Doping Control Laboratory of Athens, Olympic Athletic Centre of Athens Spiros Louis, 37 Kifissias Ave, 151 23 Maroussi, Greece.
Drug Test Anal. 2009 Aug;1(8):365-71. doi: 10.1002/dta.70.
Levodopa and dopamine have been abused as performance-altering substances in horse racing. Urinary 3-methoxytyramine is used as an indicator of dopaminergic manipulation resulting from dopamine or levodopa administration and is prohibited with a urinary threshold of 4 microg mL(-1) (free and conjugated). A simple liquid chromatographic (LC)/mass spectrometric (MS) (LCMS) method was developed and validated for the quantification and identification of 3-methoxytyramine in equine urine. Sample preparation involved enzymatic hydrolysis and protein precipitation. Hydrophilic interaction liquid chromatography (HILIC) was selected as a separation technique that allows effective retention of polar substances like 3-methoxytyramine and efficient separation from matrix compounds. Electrospray ionization (ESI) in positive mode with product ion scan mode was chosen for the detection of the analytes. Quantification of 3-methoxytyramine was performed with fragmentation at low collision energy, resulting in one product ion, while a second run at high collision energy was performed for confirmation (at least three abundant ions). Studies on matrix effects showed ion suppression depending on the horse urine used. To overcome the variability of the results originating from the matrix effects, isotopic labelled internal standard was used and linear regression calibration methodology was applied for the quantitative determination of the analyte. The tested linear range was 1-20 microg mL(-1). The relative standard deviations of intra- and inter- assay analysis of 3-methoxytyramine in horse urine were lower than 4.2% and 3.2%, respectively. Overall accuracy (relative percentage error) was less than 6.2%. The method was applied to case samples, demonstrating simplicity, accuracy and selectivity.
左旋多巴和多巴胺被滥用于赛马比赛中改变运动表现的物质。尿液中的 3-甲氧基酪胺被用作多巴胺或左旋多巴给药后多巴胺操纵的指标，规定尿液阈值为 4μg/mL（游离和共轭）时为禁用。建立并验证了一种简单的液相色谱（LC）/质谱（MS）（LCMS）方法，用于马尿中 3-甲氧基酪胺的定量和鉴定。样品制备涉及酶解和蛋白质沉淀。亲水相互作用液相色谱（HILIC）被选为一种分离技术，可有效保留 3-甲氧基酪胺等极性物质，并与基质化合物有效分离。选择电喷雾电离（ESI）正模式和产物离子扫描模式进行分析物检测。3-甲氧基酪胺的定量分析采用低碰撞能量进行碎片分析，得到一个产物离子，然后进行高碰撞能量的第二次运行以进行确认（至少三个丰度离子）。基质效应研究表明，根据所用马尿，存在离子抑制。为了克服源于基质效应的结果变异性，使用了同位素标记的内标，并应用线性回归校准方法进行分析物的定量测定。测试的线性范围为 1-20μg/mL。马尿中 3-甲氧基酪胺的日内和日间分析的相对标准偏差分别低于 4.2%和 3.2%。总体准确度（相对百分比误差）小于 6.2%。该方法应用于案例样本，证明了其简单性、准确性和选择性。
