Background: Drug Enforcement Administration confirmed that many manufacturers began adding tetramisole or its individual isomers to cocaine as an adulterant, and believed that tetramisole may augment cocaine’s effects. In recent times, there is an increasing trend in the usage of tetramisole and its individual enantiomer in race sports especially in horse and camel races. So it’s is very much required to confirm the stereochemistry of this illicit drug in the routine race day samples coming to the anti-doping labs in order to avoid legal arguments and challenges to the analytical findings.

Methods: The aim of the study was to develop a simple, rapid and accurate method for the chiral separation and determination of enantiomeric mixtures of levamisole and dexamisole using Thermo Q-Exactive High-Resolution Mass Spectrometer. In order to evaluate the suitability of the method for determining the enantiomeric purity of tetramisole, validation studies were also carried out by using equine plasma.

Results: The enantio-separation was achieved using the Lux i-cellulose-5 column. Isocratic flow was used with a 1:1 mixture of mobile phase A (10 mM ammonium acetate in water) and mobile phase B (acetonitrile), at a flow rate of 0.6 mL/min. The run time was 8.0 min, and the column temperature was 50°C. Dexamisole eluted at 5.94 min, and levamisole eluted at 6.62 min, giving the R-value of 1.50. The obtained inter-day precisions of dexamisole, levamisole were 3.16% and 2.85%, respectively. The accuracy of dexamisole was in the range of 97.78 to 102.44%, and that for levamisole was 99.16 to 102.82%. The limit of quantification value for both isomers in this method was 0.1 ng/ mL. The method was linear in the range of 0 to 50 ng/mL.

Conclusion: Chromatographic separation was achieved using the polysaccharide cellulose chiral column, and the reverse-phase separation approach was found to have the highest potential for successful chiral resolution in LC-MS. Linearity, precision, accuracy, detection limit, recovery, and the matrix effect in equine plasma were determined. Under the optimized conditions, the validated method can be applied for the identification and detection of the tetramisole enantiomers in different sources of illicit drugs of abuse.

背景：药物管理局证实，许多制造商已开始将可丁胺或它的单个异构体添加到可卡因中作为掺假品，并认为丁苯甲醚可能会增强可卡因的作用。近年来，在比赛运动中，特别是在赛马和骆驼比赛中，丁苯甲醚及其单个对映体的使用呈增加趋势。因此，非常需要在送往反兴奋剂实验室的常规比赛日样品中确认这种非法药物的立体化学，以避免法律论点和对分析结果的挑战。

方法：本研究的目的是开发一种简单，快速，准确的方法，使用Thermo Q-Exactive高分辨率质谱仪手性分离左旋咪唑和右旋咪唑的对映体混合物。为了评估该方法测定丁草醚对映体纯度的适用性，还通过使用马血浆进行了验证研究。

结果：使用Lux i-cellulose-5色谱柱完成对映体分离。等度流动与流动相A（在水中的10 mM乙酸铵）和流动相B（乙腈）的1：1混合物一起使用，流速为0.6 mL / min。运行时间为8.0分钟，柱温为50°C。地塞米索在5.94分钟处洗脱，而左旋咪唑在6.62分钟处洗脱，得出R值为1.50。所获得的右旋咪唑，左旋咪唑的日间精密度分别为3.16％和2.85％。地塞米索的准确性在97.78至102.44％的范围内，而左旋咪唑的准确性在99.16至102.82％的范围内。该方法中两种异构体的定量极限为0.1 ng / mL。该方法在0至50 ng / mL范围内是线性的。

结论：使用多糖纤维素手性色谱柱可实现色谱分离，而反相分离方法在LC-MS中成功实现手性拆分的潜力最大。测定了马血浆中的线性，精密度，准确度，检测限，回收率和基质效应。在优化的条件下，经验证的方法可用于鉴定和检测不同来源的非法滥用药物中的丁苯甲醚对映体。