Qi Ming, Morena Maria, Vecchiarelli Haley A, Hill Matthew N, Schriemer David C
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.
Department of Cell Biology & Anatomy and Psychiatry, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.
Rapid Commun Mass Spectrom. 2015 Oct 30;29(20):1889-97. doi: 10.1002/rcm.7277.
Methods for quantifying anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) are needed to support programs investigating molecular mechanisms of the central nervous system. Existing methods, while useful, are not well adapted to efficiently process large numbers of very small tissue samples. A unique challenge involves the disparity in endogenous levels of AEA (pmol/g tissue) and 2-AG (nmol/g tissue).
A simplified one-step solvent extraction procedure was developed for recovering endocannabinoids from rat brain tissues, and combined with capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS). Various multiple reaction monitoring (MRM)-based methods were evaluated for limit of detection (LOD) and robustness.
The optimized simultaneous quantitation method achieves an LOQ of 50 amol for AEA and 25 fmol for 2-AG, both with a linearity over 3 orders of magnitude, and elution times under 3 min. Accuracy, expressed as relative error (RE), is less than 12% for AEA and less than 6% for 2-AG. Precision, expressed as relative standard deviation (RSD), is less than 6% for AEA and less than 3% for 2-AG. Sample handling routines are sufficiently robust to support the automated analysis of thousands of samples from a range of tissue types.
The microscale method is a sensitive, economical and robust alternative to the larger scale LC/MS methods currently implemented for quantitation of AEA and 2-AG.
需要定量花生四烯酸乙醇胺（AEA）和2-花生四烯酸甘油酯（2-AG）的方法来支持研究中枢神经系统分子机制的项目。现有方法虽然有用，但不太适合高效处理大量非常小的组织样本。一个独特的挑战涉及AEA（皮摩尔/克组织）和2-AG（纳摩尔/克组织）内源性水平的差异。
开发了一种简化的一步溶剂萃取程序，用于从大鼠脑组织中回收内源性大麻素，并与毛细管液相色谱/串联质谱（LC/MS/MS）相结合。评估了各种基于多反应监测（MRM）的方法的检测限（LOD）和稳健性。
优化后的同时定量方法对AEA的定量下限（LOQ）为50阿托摩尔，对2-AG为25飞摩尔，两者的线性范围均超过3个数量级，洗脱时间在3分钟以内。以相对误差（RE）表示的准确度，AEA小于12%，2-AG小于6%。以相对标准偏差（RSD）表示的精密度，AEA小于6%，2-AG小于3%。样本处理程序足够稳健，能够支持对来自一系列组织类型的数千个样本进行自动化分析。
这种微量方法是目前用于定量AEA和2-AG的较大规模LC/MS方法的一种灵敏、经济且稳健的替代方法。
