The biosynthesis of prostaglandins and leukotrienes proceeds through the formation of chemically reactive intermediates leukotriene A 4 (LTA 4 ) and prostaglandin H 2 (PGH 2 ) which in aqueous solutions have chemical half-lives of 3 s and 3 min, respectively. Prostacyclin (PGI 2 ) is another chemically reactive prostanoid that has a chemical half-life of 3-4 min. The recent development of reversed phase HPLC stationary phases that are stable to elevated pH (pH 10 -12) without significant column damage has permitted direct analysis of these acid-sensitive eicosanoids. Using electrospray ionization, molecular anions [M Ϫ H] Ϫ of these compounds were observed at m/z 317 for LTA 4 and m/z 351 for both PGH 2 and PGI 2 . The mechanism of formation of ions derived from collisional activation of LTA 4 was studied using stable isotope labeled and chemical analogs of LTA 4 and found to involve formation of highly conjugated anions at m/z 261 and 163. The collisional activation of the molecular anion of PGH 2 yielded a product ion spectrum identical to that observed for the isomeric prostaglandins PGE 2 and PGD 2 . However, it was possible to baseline separate PGE 2 , PDG 2 , and PGH 2 by reversed phase HPLC using basic HPLC mobile phases. The collisional activation of PGI 2 led to a family of abundant ions including highly conjugated carbon-centered and oxygen-centered radical species (m/z 245 and 205) likely derived from the attack of the carboxylate anion on the cyclic enolether of PGI 2 as well as the most abundant product ion (m/z 215) which formed following loss of neutral hexanal and water. The structures of these product ions were consistent with high resolution measurements measured in a quadrupole time-of-flight mass

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