Simultaneous determination of fatty acids and related N-Acylethanolamines in rat plasma, rat tissue, and human plasma using UPLC-MS/MS

• A LC-MS/MS method for determination of fatty acids and related N -Acylethanolamines in rat plasma and liver. • First assay for endogenous fatty acids and N -Acylethanolamines in the liver. • Two extraction methods were used and compared. • Extensive validation using human plasma to ensure applicability. A sensitive and specific method for the simultaneous determination of palmitoyl ethanolamide (PEA), oleoyl ethanolamide (OEA), linoleoyl ethanolamide (LEA), 1-palmitoyl glycerol (1-PG), 1-oleoyl glycerol (1-OG), 1-linoleoyl glycerol (1-LG), palmitic acid (PA), oleic Acid (OA), and linoleic acid (LA) in rat plasma and liver by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed. Multiple-reaction mode (MRM) of a triple quadrupole mass spectrometer was used for detection. A reverse phase C8 column was used for chromatographic separation, and an 11.5-minute gradient elution time was used. Deuterated internal standards included 2-AG-d 5 , OEA-d 4 , LEA-d 4 , and OA-d 9 . In terms of linearity, accuracy, repeatability, intermediate precision, recovery, matrix effect, and stability, this newly designed method was approved. These nine lipids were successfully determined using this method in two sample groups: plasma from individuals with non-alcoholic fatty liver diseases (NAFLD) (n = 40) and healthy individuals (n = 40), as well as plasma and liver samples from rats with NAFLD (n = 15) and healthy ones (n = 11).