Drug Testing and Analysis
Correspondence case report Received: 31 July 2014
Revised: 6 November 2014
Accepted: 7 November 2014
Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1761
Degradation of methyltestosterone in urine samples C. Schweizer Grundisch,* N. Baume and M. Saugy Introduction
1170
Methyltestosterone is a common anabolic steroid which is rapidly metabolized in the body after consumption. Two different metabolites (17α-methyl-5β-androstane-3α,17β-diol and 17α-methyl-5αandrostane-3α,17β-diol) are usually screened to detect a misuse of this steroid by gas chromatography-mass spectrometry (GCMS) or gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS).[1] Therefore, methyltestosterone under its free form may be spiked in urine samples as an internal standard for the initial testing procedure of anabolic steroids by GC-MS or GC-MS/MS. In order to remove any ambiguity during confirmation analysis, an alternative internal standard is used. In the past two years, the Swiss Laboratory for Doping Analyses observed the conversion of spiked methyltestosterone to its metabolite 17α-methyl-5β-androstan3α,17β-diol in 5 samples (Figure 1). In some samples the steroid profiles were also affected by the degradation of testosterone and deuterated testosterone. The samples came from different sports, and had a travel time between 1 and 7 days. The samples had in common a pH over 6, a low specific gravity and signs of bacterial contamination (presence of 5α- and 5β-androstanedione). It is well known that bacteria and microorganisms can modify the concentration of urinary steroids partially or completely by degrading cholesterol and endogenous steroids[2,3] and thus influence the steroid profile.[4–7] These bacteria originate from the gastrointestinal or urogenital tracts and have the capacity to modify the steroid profile when growing is influenced and promoted by temperature.[8] Apart from endogenous steroids, microorganisms can produce others steroids, such as 19-norandrosterone by demethylation[9] or boldenone by 1-dehydrogenase activity.[10] Also, the endogenous glucocorticosteroids cortisol and cortisone may undergo transformations in prednisolone and prednisone, respectively due to dehydrogenation.[11] The indicators used by the World Anti-Doping Agency (WADA) accredited laboratories to identify activity of microorganisms in urine are the formation of 5α- and 5β-androstanenedione and the increase of free testosterone concentration.[12] To show the influence of bacteria growth, five samples with conversion of methyltestosterone into its main metabolite 17α-methyl5β-androstan-3α,17β-diol are presented herein. In doping control analyses, this biotransformation could lead to methyltestosterone false-positive cases. To solve this degradation problem of samples with presence of a methyltestosterone metabolite (17α-methyl-5α-androstan-3α,17βdiol), a confirmation procedure was performed either using an alternative internal standard or adding the internal standard after the hydrolysis step of the sample preparation. In both cases, there
Drug Test. Analysis 2014, 6, 1170–1173
was no more 17α-methyl-5α-androstan-3α,17β-diol and a clean steroid profile was obtained.
