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

Abstract

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 B 4; prostaglandin D 2 (PGD 2); and PGF 2α. The corresponding forms of PGE 2 and thromboxane B 2 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.