BIOSYNTHESIS OF ARACHIDONIC-ACID METABOLITES IN LIMULUS-POLYPHEMUS AMEBOCYTES - ANALYSIS BY LIQUID CHROMATOGRAPHY-ELECTROSPRAY IONIZATION MASS-SPECTROMETRY

Eicosanoid metabolites were generated by isolated granular amebocytes of the primitive arthropod, Limulus polyphemus, when stimulated by the calcium ionophore A23187 and/or exogenous arachidonic acid. The metab olites were isolated, identified, and the major metabolite was quantif ied using reverse-phase high pressure liquid chromatography (RP-HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS). Qualita tive examination revealed putative metabolites and the major product, 8-hydroxyeicosatetraenoic acid (8-HETE), which was quantified using st andard curves generated from extracted ion profiles of the molecular i on. Electrospray ionization of the HETEs in negative ion mode produces a base peak for all isomers which corresponded to the molecular ion [ (M-H)(-): m/z 319]. The molecular ion was accompanied by the neutral l oss of water and carbon dioxide [(M-H -H2O)(-): m/z 301; (M-H -H2O-CO2 )(-): m/z 257], as well as daughter ions which were dependent upon the position of hydroxy substitution. Standard curves were generated in f ull scan mode for standards ranging from 6.25 to 100 ng, whereas selec ted ion recording was used for the lower levels of 0.8 to 6.25 ng.