Experimental Standards and reagents 17α-methyltestosterone (4-androsten-17a-methyl-17b-ol-3-one, MeT) was purchased from Steraloids (Newport, RI, USA), androsterone-d4 glucuronide, and epitestosterone-d3 from NMIA (Pymble, Australia), testosterone-d3 from Lipomed (Arlesheim, Switzerland), 17α-methyl-5β-androstane-3α,17β-diol (MeT met) and stanozolol-d3 from Cerilliant (Round Rock, TX, USA). All reagents and solvents were of analytical grade or HPLC grade. Potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium carbonate, sodium hydrogen carbonate were provided by Merck (Darmstadt, Germany), Methyl tert-butyl ether (MTBE), methanol by Acros Organics (Geel, Belgium). β-glucuronidase from Escherichia Coli came from Roche diagnostic (Mannheim, Germany), N-Methyl-N-trimethylsilyltrifluoroacetamide from Macherey Nagel (Düren, Gremany), ammonium iodide from Sigma-Aldrich (Steinheim, Germany) and ethanethiol from Fluka (Buchs, Switzerland). Sample preparation Twenty μL of ISTDs (methyltestosterone, 200 ng/mL, testosteroned3, 80 ng/mL, epitestosterone-d3, 40 ng/mL, androsterone-d4 glucuronide, 200 ng/mL and stanozolol-d3, 40 ng/mL), 1 mL of boiled phosphate buffer (0.8 M, pH7) and 50 μL of β-glucuronidase from E.coli were added to 2.5 mL of urine. The samples were incubated for 1 hour at 50 °C or overnight at 37 °C (16 h). After hydrolysis, the urine was adjusted to pH 8.5-9 with carbonate buffer (Na2CO3/ NaHCO3 1/10, w/w) and liquid-liquid extraction performed with 5 mL of MTBE. Once the organic layer was transferred and evaporated to dryness, the residue was derivatized with 50 μL of MSTFA-NH4I-ethanethiol, (1000:2:3, v/w/v) at 60 °C for 20 min. Instrumentation The quantification for the steroid profile was performed on a Hewlett Packard 6890 GC system Plus gas chromatography system
*
Correspondence to: Carine Schweizer Grundisch, Swiss Laboratory for Doping Analyses, Ch. des Croisettes 22, 1066 Epalinges, Switzerland. E-mail: Carine. [email protected] Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Geneva and Lausanne, Ch. des Croisettes 22, 1066, Epalinges, Switzerland
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Testing and Analysis
Degradation of methyltestosterone in urine samples
Figure 1. Conversion of methyltestosterone to 17α-methyl-5β-androstan3α, 17β-diol.
coupled to a Hewlett Packard 5973 Mass Selective Detector (Palo Alto, CA, USA) mass spectrometer. The screening analyses for the detection of anabolic steroids were performed on an Agilent 7890B gas chromatography system coupled to a Triple Quadrupole 7000B (Wilmington, DE, USA).
Analytical conditions The instruments were equipped with a 17 m Agilent J&W Ultra-1 column, 0.2 mm internal diameter, 0.11 μm film thickness (Agilent Technologies, Palo Alto, CA, USA). The GC temperature program was: 180 °C, 1 min, 3 °C/min to 230 °C, then 40 °C/min to 310 °C (4 min). Injection of 2 μL was performed in split mode with a 10:1 split ratio and a temperature injector of 300 °C. Helium was used as carrier at constant flow (1.1 mL/min). Temperature of transfer line, ion source and quadrupole(s) were 280 °C, 230 °C and 150 °C, respectively. Selected ion monitoring (SIM) with a dwell time of 30 ms, multiple reaction monitoring (MRM) with a dwell time of 20 ms were used for the GC-MS and GC-MS/MS, respectively.
Results and discussion As depicted in Figure 2, various concentrations (peak area) of 17αmethyl-5β-androstan-3α,17β-diol (ranging from 1.5 to 100 ng/mL) were detected in different samples. In urines containing the highest concentrations (samples #1 and #2), almost all methyltestosterone was degraded compared to the other three samples.
