Last Month's Most Accessed Feature: GC-MS defeats the doping steroid cheats

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  • Published: Mar 1, 2018
  • Categories: Gas Chromatography
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Current anabolic steroid detection methods require improvement

Anabolic steroids continue to be used in a number of competitive sports, as well as by bodybuilders. Detection is primarily by the examination of urine samples. Many anabolic steroids have a short lifetime in the body, being metabolised relatively quickly. Current doping control methods often rely on detecting specific metabolites of the anabolic steroids, but some of the metabolites used also have fairly short lifetimes. Cheats can thus escape detection provided they stop taking the drug some days before having to give a sample.

The Ghent University scientists searched for long-lived metabolites of two anabolic steroids, oxymesterone and mesterolone. They used known metabolic pathways to predict likely metabolites and then looked for them in urine from volunteers that had taken the steroids. GC-MS was used with chemical ionisation (CI) to detect metabolites; as expected, this ionisation technique was found to give stronger molecular ions than the traditional electron ionisation (EI).

GC-MS used to detect novel long-lived anabolic steroids

Two healthy male volunteers took doses of oxymesterone while another two took mesterolone. Urine samples were taken at intervals for up to 30 or 60 days for the two subjects who took oxymesterone, and up to 15 days for the subjects who took mesterolone.

The urine samples were hydrolysed at 56 °C for 1.5 hours using β-glucuronidase from E. coli in phosphate buffer to break down any steroid glucuronides present. Deuterated testosterone was added as an internal standard and a carbonate buffer was added to bring the pH up to 9.5. The steroids were extracted with methyl tert-butyl ether (MTBE). After solvent evaporation, the steroids were silylated at 80 °C in acetonitrile with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), in the presence of ammonium iodide and ethanethiol catalysts. In order to look at sulfated species, urine samples were also treated with a mixture of β-glucuronidase and aryl sulfatase from the edible snail, Helix Pomatia, with the preparation then continuing as above.

GC employed an Agilent 7890 instrument, fitted with an Agilent HP-1MS column. The temperature was raised from 110 °C to 320 °C in a series of gradients. The carrier gas was helium, run at a flow rate of 1.0 mL/min. Mass spectrometry employed an Agilent 7000C triple quadrupole instrument using chemical ionisation with ammonia as the reagent gas. In addition, an Agilent 7200 GC-QTOF (quadrupole time-of-flight) instrument with chemical ionisation was used to help determine the structure of the longest-lived oxymesterone metabolite.

Likely metabolites of the two steroids were tabulated, assuming similar biotransformations to those seen with related compounds. The ion transitions expected for the theoretical compounds were compared to those in the mass spectrum found using selected reaction monitoring (SRM), thus identifying a number of metabolites.

By looking at urine samples collected at different times, the metabolites with the longest life were identified. For oxymesterone, a new metabolite (referred to in the paper as oxyM9) was found, which was detectable in urine samples up to 46 days after the steroid was taken, compared to a detection time of ca. 3 to 4 days with traditional methods. A structure for oxyM9 was tentatively established on the basis of its mass spectrometry. Similarly, a long-lived metabolite (referred to in the paper as mestM22) of mesterolone was found, which was detectable in urine samples in its sulfate form for 15 days after the steroid was taken.

New steroid metabolites extend doping detection time

This work has identified new long-lived anabolic steroid metabolites that allow sports cheats to be more easily caught. The use of chemical ionisation helped identify the various metabolites present in urine samples and also aided the detection of long-lived species weeks after the drug was taken. The authors noted that the identity of the new metabolites will have to be confirmed by chemical synthesis before they can be officially used.

Related Links

Drug Testing and Analysis, 2017, 9, 1673-1684. Polet et al. Identification and characterization of novel long-term metabolites of oxymesterone and mesterolone in human urine by application of selected reaction monitoring GC-CI-MS/MS.

Rapid Communications in Mass Spectrometry, 2016, 30, 511-522. Polet et al. Gas chromatography/chemical ionization triple quadrupole mass spectrometry analysis of anabolic steroids: ionization and collision-induced dissociation behaviour.

Wikipedia, Chemical Ionization

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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