Fanti Federico, Vincenti Flaminia, Imparato Giulia, Montesano Camilla, Scipioni Lucia, Ciaramellano Francesca, Tortolani Daniel, Oddi Sergio, Maccarrone Mauro, Compagnone Dario, Sergi Manuel
Department of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, 64100, Teramo, Italy.
Department of Chemistry, Sapienza University of Rome, 00185, Rome, Italy.
Talanta. 2023 May 15;257:124392. doi: 10.1016/j.talanta.2023.124392. Epub 2023 Feb 24.
The present study encompasses the development of a fast and reliable analytical method to quantify the main endocannabinoids and some of their conjugated congeners, particularly N-arachidonoyl amino acids, in brain tissue. Samples were homogenized and a micro solid phase extraction (μSPE) procedure was developed for brain homogenate clean-up. Miniaturized SPE was selected as it allowed to work with reduced sample amounts, while maintaining high sensitivity; this last feature was mandatory due to the low concentration of endocannabinoids in biological matrices that makes their determination a challenging analytical task. UHPLC-MS/MS was used for the analysis as it provided a great sensitivity, especially for conjugated forms that were detected by negative ionization. Polarity switching was applied during the run; low limits of quantification were between 0.003 ng g and 0.5 ng g. This method provided also low matrix effect (lower than 30%) and good extraction recoveries in the brain. To the best of our knowledge, this is the first time that μSPE is applied on this matrix for this class of compounds. The method was validated according to international guidelines, and then tested on real cerebellum samples from mice, which were sub-chronically treated with URB597, a well-known inhibitor of the fatty acid amide hydrolase.
本研究包括开发一种快速可靠的分析方法，用于定量脑组织中主要的内源性大麻素及其一些共轭类似物，特别是N-花生四烯酰基氨基酸。将样品匀浆，并开发了一种用于脑组织匀浆净化的微固相萃取（μSPE）程序。选择小型化固相萃取是因为它允许使用减少的样品量进行工作，同时保持高灵敏度；由于生物基质中内源性大麻素的浓度较低，使得其测定成为一项具有挑战性的分析任务，因此最后一个特性是必不可少的。采用超高效液相色谱-串联质谱（UHPLC-MS/MS）进行分析，因为它具有很高的灵敏度，特别是对于通过负离子化检测到的共轭形式。在运行过程中应用了极性切换；定量下限在0.003 ng/g至0.5 ng/g之间。该方法在脑组织中也具有较低的基质效应（低于30%）和良好的提取回收率。据我们所知，这是首次将μSPE应用于该类化合物的这种基质。该方法根据国际指南进行了验证，然后在来自小鼠的真实小脑样品上进行了测试，这些小鼠用脂肪酸酰胺水解酶的著名抑制剂URB597进行了亚慢性治疗。