When the internal standard was added after hydrolysis at 37 °C or 50 °C, no 17α-methyl-5β-androstan-3α,17β-diol was detected showing that the activity of microorganisms was clearly influenced by the temperature. The five urines shared a pH slightly elevated ranging from 6.0 to 7.3, a low specific gravity between 1.007 and 1.011 and had a clear aspect (Table 1). No typical external sign of degradation were observed. The origin of the microorganisms in these urines was investigated and apparently neither transport duration (from sample collection till the delivery to the lab) nor inappropriate temperature or storage (sample collections were done from September to January) could be the source of the bacteria growth in urine samples. In sample #1 (female athlete, beach volley) all methyltestosterone was converted into 17α-methyl5β-androstan-3α,17β-diol indicating a high microorganism activity. This might be explained by high temperature as the collection date was done on a hot day but it is also well documented that female urines contain a highest density of bacteria.[11] 5α and 5β-androstanedione were detected in a significant amounts in every of the five presented samples. Moreover, testosterone and deuterated testosterone were degraded in every sample but to a lesser extent in samples #4 and #5. Only deuterated androsterone glucuronide and deuterated stanozolol from the ISTD were not affected. Interestingly, all these samples were included in analytical batches which were incubated at 37 °C for 16 h indicating that the activity of the microorganisms was probably promoted by the duration of the incubation. Many microorganisms were listed with their degradation products obtained but in most cases endogenous steroids were primarily transformed.[13] In 2007, Grosse et al. showed that methyltestosterone can be converted in methandienone after incubation with Rhodococcus Erythropolis.[10] These bacteria are able to generate several enzymes affecting different sites of molecules only when these substances are found in free form in urine. In our screening procedure, free methyltestosterone is added in urine samples as an internal standard, making the transformation in 17α-methyl5β-androstan-3α,17β-diol possible.
Drug Test. Analysis 2014, 6, 1170–1173
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
1171
Figure 2. Chromatograms of methyltestosterone (ISTD) (upper panels) and 17α-methyl-5β-androstan-3α, 17β-diol (lower panels) in negative urine (A), positive control (B), samples #2 (C) and #3 (D).
Drug Testing and Analysis
C. Schweizer Grundisch, N. Baume and M. Saugy
Table 1. Summary of different parameters for five samples containing methyltestosterone metabolite, as highlighted with the screening procedure Samples # 1 2 3 4 5
Concentration MeT metabolite
pH
Speficic gravity
Presence of 5α- and 5β-androstanedione
Sport
Sex
Delivery delay
96 ng/mL 61 ng/mL 9 ng/mL 2 ng/mL 1.5 ng/mL
7.3 6.0 6.0 6.8 6.5
1.007 1.011 1.011 1.009 1.007
yes yes yes yes yes
Beach Volley Cyclocross Cyclocross Athletics Football
F M F M M
4 days 1 day 1 day 7 days 3 days
Figure 3. Steroid profile chromatograms of sample #1. (A) Degraded ISTD (MeT). (B) ISTD (MeT) added after the hydrolysis step, (C) MeT metabolite (m/z 435) trace present in the testosterone-d3 and epitestosterone-d3 chromatogram and (D) Normal chromatograms of testosterone and epistestosterone (m/z 432) and its deuterated ISTD (m/z 435).
1172
Prior to further investigation, the analyses were repeated for the five urine samples and production of methyltestosterone metabolite was again observed. To elucidate the origin of the presence of 17α-methyl-5β-androstan-3α,17β-diol and to obtain a clean steroid profile, the internal standard was added after the hydrolysis step of the sample preparation. The obtained chromatograms of derivatized testosterone and deuterated testosterone were clean (Figure 3). However, a steroid profile measured in a contaminated urine sample should not be used for longitudinal follow up. Different factors such as collection under non-sterile conditions, elevated temperature, transport duration, storage conditions and contaminations with microorganisms (bacteria, fungi) could impact the quality of the urinary matrix and thus the analytical results. To avoid any form of adulteration between the time of collection and analyses in the laboratory, it could be recommended to add a suitable stabilizer in the urine Berlinger kits used for sample collection. This would ensure the stability of the steroid profile without any effect on the different analyses as previously shown by Tsivou et al.[14] Nevertheless, WADA did not allow the introduction of an exogenous compound in the urine collection kits reinforcing stringent hygiene sampling and short delivery delay requirements. In addition,
wileyonlinelibrary.com/journal/dta
some experiments showed that a desired hydrolysis in the analytical sample preparation is sometimes inhibited by stabilizers.
