No abstract

AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside), is a naturally occurring substance which is part to the World AntiDoping Agency (WADA) Prohibited List. It is claimed to improve physical performance when administered as a supplement. As for other endogenous compounds such as steroids, the gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis remains an efficient tool to differentiate endogenous substances from exogenous ones. A protocol was described in the literature for the analysis of AICAR by GC-C-IRMS. The aim of the present study was to implement this protocol in our laboratory and to propose solutions to avoid the difficulties encountered. The first point discussed in this study is the derivatization step. Due to the structure of the AICAR molecule, conventional derivatization for GC-C-IRMS such as acetylation could not be applied and silylation was preferred. The improvement of the derivatives stability was achieved thanks to several derivatization conditions tested. This adjustment led to a reproducible derivatization pattern with the 3-TMS form as major derivative product. The second point discussed in this study is the diminution of extracts' background noise. Indeed, the implementation of the published protocol was not easy due to high performance liquid chromatography (HPLC) problems encountered when concentrated urine was injected into our system. Also, too many interferences in the endogenous reference compound fractions were observed. The addition of both a wash step before the HPLC purification and a HPLC purification step for the endogenous reference compound (ERC) fraction allowed us to increase the robustness of the method. This study presents the modified protocol compared to the original protocol as well as the evaluation of the whole method performances.

The impact of dehydroepiandrosterone (DHEA) administration has been widely studied for anti-doping purposes in men, whereas only a few studies have been performed in women. In the present study, the impact of DHEA on the steroid profile parameters and their carbon isotopic ratios was explored. Eleven healthy young women and 10 healthy young men received two treatments: One with 100 mg/day of DHEA for 28 days and one with a placebo according to a double-blind crossover protocol. Urine and saliva (only in females) samples were collected before and for 72 hours after each short-term treatment. In all female subjects, concentrations of the urinary parameters of the steroid profile were highly impacted by short-term DHEA administration including epitestosterone (E). Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis was performed and positive results were observed for E in the four female subjects where E concentration was adequate for such analysis, whereas men results remained negative for E. Last, the ability of the Anti-Doping Administration and Management System (ADAMS) software used for the athlete biological passport to identify such doping was assessed. Of the 11 passports generated for female subjects, 10 were automatically classified as an atypical passport finding (ATPF). For the remaining passport with normal status in one woman, the variability of the concentrations prevented the ADAMS software from adjusting individual limits. The most impacted markers in women were T/E and 5αAdiol/E, with a detection window of 36 hours for 5αAdiol/E. In addition, good correlations were observed for DHEA and T concentrations in urine and saliva in females.