Implementation of AICAR analysis by GC-C-IRMS for anti-doping purposes

Area of Science:

• Analytical Chemistry
• Biochemistry
• Sports Science

Background:

• AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) is a naturally occurring substance on the WADA Prohibited List, claimed to enhance physical performance.
• Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is crucial for distinguishing endogenous from exogenous substances like steroids.

Purpose of the Study:

• To implement and optimize a published GC-C-IRMS protocol for AICAR analysis in a laboratory setting.
• To address and resolve challenges encountered during the protocol implementation, particularly concerning derivatization and background noise.

Main Methods:

• Silylation was employed for AICAR derivatization due to the molecule's structure, optimizing conditions for derivative stability.
• High-performance liquid chromatography (HPLC) was used for sample purification, with added wash steps and specific purification for endogenous reference compounds (ERCs).

Main Results:

• A reproducible derivatization pattern was achieved, with the 3-TMS form identified as the major derivative product.
• The modified protocol, including wash steps and ERC purification, significantly reduced background noise and interferences, enhancing method robustness.

Conclusions:

• The study successfully adapted and improved a GC-C-IRMS method for AICAR detection, overcoming previous implementation difficulties.
• The enhanced protocol offers a more robust and reliable analytical tool for anti-doping efforts, ensuring accurate differentiation of AICAR.