Mass quantification of anandamide and related N-acylethanolamides in plasma for clinical investigations

The endocannabinoid (EC) anandamide and the related N-acylethanolamides (NAEs) are a family of lipid mediators involved in a wide range of biological effects. It is well known that EC plays a role in the regulation of energy homeostasis and lipid and glucose metabolism, in the regulation of the immune system [1] and in various aspects of human reproduction [2]. Furthermore the endocannabinoid system is believed to regulate energy balance and behaviour among which food intake, fear, and anxiety [3]. It became clear that an altered qualitative or quantitative production of these substances might contribute to the outcome of some pathologies demonstrating the need of a sensitive analytical method for the accurate identification and quantification of these molecules in biological fluids of healthy subjects and patients. We describe the set up of a sensitive solid phase extraction and isotope-dilution UPLC-MS/MS method for the simultaneous quantification of seven EC and NAEs in human plasma and serum for the purpose of research and clinical application. We examined effects of different protein precipitation protocols and liquid-liquid extraction and solid phase extraction on the recovery of ECs and NAEs and matrix effects. Protein precipitation with cooled acetone and extraction with acetonitrile (1% formic acid) using OASIS HLB cartridge gave better results. Separation was performed on an Waters Acquity UPLC hsst3 column using a gradient elution coupled with tripe-TOF MS in 9 minutes. Calibration curves range from 0.125 ng/ml to 200 ng/ml for each NAEs. The developed method is suitable for routine measurement of ECs and NAEs analysed in human plasma and serum samples (500 µl) in clinical settings as shown by quality control samples. [1] Nagarkatti P, Pandey R, Rieder SA, Hegde VL, (2009) Future Med Chem 1:1333-1349. [2] Taylor AH, Amoako AA, Bambang K, Karasu T, Gebeh A, Lam PM, Marzcylo TH, Konje JC (2010) Clin Chim Acta 411:921-930. [3] Di Marzo V, Matias I (2005) Nature Neurosci 8:585-589.