Conclusions These five cases showed that microorganisms could promote the conversion of methyltestosterone to its metabolite, 17α-methyl5β-androstan-3α,17β-diol, without showing typical signs of microbial degradation (elevated pH, turbidity). For scientific interest, it would be useful to identify the bacteria responsible for this biotransformation.
References [1] W. Schänzer, M. Donike. Metabolism of anabolic steroids in man: Synthesis and use of reference substances for identification of anabolic steroid metabolites. Anal. Chim. Acta 1993, 275, 23. [2] R.W. Owen, M.E. Tenneson, R.F. Bilton, A.N. Mason. The degradation of cholesterol by Escherichia coli isolated from human faeces. Biochem. Soc. Trans. 1978, 6, 377.
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Test. Analysis 2014, 6, 1170–1173
Drug Testing and Analysis
Degradation of methyltestosterone in urine samples [3] P. Talalay. Enzymatic mechanisms in steroid metabolism. Physiol. Rev. 1957, 37, 362. [4] C. Ayotte, A. Charlebois, S. Lapointe, D. Barriault, M. Sylvestre. Valitity of urine samples: Microbial degradation. Recent Advances in Doping th Analysis (4), 14 Cologne Workshop on Dope Analysis. Sport and Buch Strauss, Köln, 1996, pp. 127. [5] U. Mareck, H. Geyer, G. Opfermann, M. Thevis, W. Schänzer. Factors influencing the steroid profile in doping control analysis. J. Mass Spectrom. 2008, 43, 877. [6] X. Liu, Y. Xing, Y. Xu, Z. Yang, M. Wu. Degradation of endogenous steroids by microorganisms. Recent Advances in Doping Analysis th (19), 29 Cologne Workshop on Dope Analysis. Sport and Buch Strauss, Köln, 2011, pp. 203. [7] T. Kuuranne, M. Saugy, N. Baume. Confounding factors and genetic polymorphism in the evaluation of individual steroid profiling. Brit. J. Sports Med. 2014, 48, 848. [8] S. Ojanperä, A. Leinonen, J. Apajalahti, M. Lauraeus, S. Alaja, T. Moisander, A. Kettunen. Characterization of microbial contaminants in urine. Drug Test. Anal. 2010, 2, 576. [9] J. Grosse, P. Anielski, P. Hemmersbach, H. Lund, R.K. Mueller, C. Rautenberg, D. Thieme. Formation of 19-norsteroids by in situ
[10]
[11]
[12]
[13] [14]
demethylation of endogenous steroids in stored urine samples. Steroids 2005, 70, 499. J. Grosse, C. Rautenberg, L. Wassill, D. Ganghofner, D. Thieme. Degradation of doping-relevant steroids by Rh.Erythropolis. Recent th Advances in Doping Analysis (15), 25 Cologne Workshop on Dope Analysis. Sport and Buch Strauss, Köln, 2007, pp. 385. M. Bredehöft, R. Baginski, M.K. Paar, M. Thevis, W. Schänzer. Investigations of the microbial transformation of cortisol to prednisolone in urine samples. J. Steroid Biochem. Mol. Biol. 2012, 129, 54. M. Mazzarino, M.G. Abate, R. Alocci, F. Rossi, R. Stinchelli, F. Molaioni, X. de la Torre, F. Botrè. Urine stability and steroid profile: Towards a screening index of urine sample degradation for anti-doping purpose. Anal. Chim. Acta 2011, 683, 221. M. Tsivou, D. Livadara, D.G. Georgakopoulos, M.A. Koupparis, J. Atta-Politou, C.G. Georgakopoulos. Stabilization of human urine doping contol samples. Anal. Biochem. 2009, 388, 179. M. Tsivou, D. Georgakopoulos, H.A. Dimopoulou, M. Koupparis, J. Atta-Politou, C.G. Georgakopoulos. Stabilization of human urine doping control samples: A current opinion. Anal. Bioanal. Chem. 2011, 401, 553.
