Rapid Analysis of Urinary AICAR by Liquid Chromatography–Isotope Ratio Mass Spectrometry

5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) is prohibited in sports according to the World Anti-Doping Agency (WADA). Currently, AICAR concentrations are measured during the initial testing procedure, and urine samples showing elevated levels are flagged as suspicious and forwarded for confirmation. Here, gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) is employed to distinguish endogenous from exogenous AICAR, as synthetic AICAR exhibits a different carbon isotope ratio (CIR). However, a GC-C-IRMS method comes with multiple challenges as AICAR is very polar, making it particularly difficult to analyze using GC. It is clear that there are a number of issues related to the existing protocol, and efforts should be made to develop new, more effective, and less expensive approaches. For polar substances, liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) provides a more straightforward approach. There is no need for derivatization, AICAR can be directly analyzed with LC-IRMS, and many GC-C-IRMS related issues are avoided. LC-IRMS also allows condensing of the workflow and lowering of the turnaround time. In this study, a rapid protocol for AICAR CIR determination was developed, employing solid-phase extraction and 2D-LC cleanup prior to LC-IRMS analysis. Etiocholanolone and androsterone were used as endogenous reference compounds, and the method was fully validated according to WADA regulations. Reference population (n = 50) thresholds, used to declare an adverse analytical finding (AAF, i.e., a positive AICAR sample), were determined (6.9‰ and 5.8‰ for AICAR-etiocholanolone and AICAR-androsterone, respectively) and found to be approximately 2‰ lower than those previously reported using GC-C-IRMS, facilitating AAF identification.