AICAR(5-氨基-4-咪唑甲酰胺核糖核苷),作为代谢调节剂,被世界反兴奋剂机构(WADA)归类为S4类别。碳同位素比质谱(CIR)是区分尿液中AICAR的内源性和外源性来源的主流方法,由于浓度的显着个体差异。本研究的目的是建立基于高效二维液相色谱(2D-HPLC)的AICAR气相色谱燃烧同位素比质谱(GC/C/IRMS)分离方法。
方法:在本研究中,使用自动2D-HPLC分离技术分离和纯化尿液样品中的AICAR和内源性参考物质。然后,AICAR以3-TMS为主要衍生产品,而内源性参考化合物保持其原始形式。随后,利用已开发的GC/C/IRMS方法检测目标物质和参考物质。跟随,我们使用两名接受低剂量AICAR(3克)的亚洲男性的尿液样本评估了该方法的适用性.
结果:本研究的优点包括:1)减少了样品预处理时间:建立的2D-HPLC分离方法可以一步分离目标和内源性参考物质;2)低干扰:同位素色谱图具有低背景干扰,和内源性参考物质的分离更纯净;3)更准确的结果计算:该方法只需要对AICAR进行衍生化和结果校正,内源性参考物质以其原始形式测量,减少来自多种物质校正的偏见。检测方法效果良好,浓度限制为2500ng/mL,满足常规检测浓度的需要。来自志愿者样品的CIR结果表明,给药后16小时内收集的样品超过了文献中设定的阈值。
结论:本研究成功建立了2D-HPLC-GC/IRMS方法,该方法将CIR整合为区分AICAR的内部和外部来源的最稳定指标。在向亚洲人群施用低剂量的AICAR后,外源性药物特征在16小时内表现出来.这个观察,与文献中引用的40小时检测窗口相比,提示检测窗口的长度与试验药物的剂量呈正相关。
AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside), as a metabolic modulator, is classified in the S4 category by the World Anti-Doping Agency (WADA). Carbon Isotope Ratio Mass Spectrometry (CIR) is the mainstream method for distinguishing the endogenous and exogenous sources of AICAR in urine due to the significant individual difference in the concentration. The purpose of this study is to establish a gas chromatography combustion Isotope Ratio Mass Spectrometry (GC/C/IRMS) method for AICAR based on efficient two-dimensional liquid chromatography (2D-HPLC) separation.
METHODS: In this study, an automated 2D-HPLC separation technique 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. Subsequently, the developed GC/C/IRMS method was utilized for the detection of the target and reference substances. Followed, we evaluated the applicability of this method using urine samples from two Asian males administered a low dose of AICAR (3 grams).
RESULTS: The advantages of this study include: 1) reduced sample pretreatment time: the established 2D-HPLC separation method can separate the target and endogenous reference substances in one step; 2) low interference: the isotope chromatograms have low background interference, and the separation of endogenous reference substances is purer; 3) more accurate result calculations: this method only requires derivatization and result correction for AICAR, with the endogenous reference substances measured in their original form, reducing biases from corrections of multiple substances. The detection method performed well, with a concentriton limit of 2500 ng/mL, meeting the needs of routine detection concentrations. The CIR results from volunteer samples indicated that samples collected within 16 hours post-administration exceeded the threshold set in the literature.
CONCLUSIONS: This study successfully established a 2D-HPLC-GC/IRMS method that integrates CIR as the most stable indicator for distinguishing the internal and external sources of AICAR. After administering a low dose of AICAR to the Asian population, exogenous drug characteristics were manifested within 16 hours. This observation, when compared to the 40-hour detection window cited in the literature, suggests that the length of the detection window is positively correlated with the dosage of the test drug.