1173
Drug Test. Analysis 2014, 6, 1170–1173
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
Correspondence case report Received: 31 July 2014
Revised: 6 November 2014
Accepted: 7 November 2014
Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1761
Degradation of methyltestosterone in urine samples C. Schweizer Grundisch,* N. Baume and M. Saugy Introduction
1170
Methyltestosterone is a common anabolic steroid which is rapidly metabolized in the body after consumption. Two different metabolites (17α-methyl-5β-androstane-3α,17β-diol and 17α-methyl-5αandrostane-3α,17β-diol) are usually screened to detect a misuse of this steroid by gas chromatography-mass spectrometry (GCMS) or gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS).[1] Therefore, methyltestosterone under its free form may be spiked in urine samples as an internal standard for the initial testing procedure of anabolic steroids by GC-MS or GC-MS/MS. In order to remove any ambiguity during confirmation analysis, an alternative internal standard is used. In the past two years, the Swiss Laboratory for Doping Analyses observed the conversion of spiked methyltestosterone to its metabolite 17α-methyl-5β-androstan3α,17β-diol in 5 samples (Figure 1). In some samples the steroid profiles were also affected by the degradation of testosterone and deuterated testosterone. The samples came from different sports, and had a travel time between 1 and 7 days. The samples had in common a pH over 6, a low specific gravity and signs of bacterial contamination (presence of 5α- and 5β-androstanedione). It is well known that bacteria and microorganisms can modify the concentration of urinary steroids partially or completely by degrading cholesterol and endogenous steroids[2,3] and thus influence the steroid profile.[4–7] These bacteria originate from the gastrointestinal or urogenital tracts and have the capacity to modify the steroid profile when growing is influenced and promoted by temperature.[8] Apart from endogenous steroids, microorganisms can produce others steroids, such as 19-norandrosterone by demethylation[9] or boldenone by 1-dehydrogenase activity.[10] Also, the endogenous glucocorticosteroids cortisol and cortisone may undergo transformations in prednisolone and prednisone, respectively due to dehydrogenation.[11] The indicators used by the World Anti-Doping Agency (WADA) accredited laboratories to identify activity of microorganisms in urine are the formation of 5α- and 5β-androstanenedione and the increase of free testosterone concentration.[12] To show the influence of bacteria growth, five samples with conversion of methyltestosterone into its main metabolite 17α-methyl5β-androstan-3α,17β-diol are presented herein. In doping control analyses, this biotransformation could lead to methyltestosterone false-positive cases. To solve this degradation problem of samples with presence of a methyltestosterone metabolite (17α-methyl-5α-androstan-3α,17βdiol), a confirmation procedure was performed either using an alternative internal standard or adding the internal standard after the hydrolysis step of the sample preparation. In both cases, there
Drug Test. Analysis 2014, 6, 1170–1173
was no more 17α-methyl-5α-androstan-3α,17β-diol and a clean steroid profile was obtained.
