Determination Of Endocannabinoids And Its Application

The previous method for the determination of endocannabinoids in hair and saliva showed some limitations as follows.Firstly,purification in alkaline environment heightens the isomerization rate of 2-AG to 1-AG or 3-AG and enhances the conversion of o-arachidonyl ethanolamine(o-AEA)to AEA.Secondly,external standard method used previously for the quantification of AEA and 2-AG was easily affected by matrix effect,resulting in the heavy deviation from the real contents.Thirdly,the previous method ultilized larger sample amount and complex and time-consuming pre-treatment process and showed low sensitivity.Recent study shows that purification of hair endocannabinoids under a neutral incubation solution can effectively reduce the 2-AG isomerization rate and conversion of o-AEA into AEA.Therefore,based on an internal standard method with simple sample pretreatment,lower detection limit and smaller sample amount,the present study aims to develop a suitable assay for simultaneous determination of AEA and 2-AG in hair and saliva and for exploreing correlation of endocannabinoid contents with PTSD,obesity.Firstly,AEA and 2-AG in 20-mg and 15-mg hair were extracted with methanol and were purified with solid phase extraction.They were ionized with atmosphere pressure chemical ionization in positive mode and indentified with multiple reaction monitoring in positive mode and quantified with internal standard method.The assay methods on both 20-mg and 15-mg hair showed that the recoveries ranged between 100.0 and 109.0%and the intra-day and inter-day coefficients were less than 10%.Their accuracy and reproducibility were in accordance with the requirements of the standard for quantitative analysis.Secondly,AEA and 2-AG in saliva were extracted with liquid-liquid extraction with ethyl acetate.They were ionized with atmosphere pressure chemical ionization in positive mode and indentified with multiple reaction monitoring in positive mode and quantified with internal standard method.The assay method showed the intra-day and inter-day CVs at less than 10.0%and recovery ranging from 95.9 to 109.0%.Thirdly,population analysis revealed that there were no significant differences in hair AEA and 2-AG between PTSD patients and non-PTSD 4.5,5.5 and 6.5 years after Wenchuan earthquake(ps>0.05).Obese individuals had significantly higher levels of salivary and hair AEA and 2-AG than the non-obese ones(ps<0.01).In addition,hair 2-AG level has moderate intraindividual relative stability across a two-year period,but hair AEA level showed weak intraindividual stability.