Reference interval values of four plasmatic endocannabinoids measured by isotopic dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)
Flaminia Fanelli, Gaia Cuomo, Valentina Diana Di Lallo, Ilaria Belluomo, Margherita Baccini, Alessandra Gambineri, Valentina Vicennati, Renato Pasquali & Uberto Pagotto
The endocannabinoid system is involved in modulating many metabolic and behavioural processes. Due to the instable nature of endocannabinoids in biological fluids and to the poor sensitivity of traditional analytical technologies, little is known about physiological circulating levels of these lipid mediators. We developed and validated an ID-LC-MS/MS method for the simultaneous measurement of four endocannabinoids: anandamide (AEA), 2-arachidonoylglycerol (2-AG), palmitoyl-ethanolamide (PEA) and oleoyl-ethanolamide (OEA). Endocannabinoids were extracted by protein precipitation and SPE purification from 1 ml of plasma added to phenylmethanesulfonyl-fluoride 10 μM immediately after blood withdrawal. Eluate was injected into an HPLC system, and separated on a C18 column. Along 32 min isocratic run, complete resolution of 2-AG and its inactive isomer 1-AG was achieved. Analytes underwent atmospheric pressure chemical ionization before spectrometric revelation in multiple reaction monitoring mode by a 4000Q-Trap triple quadrupole (Applied Biosystems). Quantitative and qualitative transitions were selected for each analyte in order to ensure specificity of measurement. The calibration curve showed linearity across four orders of magnitude. Limit of quantification was 0.224, 0.153, 0.260 and 0.157 pmol/ml for AEA, 2AG, PEA and OEA, respectively. Imprecision was below 8.5 and 14.1% in intra-assay and inter-assay, respectively. Acceptable accuracy between 85 and 115% was obtained. After given informed consent, 116 male and 61 female healthy subjects were recruited. The study was approved by the local ethics committee. Central 95th percentile reference intervals estimated in 1869 y.o. male and female groups were: AEA: 1.2283.725 and 0.9982.949 pmol/ml; 2-AG: 0.5502.994 and 0.4662.389 pmol/ml; PEA: 10.6527.08 and 8.5523.18 pmol/ml and OEA: 3.099.81 and 2.449.33 pmol/ml. We have therefore generated for the first time the reference intervals of the haematic circulating levels of four endocannabinoids in a large number of subjects in view of further approaches to various diseases in which endocannabinoids are known to play a role in their pathogenesis.