Implementation and validation of a UHPLC-MS/MS method for quantification of the endocannabinoids AEA and 2-AG in cerebral interstitial fluid and plasma

Endogenous endocannabinoids such as N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) are involved in the patho-biochemistry of several neurological diseases and have been associated with moodenhancing phenomena. Although they have been intensively studied in recent years, accurate and reliable quantification of these analytes in cerebral interstitial fluid (cISF) to elucidate their neuro-modulatory role is still challenging. Moreover, there is a need for an analytical method that can analyze plasma in addition to cISF and is thus able to address research questions in both preclinical and clinical studies. Aim was to implement a method for simultaneous quantification of AEA and 2-AG in cISF and plasma, to validate it by taking the requirements of the U.S. Food and Drug Administration into account, and to test its usability in three different case studies. A UHPLC-MS/MS method with preceding liquid-liquid extraction to determine AEA and 2-AG in cISF and plasma was successfully implemented, and the parameters selectivity, specificity, linearity, accuracy, precision, sensitivity, carry-over and stability met the validation criteria. The usability of the analytical method was demonstrated in an in vitro study with cerebral open flow microperfusion (cOFM), an in vivo cOFM study in rats, and a clinical study in human plasma. The developed method allowed quantification of AEA and 2-AG in the biologically relevant concentration ranges in cISF and plasma. The availability of a reliable, complementary, timeresolved dataset of endocannabinoid concentrations in both matrices can be of considerable future importance for the evaluation of drug efficacy.