Experimental Standards and reagents 17α-methyltestosterone (4-androsten-17a-methyl-17b-ol-3-one, MeT) was purchased from Steraloids (Newport, RI, USA), androsterone-d4 glucuronide, and epitestosterone-d3 from NMIA (Pymble, Australia), testosterone-d3 from Lipomed (Arlesheim, Switzerland), 17α-methyl-5β-androstane-3α,17β-diol (MeT met) and stanozolol-d3 from Cerilliant (Round Rock, TX, USA). All reagents and solvents were of analytical grade or HPLC grade. Potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium carbonate, sodium hydrogen carbonate were provided by Merck (Darmstadt, Germany), Methyl tert-butyl ether (MTBE), methanol by Acros Organics (Geel, Belgium). β-glucuronidase from Escherichia Coli came from Roche diagnostic (Mannheim, Germany), N-Methyl-N-trimethylsilyltrifluoroacetamide from Macherey Nagel (Düren, Gremany), ammonium iodide from Sigma-Aldrich (Steinheim, Germany) and ethanethiol from Fluka (Buchs, Switzerland). Sample preparation Twenty μL of ISTDs (methyltestosterone, 200 ng/mL, testosteroned3, 80 ng/mL, epitestosterone-d3, 40 ng/mL, androsterone-d4 glucuronide, 200 ng/mL and stanozolol-d3, 40 ng/mL), 1 mL of boiled phosphate buffer (0.8 M, pH7) and 50 μL of β-glucuronidase from E.coli were added to 2.5 mL of urine. The samples were incubated for 1 hour at 50 °C or overnight at 37 °C (16 h). After hydrolysis, the urine was adjusted to pH 8.5-9 with carbonate buffer (Na2CO3/ NaHCO3 1/10, w/w) and liquid-liquid extraction performed with 5 mL of MTBE. Once the organic layer was transferred and evaporated to dryness, the residue was derivatized with 50 μL of MSTFA-NH4I-ethanethiol, (1000:2:3, v/w/v) at 60 °C for 20 min. Instrumentation The quantification for the steroid profile was performed on a Hewlett Packard 6890 GC system Plus gas chromatography system
*
Correspondence to: Carine Schweizer Grundisch, Swiss Laboratory for Doping Analyses, Ch. des Croisettes 22, 1066 Epalinges, Switzerland. E-mail: Carine. [email protected] Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Geneva and Lausanne, Ch. des Croisettes 22, 1066, Epalinges, Switzerland
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Testing and Analysis
Degradation of methyltestosterone in urine samples
Figure 1. Conversion of methyltestosterone to 17α-methyl-5β-androstan3α, 17β-diol.
coupled to a Hewlett Packard 5973 Mass Selective Detector (Palo Alto, CA, USA) mass spectrometer. The screening analyses for the detection of anabolic steroids were performed on an Agilent 7890B gas chromatography system coupled to a Triple Quadrupole 7000B (Wilmington, DE, USA).
Analytical conditions The instruments were equipped with a 17 m Agilent J&W Ultra-1 column, 0.2 mm internal diameter, 0.11 μm film thickness (Agilent Technologies, Palo Alto, CA, USA). The GC temperature program was: 180 °C, 1 min, 3 °C/min to 230 °C, then 40 °C/min to 310 °C (4 min). Injection of 2 μL was performed in split mode with a 10:1 split ratio and a temperature injector of 300 °C. Helium was used as carrier at constant flow (1.1 mL/min). Temperature of transfer line, ion source and quadrupole(s) were 280 °C, 230 °C and 150 °C, respectively. Selected ion monitoring (SIM) with a dwell time of 30 ms, multiple reaction monitoring (MRM) with a dwell time of 20 ms were used for the GC-MS and GC-MS/MS, respectively.
Results and discussion As depicted in Figure 2, various concentrations (peak area) of 17αmethyl-5β-androstan-3α,17β-diol (ranging from 1.5 to 100 ng/mL) were detected in different samples. In urines containing the highest concentrations (samples #1 and #2), almost all methyltestosterone was degraded compared to the other three samples.
