Quantification of AICAR and study of metabolic markers after administration.

: AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside) was reported as the first pharmacological AMPK (adenosine 5'-monophosphate (AMP)-activated protein kinase) activator, and it has been confirmed to exhibit a significant endurance enhancement effect and prohibited for doping by the World Anti-Doping Agency. Due to the fact that the human body can produce such substances, in order to ensure fairness in sports competition, methods for rapid detection and multi-type identification of AICAR drugs taken orally should be established. : to assess AICAR levels, a new rapid, sensitive, efficient, and selective method was reported for the quantitative detection of AICAR in urine using LC-MS/MS. The method was validated for quantitative purposes based on the elemental selectivity, intra- (1.0-15.6%) and inter-day precision (1.3-16.3%), accuracy (99.9-112.8%), matrix effects (88.9-103.6%), recovery (87.4-106.5%), and stability at four different concentrations. The calibration curve was linear over a wide concentration range of 10-10,000 ng mL with a high coefficient of determination ( > 0.998). The limit of detection (LOD) and limit of quantification (LOQ) for the experiment were determined to be 1 and 10 ng mL, respectively. Simultaneously, metabolomics analysis was used to obtain the metabolic fingerprint of different populations and biomarkers to distinguish administration cases through partial least squares discriminant analysis (PLS-DA) and a receiver operating characteristic (ROC) curve. : the method enables easy quantitation for LC-MS/MS analysis with the best recovery yield maintained, and the method was applied to 122 Asian biological samples with an average concentration of 1310.5 ± 1031.4 ng mL. Through drug metabolism research, 734 and 294 variables were extracted for data analysis respectively in the positive and negative ion modes, and more than 100 metabolites with significant up- and down-regulation were found after the test. : this research developed a fast, precise, effective, and specific approach for the qualitative and quantitative identification of AICAR in urine. Meanwhile, administration metabolism studies found that there were significant changes in AICAR levels and other compounds, such as PC types PC(18:1/16:0), PC(16:0/18:0), and PC(16:0/16:0), PE types PE(18:0/20:4), and LPE-type 18:1, which could better distinguish samples before and after AICAR administration. The analysis provides a multi-perspective reference for WADA to determine a positive criterion.