GC defeats AICAR sports cheats

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  • Published: Dec 14, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Gas Chromatography
thumbnail image: GC defeats AICAR sports cheats

Accurate methods needed to combat AICAR doping

5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR or acadesine) occurs naturally at low levels in the human body, but is also used as a performance-enhancing drug in sport. It is believed to increase endurance and was suspected to have been used in the 2009 Tour de France. AICAR has been banned by the World Anti-Doping Agency (WADA), so effective means of differentiating material arising from doping from the natural background substance are needed. The normal range of AICAR in athletes has been determined, and values obtained outside of this should indicate doping. However, any accusations based on AICAR levels are open to claims that a particular athlete has an ‘unusual metabolism’, etc. Since natural compounds have different isotope ratios to synthetic drugs, these ratios can be employed as a definite sign of doping. GC-C-IRMS (gas chromatography, combustion, isotope ratio mass spectrometry) is a useful technique to determine isotope ratios; in this technique, the GC output undergoes combustion, and the isotope ratio of the resulting carbon dioxide is then determined.

The Paris researchers developed a method for AICAR determination based on an earlier GC-C-IRMS method (Piper et al.). Urine samples were initially purified by HPLC, prior to GC-C-IRMS. Naturally occurring steroids were also isolated from the urine samples; these were used as endogenous reference compounds (ERC), giving a measure of the natural isotope ratio for comparison.

AICAR analysed by HPLC, derivatisation and GC-C-IRMS

Urine samples were taken from volunteers, with some being spiked with synthetic AICAR to mimic doping. The pH of the samples was adjusted to 9–10, and solids were removed by centrifugation. The samples were then absorbed onto HLB solid phase extraction (SPE) cartridges, which were eluted with ethanol. The ethanol was evaporated and the residue was taken up in water. Solids were removed by centrifugation. The solution was filtered through a PTFE membrane and centrifuged again prior to HPLC. This sample preparation was needed to prevent clogging of the HPLC.

HPLC employed a Thermo Scientific UltiMate 3000 system with a phenyl hexyl column. This separated the AICAR from the mixture of steroids. The fractions containing the AICAR were evaporated, and the residue was taken up into methanol:water 75:25 v/v. A second HPLC was carried out using a NH2-RP column on the crude AICAR material. Meanwhile, the steroid fractions were treated with β-glucuronidase to hydrolyse any conjugates, and then further purified by HPLC.

The AICAR was then derivatised to give a triple trimethsilyl derivative using ‘MSTFA activated III’ (N-methyl-N-trimethylsilyltrifluoroacetamide activated with imidazole) at 60 °C. The conditions had to be carefully optimised in order to only silylate the three hydroxyl groups, not the amine or amide groups (the silylation of the amide group gives an unstable derivative, and it is difficult to silylate the amine without derivatisation of the amide). The steroid reference compounds were derivatised under the same conditions.

GC used an HP6890N instrument fitted with an Agilent J&W DB-17MS column. The oven temperature was raised from 70 to 300 °C in two gradients. The presence of the trimethylsilyl derivative with three silyl groups present was confirmed by mass spectrometry using an HP5973 quadrupole instrument. For combustion, the AICAR from the GC was sent to an Isoprime GC-V combustion interface and then on to an Isoprime isotope ratio mass spectrometer. The corrected δ 13C values (a measure of the amount of 13C) for AICAR ranged from -22.5 to 25.1‰ for unspiked samples; the reference steroids gave similar results. In contrast, the spiked urine samples containing synthetic AICAR gave values ranging from -8.96 to -10.54‰, showing a clear difference.

New GC-C-IRMS method detects AICAR doping

The improved AICAR detection method can clearly detect AICAR doping. The authors note that further studies are needed to fully validate the system and to determine the concentration threshold of AICAR that should trigger an isotopic investigation of a urine sample.

Related Links

Drug Testing and Analysis, 2017, Early View Paper. Buisson et al. Implementation of AICAR analysis by GC-C-IRMS for anti-doping purposes.

Rapid Communications in Mass Spectrometry, 2014, 28, 1194-1202. Piper et al. Determination of 13C/12C ratios of endogenous urinary 5-amino-imidazole-4-carboxamide 1β-D-ribofuranoside (AICAR).

Wikipedia, Acadesine

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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