Separation of unlabeled metabolites of arachidonic acid from their deuterium- and tritium-labeled analogs by argentation high-pressure liquid chromatography.

Deuterium- and tritium-labeled analogs of high isotopic purity are required for the analysis of metabolites of arachidonic acid (20:4) by mass spectrometry and radioimmunoassay. We prepared a number of 2H-labeled standards by incubating [5,6,8,9,11,12,14,15-2H]20:4 with soybean lipoxygenase, polymorphonuclear leukocytes, or homogenates of rat spleen or bovine lung. The unlabeled and 2H-labeled forms of the following products were completely resolved by argentation high-pressure liquid chromatography: 20:4; 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5h-20:4); 11h-20:4; 12h-20:4; 15h-20:4; 12-hydroxy-5,8,10-heptadecatrienoic acid; leukotriene B4; prostaglandin D2 (PGD2); and PGF2 alpha. The corresponding forms of PGE2 and thromboxane B2 were also separated, but not quite as well as the above compounds. Analysis by mass spectrometry indicated that the amounts of unlabeled material in approx 1:1 mixtures of unlabeled and 2H-labeled products could be reduced to 0.1% or less after two steps of argentation high-performance liquid chromatography. Metabolites of 20:4 labeled with tritium were also separated from their unlabeled counterparts and had retention times longer than those of the corresponding 2H-labeled analogs.