Development and validation of a chiral LC-MS method for the enantiomeric resolution of (+) and (-)-medetomidine in equine plasma by using polysaccharide-based chiral stationary phases.

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.

Investigation of the complexation between cyclodextrins and medetomidine enantiomers by capillary electrophoresis, NMR spectroscopy and molecular modeling.

The migration order of the enantiomers of medetomidine in the presence of cyclodextrins studied by capillary electrophoresis in phosphate buffer, pH 2.5, depended on the cavity size and the substitution pattern of the cyclodextrins. Opposite migration order was observed in the presence of ß-cyclodextrin (ß-CD) and γ-cyclodextrin (γ-CD) as well as randomly sulfated ß-CD (S-ß-CD) and heptakis(6-O-sulfo)-ß-CD (HS-ß-CD). This could be rationalized by the fact that dexmedetomidine formed more stable complexes with ß-CD and S-ß-CD, while levomedetomidine interacted stronger with γ-CD and HS-ß-CD. The structure of the complexes was derived from rotating frame nuclear Overhauser (ROESY) experiments for ß-CD, γ-CD and HS-ß-CD. In the case of the native CDs, the phenyl ring of medetomidine entered the cavity through the wider secondary rim of the CDs, whereas the protonated imidazole ring was positioned inside the CD cavity interacting with the sulfate groups of HS-ß-CD. Furthermore, molecular dynamics calculations also suggested opposite affinities of the medetomidine enantiomers toward ß-CD and γ-CD.