Forensic Equine Drug Testing, Bureau Veritas Laboratories, 8577 Commerce Court, Burnaby, BC, V5A 4N5, Canada.
Anal Bioanal Chem. 2021 Mar;413(8):2147-2161. doi: 10.1007/s00216-021-03182-1. Epub 2021 Jan 31.
Resolution of cathinone enantiomers in equine anti-doping analysis is becoming more important to distinguish the inadvertent ingestion of plant-based products from those of deliberate administration of designer synthetic analogs. With this in mind, a rapid and sensitive method was developed and validated for the detection, resolution and quantitative determination of cathinone enantiomers in horse blood plasma and urine. The analytes were recovered from the blood plasma and urine matrices by using a liquid-liquid extraction after adjusting the pH to 9. The recovered analytes were derivatized with N-(2,4-dinitro-5-fluorophenyl)-L-valinamide, a chiral derivatizing agent analogous to Marfey's reagent. The resulting diastereoisomers were baseline resolved under a reversed-phase liquid chromatographic condition. Derivatization of the analytes not only allowed the separation of the enantiomers using cost-effective traditional liquid chromatography conditions and reversed-phase columns but also increased the sensitivity, at least to an order of magnitude, when tandem mass spectrometry is used for the detection. A limit of detection of 0.05 ng/mL was achieved for cathinone enantiomers for both matrices. Acceptable intraday and interday precision and accuracy along with satisfactory dilution accuracy and precision were observed during the method validation. The method suitability was tested using the post administration urine samples collected after single doses of cathinone and ephedrine as single-enantiomeric form and methcathinone as racemic form. Finally, a proof of concept of the isomeric ratio in urine samples to distinguish the presence of cathinone as a result of accidental ingestion of plant-based product from that of an illicit use of a designer product is demonstrated. To the best of our knowledge, this is the first such work where cathinone enantiomers were resolved and quantified in horse blood plasma and urine at sub nanogram levels.
手性拆分和定量检测马血和尿液中的卡西酮对马属动物兴奋剂分析变得越来越重要，这有助于区分误食植物源产品与故意使用合成类似物的情况。考虑到这一点，建立并验证了一种灵敏、快速的方法，用于检测、拆分和定量检测马血和尿液中的卡西酮对映异构体。通过将 pH 值调至 9 后用液液萃取从血和尿基质中提取分析物。然后用 N-（2,4-二硝基-5-氟苯甲酰基）-L-缬氨酸酰胺（类似于马菲试剂的手性衍生化试剂）对回收的分析物进行衍生化。在反相液相色谱条件下，衍生化的非对映异构体可以实现基线分离。分析物的衍生化不仅允许使用经济高效的传统液相色谱条件和反相柱分离对映异构体，而且在串联质谱检测时，灵敏度至少提高了一个数量级。在两种基质中，卡西酮对映异构体的检测限达到 0.05 ng/mL。在方法验证过程中，观察到可接受的日内和日间精密度和准确度以及令人满意的稀释精密度和准确度。使用卡西酮和麻黄碱作为单一对映异构体和作为外消旋体的甲卡西酮单次给药后收集的尿液样本对方法适用性进行了测试。最后，证明了在尿液样本中检测到的对映体比值可用于区分误食植物源产品还是非法使用设计药物导致的卡西酮存在。据我们所知，这是首次在亚纳克级水平在手性拆分和定量检测马血和尿液中的卡西酮对映异构体。
