The detection and separation of medetomidine enantiomers from the complex biological matrices poses a great analytical challenge, especially in the field of forensic toxicology and pharmacology. Couple of researchers reported resolution of medetomidine using protein‐based chiral columns, but the reported method is quiet challenging and tedious to be employed for routine analysis. This research paper reported a method that enables the enantio‐separation of medetomidine by using polysaccharide cellulose chiral column. The use of chiralcel OJ‐3R column was found to have the highest potential for successful chiral resolution. Ammonium hydrogen carbonate was the ideal buffer salt for chiral liquid chromatography (LC) with electrospray ionization (ESI)+ mass spectrometry (MS) detection for the successful separation and detection of racemic compound. The method was linear over the range of 0 to 20 ng/mL in equine plasma and the inter‐day precisions of levomedetomidine, dexmedetomidine were 1.36% and 1.89%, respectively. The accuracy of levomedetomidine was in the range of 99.25% to 101.57% and that for dexmedetomidine was 99.17% to 100.99%. The limits of quantification for both isomers were 0.2 ng/mL. Recovery and matrix effect on the analytes were also evaluated. Under the optimized conditions, the validated method can be adapted for the identification and resolution of the medetomidine enantiomers in different matrices used for drug testing and analysis.
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 increased 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 50ng/ml.
CONCLUSION:
Chromatographic separation was achieved using 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.
Chiral considerations are found to be very much relevant in various aspects of forensic toxicology and pharmacology. In forensics, it has become increasingly important to identify the chirality of doping agents to avoid legal arguments and challenges to the analytical findings. The scope of this study was to develop an liquid chromatography–mass spectrometry (LCMS) method for the enantiomeric separation of typical illicit drugs such as ephedrines (ie, 1S,2R(+)‐ephedrine and 1R,2S(−)‐ephedrine) and pseudoephedrine (ie, R,R(−)‐pseudoephedrine and S,S(+)‐pseudoephedrine) by using normal phase chiral liquid chromatography–high‐resolution mass spectrometry technique. Results show that the Lux i‐amylose‐1 stationary phase has very broad and balancing‐enantio‐recognition properties towards ephedrine analogues, and this immobilized chiral stationary phase may offer a powerful tool for enantio‐separation of different types of pharmaceuticals in the normal phase mode. The type of mobile phase and organic modifier used appear to have dramatic influences on separation quality. Since the developed method was able to detect and separate the enantiomers at very low levels (in pico grams), this method opens easy access for the unambiguous identification of these illicit drugs and can be used for the routine screening of the biological samples in the antidoping laboratories.
The major challenge in identifying dexamethasone, betamethasone, and paramethasone from a mixture of these corticosteroids is difficulty in achieving an efficient separation. In this study, we aimed to develop an efficient technique to identify these co-eluting isomers based on the mass spectral patterns of them and their corresponding phase II metabolites after electrospray ionization. Fragmentation pathways in tandem mass spectrometry revealed acceptable specificity within the groups of conjugates. The method was validated using individual isomers and mixtures at various compositions. The effects of concentration and collision energies on fragmentation patterns were also studied extensively. Matrix-fortified equine urine and plasma samples were also included so that matrix effects and interferences on fragmentation ratios could be elucidated. Preliminary results using biological samples demonstrated the suitability of this analytical strategy for direct measurement from their fragmentation patterns. Possible fragmentation pathways for each isomer were proposed.
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