High-Pressure Liquid Chromatography of Oxo-Eicosanoids Derived from Arachidonic Acid

• Published in: Analytical biochemistry 1997-04, Vol.247 (1), p.17-24
• Main Authors: Powell, William S., Wang, Lei, Khanapure, Subhash P., Manna, Sukumar, Rokach, Joshua
• Format: Article
• Language: English
• Subjects: Animals; Arachidonic Acids - isolation & purification; Arachidonic Acids - metabolism; Chromatography, High Pressure Liquid - methods; Eicosanoids - chemistry; Eicosanoids - isolation & purification; Eicosanoids - metabolism; Hydroxyeicosatetraenoic Acids - isolation & purification; Hydroxyeicosatetraenoic Acids - metabolism; In Vitro Techniques; Leukotriene B4 - isolation & purification; Leukotriene B4 - metabolism; Molecular Structure; Neutrophils - metabolism; Prostaglandins - isolation & purification; Prostaglandins - metabolism; Rats
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1997.2024

Eicosanoids are a large group of biologically active metabolites of arachidonic acid and related C20fatty acids. Many of these compounds contain hydroxyl groups which can be converted to oxo groups by a variety of substrate-specific dehydrogenases. In many cases, this results in a reduction in potency, but in others, such as the oxidation of 5-hydroxy-eicosatetraenoic acid to its oxo metabolite 5-oxo-eicosatetraenoic acid, there is a dramatic increase in biological activity. Thus, it is often very important to analyze the relative amounts of oxo- and hydroxy-eicosanoids formed by various cells and tissues. The present study was designed to compare the chromatographic behavior of oxo-eicosanoids and their hydroxy counterparts in commonly used mobile phases for reversed-phase and normal-phase HPLC. We examined three groups of eicosanoids: prostaglandins, leukotriene B4and some of its metabolites, and monohydroxy-eicosanoids and their oxo metabolites. We found that in reversed-phase HPLC, the retention times of oxo-eicosanoids were longer than those of the corresponding hydroxy-eicosanoids in mobile phases containing acetonitrile as the major organic component, whereas the reverse was true for mobile phases containing methanol. Normal-phase HPLC using mobile phases containing hexane, isopropanol, and acetic acid gave excellent separation of oxo- and hydroxy-eicosanoids. Increasing the concentration of acetic acid in the mobile phase selectively reduced the retention times of oxo-eicosatetraenoic acids compared to monohydroxy-eicosatetraenoic acids, whereas the reverse was true for isopropanol. Differences in the chromatographic behavior of oxo- and hydroxy-eicosanoids can be useful clues in the structural characterization of these compounds, as illustrated by the chromatographic properties of a complex series of LTB4metabolites formed by rat neutrophils.