Temperature-programmed packed capillary liquid chromatography separation with large volume on-column focusing of retinyl esters

A non-aqueous isocratic reversed-phase packed capillary high performance liquid chromatography method for the determination of retinyl esters, utilizing temperature programming and on-column focusing large volume injection, has been developed. The stationary phase material was C-30, and the mobile phase consisted of acetonitrile-dichloromethane (70:30, v/v). A three-step temperature program, starting at 10 degrees C for 10 min, then 1 degrees/min to 30 degrees C, and finally 2.5/min to 70 degrees C, was found most appropriate. Compared to an isothermal separation at 25 degrees C, this temperature program provided improved peak resolution, enhanced peak shapes of the last eluting compounds, and a reduction of the overall elution time. A mass limit of detection of 27 pg was found with respect to retinyl palmitate, using UV detection with an "U" shaped flow cell at 327 mm. This corresponds to a concentration limit of detection of 2.7 pg/mu L, when utilizing an injection volume of 10 mu L The concentration of retinyl palmitate in arctic seal If ver samples was estimated to be 62.6 mu g/g liver.