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Evaluation of several thin-layer chromatographic procedures for the separation of various labeled arachidonic acid metabolites (including 6-keto-prostaglandin F1 alpha) produced in the biological system is described. Manual scanning and autoradiography of the plates developed by two-dimensional thin-layer chromatography was also done for locating the radioactivities due to arachidonic acid metabolites other than thromboxane B2 and the classical prostaglandins (PGF2 alpha, PGE2, and PGD2).

A major limitation of high-performance liquid chromatographic techniques for measuring biologically active eicosanoids has been the inadequate sensitivity of most on-line detection systems. In addition, the availability of a technique suitable for measuring small quantities of non-esterified fatty acids (NEFAs) in plasma would allow longitudinal studies of plasma levels of these lipids in small animals. To improve the sensitivity of detection, the compounds with acyl groups containing carboxylic acids were derivatized with the highly fluorescent compound, 4-bromomethyl-7-acetoxycoumarin. All classes of NEFA and arachidonic acid metabolites, including the cyclooxygenase and lipoxygenase products, and hydroxy acid compounds could be derivatized with this reagent. The derivatized metabolites were separated with a reversed-phase high-performance liquid chromatographic system using a radial compression column and a gradient elution technique. Reproducible measurements of plasma NEFAs from as little as 5 microliters of plasma, and femtomolar concentrations of eicosanoids, could be detected using an on-line fluorescent spectrometer. This improvement in sensitivity should permit the quantification of all eicosanoids, including the leukotrienes, in biologic fluids and the longitudinal measurement of changes in plasma NEFA levels in small animals.

Gas chromatographic analysis revealed that polyunsaturated fatty acids such as arachidonic acid and total tissue free fatty acids isolated from an aminopropyl bonded-phase column yield a two- to three-fold higher recovery of arachidonic acid as compared to those isolated from thin-layer chromatographic plates. This method was further improved by packing the aminopropyl bonded phase in glass columns, since the glass column significantly eliminated the other contaminants (from polypropylene columns) coeluting with fatty acids in both a neutral lipid thin-layer chromatographic system and on a 5% DEGS-PS column of gas chromatographic analysis. In aminopropyl bonded-phase columns, the standard triglycerides and phospholipids were completely separated from free fatty acids as judged by gas chromatographic analysis. These results warrant the use of an aminopropyl bonded-phase column for the isolation of free fatty acids to obtain better recovery of polyunsaturated fatty acids.