Determination of Carotenoid Pigments, Retinol, and α-Tocopherol in Feeds, Tissues, and Blood Serum by Normal Phase High Performance Liquid Chromatography

A normal phase HPLC method employing a hexane:ethyl acetate mobile phase solvent gradient for the simultaneous separation and quantification of carotenoid pigments and vitamins A and E has been developed. The gradient consists of a 10-min linear change from 1 to 2 mL/min and 95:5 to 70:30 hexane:ethyl acetate mobile phase, which is followed by 7 min of isocratic mobile phase of 70:30 hexane:ethyl acetate at 2 mL/min. The stationary phase consisted of a 4-mu silica column. A photodiode array detector generated simultaneous chromatogram data at three wavelengths (294, 325, and 445 nm) with broad spectrum data at peaks. The method produced good separation of trans-lutein and zeaxanthin.