjugated) methylstenbolone. The discrepancy between the herein accomplished detection window and earlier published data might be explained when analyzing the excretion study urine sample data collected after the administration of non-deuterated methylstenbolone.
Here, samples were prepared strictly according to routine methods, i.e. without an additional HPLC purification step. Employing high resolution mass spectrometry together with parallel reaction monitoring

| Novel reduced long term metabolites
The HPLC fraction containing methylstenbolone also furnished two additional peaks of different retention times and different relative intensities but highly comparable mass spectra. These analytes were detected, alongside MSTEN, until the end of the elimination study.
The more abundant compound was referred to as Epi-NEW_LTM (Table 1, metabolites 23 and 40) and the less abundant metabolite was termed NEW_LTM (Table 1,  Interestingly, both metabolites were predominantly found in the urinary fraction of glucuronic acid conjugates. This is particularly noteworthy as steroids exhibiting a 3β-OH-functionality preferentially undergo sulfoconjugation. A potential explanation would be the directing of phase-II-metabolism towards position C-17 due to the additional methyl group in position C-2. Another unexpected aspect concerns the detected relative urinary concentrations of the two metabolites. Throughout the entire elimination study, the 3β-epimer (Epi-NEW_LTM) is excreted at higher concentrations than the 3αepimer (NEW_LTM). In contrast to this, the endogenously produced androsterone (3α-hydroxy-5α-androstan-17-one) always prevails over epiandrosterone (3β-hydroxy-5α-androstan-17-one) in human urine and androsterone is excreted predominantly as glucuronide while epiandrosterone is exclusively found in the fraction of sulfoconjugated steroids.
In the second excretion study employing non-deuterated methylstenbolone, both metabolites proved to be suitable for sports drug testing. Regarding Epi-NEW_LTM, the peaks were visible for 20 and 23 days (glucuronidated and sulfoconjugated, respectively) and NEW_LTM for 27 and 12 days (glucuronidated and sulfoconjugated, respectively). The results obtained for the glucuronoconjugated NEW_LTM are depicted in Figure 6.

| Novel oxygenated long term metabolite
Among the numerous hydroxylated metabolites found in this study, only one candidate showed the potential to prolong the detection time (Table 1,

| CONCLUSION
The combination of hydrogen isotope ratio mass spectrometry with high resolution/high accuracy mass spectrometry was successfully applied to the investigation of methylstenbolone metabolism. Even though methylstenbolone appears to be comparably resistant to metabolic enzymes, up to 40 metabolites were identified. The metabolic inertness was reflected by the fact that deuterated MSTEN itself was detected as intact (whilst conjugated) drug until the end of the elimination study. Two novel long-term metabolites NEW_LTM and Epi-NEW_LTW exhibited similar detection windows (27 and 20 days respectively) for sports drug testing purposes, and especially NEW_LTM was found to represent a promising complement for doping controls. As Epi-NEW_LTM showed comparable detection windows, it should also be monitored taking into account the probability of matrix interferences on the one or the other target compound. The third new long-term metabolite OXO_LTM was found to be less suitable for doping control analysis as it was largely indistinguishable from an endogenous substance eluting at the same retention time under the chosen chromatographic conditions.