Bardhi Anisa, Zaghini Anna, Levionnois Olivier, Barbarossa Andrea
Department of Veterinary Medical Sciences, Alma Mater Studiorum - University of Bologna, Bologna, Italy.
Unit of Anaesthesiology and Pain therapy, Department for Clinical Veterinary Science, Faculty Vetsuisse, University of Bern, Bern, Switzerland.
Drug Test Anal. 2021 Jul;13(7):1249-1255. doi: 10.1002/dta.3015. Epub 2021 Feb 23.
In the present study, a rapid, sensitive and high-throughput liquid chromatography-tandem mass spectrometry (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 LC. 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 MS/MS and the use of racemic medetomidine-d3 as internal standard prevented potential matrix effect. Linearity was satisfying (R > 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 pharmacokinetic (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, makes this technique a useful tool for this kind of applications even when small animals are involved, due to the small amount of sample required.
在本研究中，开发并验证了一种用于测定犬血浆中美托咪定对映体的快速、灵敏且高通量的液相色谱-串联质谱（LC-MS/MS）方法。手性化合物的分离和单独定量在液相色谱中可能是一项棘手的任务。当目标分析物质量相对较小时，情况尤其如此，美托咪定就是这样一种强效且高度特异性的α2肾上腺素能受体激动剂，广泛应用于人类和兽医学。所提出的方法基于用乙酸乙酯进行快速液-液萃取，并在进样前进行过滤。优化后的流动相组成能够在短色谱运行时间内，使用基于三（4-甲基苯甲酸）纤维素的手性柱完美分离美托咪定的两种对映体。由于MS/MS的高灵敏度和选择性，两种分析物的定量下限均达到0.1 ng/mL，并且使用外消旋美托咪定-d3作为内标可防止潜在的基质效应。在0.1-25 ng/mL范围内线性良好（R＞0.99），批内和批间的准确度及精密度均始终＜15%。该方法还成功应用于一项药代动力学（PK）研究的一系列样品，该研究旨在比较犬静脉注射外消旋混合物后右美托咪定和左美托咪定的行为。这种简单的萃取程序能够减少溶剂和时间消耗，同时不影响分析性能，即使涉及小动物，由于所需样品量少，该技术也是此类应用的有用工具。
