GC/C/IRMS Applied to Develop Doping Control Analysis Method

Scientists from the Shanghai University of Sport in Shanghai, China recently developed a new gas chromatography (GC) combustion isotope ratio mass spectrometry (GC/C/IRMS)-based procedure for assisting doping control procedures involving urine samples. Their findings were published in the Journal of Chromatography A (1).

Sports doping, anabolic capsule - shape of a male muscular upper body - symbol for medical drug abuse. | Image Credit: © Andy Shell - stock.adobe.com

5-amino-4-imidazolecarboxyamide ribonucleoside, more commonly known as AICAR, is an endogenous substance and intermediate metabolite in the purine de novo synthesis pathway. In organisms, AICAR is an adenosine analogue that is taken into a cell via adenosine transporter proteins and phosphorylated by adenosine kinase. This produces AICAR monophosphate (ZMP), which mimics the action of amphetamines (AMP), thus acting as a direct activator of AMP activated protein kinase (AMPK). When activated, AMPK leads to increased glucose uptake and enhanced insulin sensitivity, which can stimulate fatty acid oxidation and promote muscle remodeling, among other benefits.

AICAR has been viewed as a performance-enhancing substances for athletes and it has been prohibited in sport and viewed as a health risk, since it is not approved for therapeutic use in humans anywhere in the world. In fact, according to the United States Anti-Doping Agency (USADA), AICAR is on the World Anti-Doping Agency (WADA) Prohibited List “because it’s an AMPK activator, which are prohibited at all times under the category of Hormone and Metabolic Modulators on the WADA Prohibited List because of their potential performance-enhancing effects” (2).

Isotope ratio mass spectrometry (MS) technology is effective for identifying the source of substances, being more commonly known as gas chromatography combustion isotope ratio mass spectrometry (GC/C/IRMS). Urine is a non-invasive and readily available matrix, thus making it a more desirable sample source for IRMS. However, urine matrices are very complex, containing substances like glucose, proteins, and inorganic salts; therefore, effective separation and purification must be conducted before IRMS. Further, IRMS detection of AICAR can come with many challenges. AICAR’s molecular structure can make it difficult to analyze with either gas chromatography–mass spectrometry (GC–MS) or GC/C/IRMS using conventional anti-doping chromatographic protocols. Five derivatization sites being present in AICAR molecules also present selectivity problems as well.

Carbon isotope ratio (CIR) mass spectrometry is the most common method for distinguishing the endogenous and exogenous source of AICAR in urine due to significant individual differences in concentration. In this study, the scientists hoped to establish a GC/C/IRMS method for analyzing AICAR based on two-dimensional high-performance liquid chromatography (2D-HPLC) separation. First, 2D-HPLC was used to separate and purify AICAR and endogenous reference substances in urine samples. Then, AICAR was derivatized with 3-TMS as the main derivative product, while the endogenous reference compounds remained in their original form. Then the developed GC/C/IRMS method was used to detect the target and reference substances. Finally, the method’s applicability was evaluated using urine samples from two subjects that were each administered 3 grams of AICAR.

Throughout the procedures, the scientists noted various advantages to the approach. Since 2D-HPLC can separate target and endogenous reference substances in one step, sample pretreatment time is reduced. Further, isotope chromatograms have low background interference with endogenous reference substance separation being purer, with the method also having more accurate result calculations as well. Overall, the detection method met the needs of routine detection concentrations with a concentration of 2500 ng/mL. CIR results from the volunteer samples show that samples collected within 16 hours post-administration exceeding the threshold set in the literature.

Overall, this method showed a new and effective 2D-HPLC-GC/IRMS method that integrates CIR as the most stable indicator for distinguishing between internal and external AICAR sources. Further, exogenous drug characteristics were manifested from the volunteer samples, showing that the length of detection windows are positively correlated with test drug dosages.

References

(1) Wang, Y.; Fan, J.; Fang, X.; et al. Two-Dimensional Liquid Chromatography Purified GC/C/IRMS Doping Control Method: Analysis of Endogenous and Exogenous Sources in Urine Samples from Asian Subjects Administered a Low Dose of AICAR. J. Chromatogr. A 2024, 1735, 465312. DOI: 10.1016/j.chroma.2024.465312

(2) What Athletes Should Know About AICAR and Other Prohibited AMP Activated Protein Kinase Activators. USADA 2019. https://www.usada.org/spirit-of-sport/education/aicar-and-other-prohibited-amp-activated-protein-kinase-activators/ (accessed 2024-12-2)