Steric analysis of hydroxyeicosatetraenoates (HETEs) 3 and other fatty acid derivatives plays a vital part in the characterization of lipoxygenase, cyclooxygenase, and P450 reactions, and there is also often a need for preparative resolution of oxylipin and eicosanoid enantiomers. Assignment of the absolute configuration can be accomplished using spectroscopic techniques such as NMR (1) and circular dichroism (2). However, chiral column chromatography represents one of the most straightforward methods, and it can be performed with basic HPLC equipment. The first generation of chiral columns gave marginal resolution of HETE enantiomers (3), and for many compounds derivatization was required to achieve baseline resolution (e.g., ( 4)). More recent chiral columns, particularly of the Chiralcel class of derivatized cellulosebased supports, can achieve much improved resolution of individual hydroxy eicosanoids (5). A problem is that an individual column type is capable of separating a limited range of racemic eicosanoids. For example, the Chiralcel OD column, perhaps the most versatile of the Chiralcel class, will resolve 8-HETE and 12-HETE, whereas 15-HETE is not well resolved. Also, preparative separations require more than the minimal baseline resolution that is adequate for analytical work. Overall, a battery of chiral columns is required to allow the investigator to deal with a wide variety of fatty acid derivatives.
After we had been unable to resolve a tosylated hydroxystearate using chiral-phase HPLC (including testing methyl and pentafluorobenzyl ester derivatives (6, 7)), the technical service at Chiral Technologies (Exton, PA) screened the sample on their selection of columns and reported baseline resolution of the methyl ester using a Chiralpak AD column and a solvent of hexane/ethanol (100/2, by vol). Subsequently, we found that this separation was completely dependent on the use of ethanol as opposed to isopropanol as the alcohol modifier. Effects of the alcohol modifier on chiral separations have been reported for several classes of aromatics and pharmaceuticals (e.g., (8, 9)). Here we report that with the Chiralpak AD column there are striking improvements in separation with a wide variety of lipoxygenase products and other hydroxy derivatives on changing the alcohol modifier from isopropanol to ethanol or to methanol.
Racemic HETEs and HODEs were synthesized by autooxidation of arachidonic acid or linoleic acid, respectively, in the presence of β£-tocopherol (10). Follow-