When the internal standard was added after hydrolysis at 37 °C or 50 °C, no 17α-methyl-5β-androstan-3α,17β-diol was detected showing that the activity of microorganisms was clearly influenced by the temperature. The five urines shared a pH slightly elevated ranging from 6.0 to 7.3, a low specific gravity between 1.007 and 1.011 and had a clear aspect (Table 1). No typical external sign of degradation were observed. The origin of the microorganisms in these urines was investigated and apparently neither transport duration (from sample collection till the delivery to the lab) nor inappropriate temperature or storage (sample collections were done from September to January) could be the source of the bacteria growth in urine samples. In sample #1 (female athlete, beach volley) all methyltestosterone was converted into 17α-methyl5β-androstan-3α,17β-diol indicating a high microorganism activity. This might be explained by high temperature as the collection date was done on a hot day but it is also well documented that female urines contain a highest density of bacteria.[11] 5α and 5β-androstanedione were detected in a significant amounts in every of the five presented samples. Moreover, testosterone and deuterated testosterone were degraded in every sample but to a lesser extent in samples #4 and #5. Only deuterated androsterone glucuronide and deuterated stanozolol from the ISTD were not affected. Interestingly, all these samples were included in analytical batches which were incubated at 37 °C for 16 h indicating that the activity of the microorganisms was probably promoted by the duration of the incubation. Many microorganisms were listed with their degradation products obtained but in most cases endogenous steroids were primarily transformed.[13] In 2007, Grosse et al. showed that methyltestosterone can be converted in methandienone after incubation with Rhodococcus Erythropolis.[10] These bacteria are able to generate several enzymes affecting different sites of molecules only when these substances are found in free form in urine. In our screening procedure, free methyltestosterone is added in urine samples as an internal standard, making the transformation in 17α-methyl5β-androstan-3α,17β-diol possible.
Drug Test. Analysis 2014, 6, 1170–1173
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
1171
Figure 2. Chromatograms of methyltestosterone (ISTD) (upper panels) and 17α-methyl-5β-androstan-3α, 17β-diol (lower panels) in negative urine (A), positive control (B), samples #2 (C) and #3 (D).
Drug Testing and Analysis
C. Schweizer Grundisch, N. Baume and M. Saugy
Table 1. Summary of different parameters for five samples containing methyltestosterone metabolite, as highlighted with the screening procedure Samples # 1 2 3 4 5
Concentration MeT metabolite
pH
Speficic gravity
Presence of 5α- and 5β-androstanedione
Sport
Sex
Delivery delay
96 ng/mL 61 ng/mL 9 ng/mL 2 ng/mL 1.5 ng/mL
7.3 6.0 6.0 6.8 6.5
1.007 1.011 1.011 1.009 1.007
yes yes yes yes yes
Beach Volley Cyclocross Cyclocross Athletics Football
F M F M M
4 days 1 day 1 day 7 days 3 days
Figure 3. Steroid profile chromatograms of sample #1. (A) Degraded ISTD (MeT). (B) ISTD (MeT) added after the hydrolysis step, (C) MeT metabolite (m/z 435) trace present in the testosterone-d3 and epitestosterone-d3 chromatogram and (D) Normal chromatograms of testosterone and epistestosterone (m/z 432) and its deuterated ISTD (m/z 435).
1172
Prior to further investigation, the analyses were repeated for the five urine samples and production of methyltestosterone metabolite was again observed. To elucidate the origin of the presence of 17α-methyl-5β-androstan-3α,17β-diol and to obtain a clean steroid profile, the internal standard was added after the hydrolysis step of the sample preparation. The obtained chromatograms of derivatized testosterone and deuterated testosterone were clean (Figure 3). However, a steroid profile measured in a contaminated urine sample should not be used for longitudinal follow up. Different factors such as collection under non-sterile conditions, elevated temperature, transport duration, storage conditions and contaminations with microorganisms (bacteria, fungi) could impact the quality of the urinary matrix and thus the analytical results. To avoid any form of adulteration between the time of collection and analyses in the laboratory, it could be recommended to add a suitable stabilizer in the urine Berlinger kits used for sample collection. This would ensure the stability of the steroid profile without any effect on the different analyses as previously shown by Tsivou et al.[14] Nevertheless, WADA did not allow the introduction of an exogenous compound in the urine collection kits reinforcing stringent hygiene sampling and short delivery delay requirements. In addition,
wileyonlinelibrary.com/journal/dta
some experiments showed that a desired hydrolysis in the analytical sample preparation is sometimes inhibited by stabilizers.
