ABSTRACT: A method for the detection and quantification of hydroxyl and epoxy arachidonic acid (AA) metabolites in human plasma was developed using liquid-liquid extraction, phospholipid saponification followed by derivatization of the acid moiety and liquid chromatographic tandem mass spectrometric detection. Derivatization with a pyridinium analog allowed for detection in the positive ion mode, greatly improving sensitivity and the stability of the more labile AA metabolites. The entire method utilizes a 96-well plate format, increasing sample throughput, and was optimized to measure 5-, 8-, 9-, 11-, 12-, 15-, 19-, and 20- hydroxyeicosatetraenoic acid (HETE), 5,6-, 8,9-, 11,12-, and 14,15- dihydroxyeicosatrienoic acid (DHET), and the regio- and cis-/ trans- isomers of 5,6-, 8,9-, 11,12-, and 14,15
