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
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
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.