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 metabolites were isolated, identified, and the major metabolite was quantified using reverse-phase high pressure liquid chromatography (RP-HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS). Qualitative examination revealed putative metabolites and the major product, 8-hydroxyeicosatetraenoic acid (8-HETE), which was quantified using standard curves generated from extracted ion profiles of the molecular ion. 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 loss 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 full scan mode for standards ranging from 6.25 to 100 ng, whereas selected ion recording was used for the lower levels of 0.8 to 6.25 ng.