A UHPLC–MS/MS method for determination of N-acylethanolamines in cerebrospinal fluid

N-acylethanolamines (NAEs) and primary fatty amides (PFAMs) are of a great interest due to the range of physiological effects they exhibit, potentially serving as neuromodulators. However, they are present at nano and picomolar concentrations in human cerebrospinal fluid (CSF) samples, posing challenges for detection and measurement using conventional Ultra-high performance liquid chromatography systems coupled to tandem mass spectrometry (UHPLC-MS). UHPLC-MS was used in dynamic multiple reaction monitoring (dMRM) mode. Seven deuterated NAEs internal standards were used to develop the method. Six solvent combinations were tested for extraction efficiency, accuracy, precision, matrix effect, linearity, limits of detection. Lastly the method was applied to CSF from healthy individuals (n=33) to estimate their natural range of concentrations. Extraction with acetonitrile/acetone showed the highest efficiency and recovery. The presented method was able to measure the following 17 NAEs and PFAMs in human CSF: linoleoyl ethanolamide, heptadecanoyl ethanolamide, stearoyl ethanolamide, palmitoyl ethanolamide, dihomolinolenoyl ethanolamide, eicosatrienoic acid ethanolamide, behenamide, octadecanamide, lauramide, tetradecanamide, erucamide, linoleamide, palmitamide, myristic monoethanolamide, pentadecanoyl ethanolamide, oleamide and palmitoleoyl ethanolamide. In healthy individuals the concentrations ranged three-fold from pg/mL to mg/mL. Further studies could apply this method to clinical CSF samples. ### Competing Interest Statement The authors have declared no competing interest. * (ACN) : Acetonitrile (CSF) : cerebrospinal fluid (dMRM) : dynamic multiple reaction monitoring (ESI) : electrospray ionization (FA) : formic acid (FDA) : food and drug administration (GC-MS) : gas chromatography mass spectrometry (IPA) : isopropanol (LOD) : limit of detection (LOQ) : limit of quantification (MeOH) : methanol (NAE) : n-acylethanolamine (PFAM) : primary fatty amide (RSD) : relative standard deviation (ISTD) : internal standard (QqQ) : triple quadrupole (UHPLC-MS) : ultra-high performance liquid chromatography mass spectrometry (AEA) : Anandamide (BEH) : Behenamide (DLEA) : Dihomolinolenoyl ethanolamide (DHEA) : Docosahexaenoyl ethanolamide (EPEA) : Eicosapentaenoyl ethanolamide (ERU) : Erucamide (HPEA) : Heptadecanoyl Ethanolamide (LAU) : Lauramide (LIN) : Linoleamide (LEA) : Linoleoyl ethanolamide (MEA) : Myristic monoethanolamide (OCT) : Octadecanamide (OLE) : Oleamide (OEA) : Oleoyl ethanolamide (PAL) : Palmitamide (POEA) : Palmitoleoyl Ethanolamide (PEA) : Palmitoyl ethanolamide (PTEA) : Pentadecanoyl Ethanolamide (SEA) : Stearoyl ethanolamide (TET) : Tetradecanamide