Abstract

In the last 20 years, isotopic ratio mass spectrometry coupled with gas chromatography (GC/C/IRMS) applied to carbon stable isotope (13C/12C) ratios (CSIR) has been a fundamental tool in the field of anti-doping analysis. Some compounds, such as testosterone, can be found in human urine as the result of natural metabolism or exogenous intake, sometimes leaving its carbon isotopic signature as the only proof of an antidoping violation. More recently, the appearance of a new non-steroidal compound named 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) has been of major concern. Experiments made on mice, combined with rumors in the world of sport, has made the compound a suspect for anti-doping authorities. As an intermediate in the purine synthesis, AICAR is also present in urine. The development of a GC/C/IRMS method to distinguish between natural and synthetic AICAR therefore appears necessary, but is not done without issues as AICAR is a non-volatile and unstable molecule at high temperatures. Existing methods use trimethylsilyl (TMS) derivatization, which damages combustion furnaces and adds 9 carbon atoms to the AICAR molecule.
This work therefore proposes a new GC/C/IRMS method that uses acetylation as an alternative route to analyze the CSIR of AICAR for anti-doping purposes. The different issues encountered when developing such method are described as well as the data obtained from its validation and from the analysis of 46 urines samples. A comparison of the results from this work with the existing literature is also made. The results suggest that the use of CSIR to determine the origin of AICAR in urine is more complex than previously reported.