向世勰+徐颖+朱晶晶+王智民+张东+陈两绵+冯伟红

[摘要]该文旨在建立一种超高效液相色谱-串联质谱（UPLC-MS/MS）同步检测去卵巢大鼠血浆中22种内源性大麻素类物质的新方法。首先血浆样品经固相小柱（SPE柱）萃取，然后利用UPLC-MS/MS进行检测。采用ACQUITY UPLC BEH C18色谱柱，以0.1%乙酸溶液为流动相A，乙腈-异丙醇（9∶1）溶液为流动相B，进行梯度洗脱，采用正离子多反应离子监测模式，以氘代试剂作为对照品的替代物计算回收率，同步定量22种内源性大麻素。其中，22种内源性大麻素类物质的线性相关系数均大于0.99，定量限（LOQs）为0.089 6～1.965 2 nmol·L^-1；5个氘代替代物的相对回收率为11.40%～129.9%；重复性考察结果显示，RSD均小于8.0%。所建立的方法，可以用于去卵巢大鼠血浆样品中PGF2α EA，AEA等多种内源性大麻素类物质的定性与定量分析，验证了该方法的可行性及实用性。总之，该方法灵敏度高、重复性好、实用性强，可以用于大鼠血浆样品脂质代谢组学研究。

[关键词]高效液相色谱-串联质谱； 内源性大麻素； 氘代替代物

[Abstract]A new method based on ultraperformance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS） was developed for the simultaneous determination of 22 endocannabinoids（eCBs） and relevant compounds in ovariectomized rat plasma. After being extracted by solid-phase column（SPE）， the plasma samples were detected by using UPLC-MS/MS. Analysis was carried out with ACQUITY UPLC BEH C18 column. The mobile phase was 0.1% acetic acid solution（A）-acetonitrile and isopropanol（9∶1， B） for the gradient elution. In the positive ion multiple reaction monitoring（MRM） mode， deuterated reagents were taken as standard alternatives to calculate recoveries and simultaneously quantify 22 endocannabinoids. The established method provided a good linearity for the 22 eCBs， and their linearly dependent coefficients were all higher than 0.99. The limits of quantitation（LOQs） ranged from 0.089 6 to 1.965 2 nmol·L^-1. Relative recoveries of 5 deuterated surrogates ranged between 11.40% and 129.9%. The repeatability study results showed that RSD was all less than 8.0%. The established method could be used to analyze PGF2a EA， AEA and other endogenous cannabinoids in plasma samples of ovariectomized rats. In summary， this method was proved to boast a high sensitivity， repeatability and practicability， and thus could be used in rat plasma lipid metabolomics study.

[Key words]UPLS-MS/MS； endocannabinoids； deuterated surrogate

doi：10.4268/cjcmm20162122

內源性大麻素（endocannabinoids，eCBs）作为内源性脂质介质，能结合和激活大麻素受体（CB1与CB2），在各种不同的生理和病理过程中具有重要作用。与神经保护、记忆、肿瘤、肥胖、免疫功能调节和心血管系统疾病等多种病理、药理作用均有关^[1]。eCBs主要是N-花生四烯酸氨基乙醇（anandamine）和2-花生四烯酸甘油（2-AG）2种（化学结构见图1），还包括长链饱和或不饱和脂肪酰胺、酯和醚等不同种类，如：O-花生四烯酸乙醇胺（O-arachidonoyl ethanolamine，O-AEA）^[2]，2-花生四烯酸甘油酯（2-arachidonoyl glycerol ether，2-AGE）^[3]和N-花生四烯酸多巴胺（N-arachidonoyl dopamide，NADA）^[4]等。其中AEA和2-AG的生物活性最强，且两……