A quick approach for medetomidine enantiomers determination in dog plasma by chiral LC-MS/MS and application to a pharmacokinetic study.

Abstract

In the present study, a rapid, sensitive and high-throughput LC-MS/MS method for the determination of medetomidine enantiomers in dog plasma was developed and validated. The separation and individual quantification of chiral compounds can be a tricky task in liquid chromatography. This is particularly true when target analytes have a relatively small mass, as is the case with medetomidine, a potent and highly specific α2-adrenoceptor agonist widely used in both human and veterinary medicine. The proposed approach is based on a quick liquid-liquid extraction with ethyl acetate and filtration prior to injection. The optimized mobile phase composition allowed to perfectly separate the two enantiomers of medetomidine in a short chromatographic run time, using a cellulose tris(4-methylbenzoate) based chiral column. A lower limit of quantification of 0.1 ng/mL was reached for both analytes thanks to the high sensitivity and selectivity of tandem mass spectrometry, and the use of racemic medetomidine-d3 as internal standard prevented potential matrix effect. Linearity was satisfying (R2>0.99) over the range 0.1-25 ng/mL, as well as within- and between-session accuracy and precision, both always <15%. This method was also applied with success to a series of samples from a PK study aimed at comparing dex- and levomedetomidine behaviour after administration of the racemic mixture in dogs. The simple extraction procedure, which allows reduced solvent and time consumption without compromising analytical performances, make this technique a useful tool for this kind of applications even when small animals are involved, due to the small amount of sample required.