Determination of vitamins A, E and K1in milk by high-performance liquid chromatography with dual amperometric detection

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Abstract
A high-performance liquid chromatographic method with dual-electrochemical detection (HPLC–DEC) for the simultaneous determination of vitamins A, E and K1 is described. Separation was carried out using a C18 reversed-phase column and 2.5 mmol l–1 acetic acid–sodium acetate in methanol–water (99 + 1, v/v) as the mobile phase. The compounds eluted with good resolution in the above mentioned order within about 20 min and were quantified by dual-amperometric detection with a glassy carbon electrode at –1100 mV (E1) and + 700 mV (E2)(versus Ag/AgCl). This reductive–oxidative detection mode gave reproducible results and the detection limits were of the order of 0.06, 0.19 and 3.1 ng for vitamins A, E and K1, respectively. The HPLC–DEC method was successfully applied to the analysis of vitamins A, E and K1 in liquid cows' milk and infant-formula powdered-milk samples after applying alkaline hydrolysis of the fatty material and extraction of the vitamins with hexane for the analysis of vitamins A and E. In the case of vitamin K1 it was necessary to carry out enzymic hydrolysis, since this vitamin is degraded in basic medium, followed by cleaning with a Sep-Pak silica cartridge to isolate it from the other vitamins. Good recoveries (between 81 and 106%) were obtained.

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