Separation and Determination of the Enantiomeric Levamisole and Dexamisole in Equine Plasma Samples Using Chiral Polysaccharide Column/LC-MS/MS

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 degrees 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.