Keen Bethany, Cawley Adam, Reedy Brian, Noble Glenys, Loy Jaymie, Fu Shanlin
Centre for Forensic Science, University of Technology Sydney, Broadway, NSW 2007, Australia.
Australian Racing Forensic Laboratory, Racing NSW, Sydney, NSW 2000, Australia.
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Apr 1;1220:123652. doi: 10.1016/j.jchromb.2023.123652. Epub 2023 Mar 7.
The use of catechol-O-methyltransferase inhibitors may mask doping agents, primarily levodopa, administered to racehorses and prolong the stimulating effects of dopaminergic compounds such as dopamine. It is known that 3-methoxytyramine is a metabolite of dopamine and 3-methoxytyrosine is a metabolite of levodopa thus these compounds are proposed to be potential biomarkers of interest. Previous research established a urinary threshold of 4,000 ng/mL for 3-methoxytyramine to monitor misuse of dopaminergic agents. However, there is no equivalent biomarker in plasma. To address this deficiency a rapid protein precipitation method was developed and validated to isolate target compounds from 100 µL equine plasma. A liquid chromatography-high resolution accurate mass (LC-HRAM) method using an IMTAKT Intrada amino acid column provided quantitative analysis of 3-methoxytyrosine (3-MTyr) with lower limit of quantification of 5 ng/mL. Reference population profiling (n = 1129) investigated the expected basal concentrations for raceday samples from equine athletes and showed a right-skewed distribution (skewness = 2.39, kurtosis = 10.65) which resulted from large variation (RSD = 71%) within the data. Logarithmic transformation of the data provided a normal distribution (skewness = 0.26, kurtosis = 3.23) resulting in the proposal of a conservative threshold for plasma 3-MTyr of 1,000 ng/mL at a 99.995% confidence level. A 12-horse administration study of Stalevo® (800 mg L-DOPA, 200 mg carbidopa, 1600 mg entacapone) revealed elevated 3-MTyr concentrations for 24-hours post-administration.
儿茶酚-O-甲基转移酶抑制剂的使用可能会掩盖给予赛马的主要兴奋剂左旋多巴，并延长多巴胺等多巴胺能化合物的刺激作用。已知3-甲氧基酪胺是多巴胺的代谢产物，3-甲氧基酪氨酸是左旋多巴的代谢产物，因此这些化合物被认为是潜在的感兴趣生物标志物。先前的研究确定了3-甲氧基酪胺的尿液阈值为4000 ng/mL，以监测多巴胺能药物的滥用情况。然而，血浆中没有等效的生物标志物。为了解决这一缺陷，开发并验证了一种快速蛋白质沉淀方法，用于从100 μL马血浆中分离目标化合物。使用IMTAKT Intrada氨基酸柱的液相色谱-高分辨率精确质量（LC-HRAM）方法对3-甲氧基酪氨酸（3-MTyr）进行了定量分析，定量下限为5 ng/mL。参考人群分析（n = 1129）研究了赛马运动员比赛日样本的预期基础浓度，结果显示分布呈右偏态（偏度 = 2.39，峰度 = 10.65），这是由数据中的较大变异（相对标准偏差 = 71%）导致的。对数据进行对数转换后得到正态分布（偏度 = 0.26，峰度 = 3.23），从而提出在99.995%置信水平下血浆3-MTyr的保守阈值为1000 ng/mL。一项对Stalevo®（800 mg左旋多巴、200 mg卡比多巴、1600 mg恩他卡朋）进行的12匹马给药研究显示，给药后24小时3-MTyr浓度升高。