Conclusions These five cases showed that microorganisms could promote the conversion of methyltestosterone to its metabolite, 17α-methyl5β-androstan-3α,17β-diol, without showing typical signs of microbial degradation (elevated pH, turbidity). For scientific interest, it would be useful to identify the bacteria responsible for this biotransformation.
References [1] W. Schänzer, M. Donike. Metabolism of anabolic steroids in man: Synthesis and use of reference substances for identification of anabolic steroid metabolites. Anal. Chim. Acta 1993, 275, 23. [2] R.W. Owen, M.E. Tenneson, R.F. Bilton, A.N. Mason. The degradation of cholesterol by Escherichia coli isolated from human faeces. Biochem. Soc. Trans. 1978, 6, 377.
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Test. Analysis 2014, 6, 1170–1173
Drug Testing and Analysis
Degradation of methyltestosterone in urine samples [3] P. Talalay. Enzymatic mechanisms in steroid metabolism. Physiol. Rev. 1957, 37, 362. [4] C. Ayotte, A. Charlebois, S. Lapointe, D. Barriault, M. Sylvestre. Valitity of urine samples: Microbial degradation. Recent Advances in Doping th Analysis (4), 14 Cologne Workshop on Dope Analysis. Sport and Buch Strauss, Köln, 1996, pp. 127. [5] U. Mareck, H. Geyer, G. Opfermann, M. Thevis, W. Schänzer. Factors influencing the steroid profile in doping control analysis. J. Mass Spectrom. 2008, 43, 877. [6] X. Liu, Y. Xing, Y. Xu, Z. Yang, M. Wu. Degradation of endogenous steroids by microorganisms. Recent Advances in Doping Analysis th (19), 29 Cologne Workshop on Dope Analysis. Sport and Buch Strauss, Köln, 2011, pp. 203. [7] T. Kuuranne, M. Saugy, N. Baume. Confounding factors and genetic polymorphism in the evaluation of individual steroid profiling. Brit. J. Sports Med. 2014, 48, 848. [8] S. Ojanperä, A. Leinonen, J. Apajalahti, M. Lauraeus, S. Alaja, T. Moisander, A. Kettunen. Characterization of microbial contaminants in urine. Drug Test. Anal. 2010, 2, 576. [9] J. Grosse, P. Anielski, P. Hemmersbach, H. Lund, R.K. Mueller, C. Rautenberg, D. Thieme. Formation of 19-norsteroids by in situ
[10]
[11]
[12]
[13] [14]
demethylation of endogenous steroids in stored urine samples. Steroids 2005, 70, 499. J. Grosse, C. Rautenberg, L. Wassill, D. Ganghofner, D. Thieme. Degradation of doping-relevant steroids by Rh.Erythropolis. Recent th Advances in Doping Analysis (15), 25 Cologne Workshop on Dope Analysis. Sport and Buch Strauss, Köln, 2007, pp. 385. M. Bredehöft, R. Baginski, M.K. Paar, M. Thevis, W. Schänzer. Investigations of the microbial transformation of cortisol to prednisolone in urine samples. J. Steroid Biochem. Mol. Biol. 2012, 129, 54. M. Mazzarino, M.G. Abate, R. Alocci, F. Rossi, R. Stinchelli, F. Molaioni, X. de la Torre, F. Botrè. Urine stability and steroid profile: Towards a screening index of urine sample degradation for anti-doping purpose. Anal. Chim. Acta 2011, 683, 221. M. Tsivou, D. Livadara, D.G. Georgakopoulos, M.A. Koupparis, J. Atta-Politou, C.G. Georgakopoulos. Stabilization of human urine doping contol samples. Anal. Biochem. 2009, 388, 179. M. Tsivou, D. Georgakopoulos, H.A. Dimopoulou, M. Koupparis, J. Atta-Politou, C.G. Georgakopoulos. Stabilization of human urine doping control samples: A current opinion. Anal. Bioanal. Chem. 2011, 401, 553.
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Drug Test. Analysis 2014, 6, 1170–1173
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
