Quantification of β-carotene, retinol, retinyl acetate and retinyl palmitate in enriched fruit juices using dispersive liquid–liquid microextraction coupled to liquid chromatography with fluorescence detection and atmospheric pressure chemical ionization-mass spectrometry

Abstract

A detailed optimization of dispersive liquid–liquid microextraction (DLLME) was carried out for developing liquid chromatographic (HPLC) techniques, using both fluorescence and atmospheric pressure chemical ionization mass spectrometric (APCI-MS) detection, for the simultaneous analysis of preforms of vitamin A: retinol (R), retinyl acetate (RA), retinyl palmitate (RP) and β-carotene (β-C). The HPLC analyses were carried out using a mobile phase composed of methanol and water, with gradient elution. The APCI-MS and fluorescence spectra permitted the correct identification of compounds in the analyzed samples. Parameters affecting DLLME were optimized using 2mL of methanol (disperser solvent) containing 150μL carbon tetrachloride (extraction solvent). The precision ranged from 6% to 8% (RSD) and the limits of detection were between 0.03 and 1.4ngmL−1, depending on the compound. The enrichment factor values were in the 21–44 range. Juice samples were analyzed without saponification and no matrix effect was found when using fluorescence detection, so calibration was possible with aqueous standards. However, a matrix effect appeared with APCI-MS, in which case it was necessary to apply matrix-matched calibration. There was great variability in the forms of vitamin A present in the juices, the most abundant ester being retinyl acetate (0.04 to 3.4μgmL−1), followed by the amount of retinol (0.01 to 0.16μgmL−1), while retinyl palmitate was not detected, except in the milk-containing juice, in which RP was the main form. The representative carotenoid β-carotene was present in the orange, peach, mango and multifruit juices in high amounts. The method was validated using two certified reference materials.