The Reversed-Phase HPLC Behavior of Retinyl Esters

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Abstract
Fifteen retinyl esters were synthesized, including a number of esters containing naturally occurring polyunsaturated fatty acids. The esters were purified by HPLC and their chromatographic behavior on reversed phase HPLC was studied. A plot of Log k' versus % water in the eluting solvent for each ester was very close to linear in the range from 1% to 3.5% water in methanol. Retinyl esters containing a monounsaturated fatty acyl group had an effective carbon number (ECN) about 1.85 less than the corresponding ester with a saturated acyl group with the same chain length. Thus the monoenes were relatively difficult to separate from esters with saturated acyl groups with two fewer carbons. Retinyl esters with polyene acyl groups showed decreased ECNs of: 3.32–3.47 for dienes; 4.64–4.84 for trienes; and 5.92 for the one tetraene tested. From these data it is expected that even chain length trienes would chromatograph very close to the corresponding saturated odd chain ester with five fewer carbons. The usefulness of ECN is discussed. Retinyl arachidonate (R20:4) was not separated from both and retinyl palmitoleate (R16:1) and retinyl myristate (R14:0), using any one condition. Under the chromatography conditions used, the complex lipids eluted soon after the solvent front. Thus the Folch extract of liver or plasma could be used directly for analysis of retinyl esters monitored at 325 nm without preliminary separation from other lipids.