Simultaneous determination of six vitamin A substances by multi-wavelength HPLC and application to different marine shellfishes

Abstract

An HPLC-DAD method was developed for the simultaneous analytical determination of six vitamin A substances (retinol, retinyl acetate, retinyl palmitate, retinoic acid, retinal and β-carotene) and applied to the analysis of vitamin A substances in five marine shellfishes. The chromatographic column was a YMC-β carotene C30 column (250 × 4.6 mm, 3 µm), and the mobile phases were selected as A (methanol), B (ultrapure water) and C (methyl tert-butyl ether). The initial ratio of the mobile phase was A:B:C= 90:1:9, which was maintained for 8 min, while the ratio of the three was changed to A:B:C= 35:0:65 and continued for 8 min, and finally returned to the initial ratio within 0.1 min. Ultrasound-assisted direct extraction was investigated, and the optimal extraction conditions were determined by exploring different sample-to-liquid ratios, extractants, extraction times and extraction temperatures. The method was validated and the established assay showed good linearity in the range of 0.5–50 μg/mL with good precision and accuracy. The experimental results showed that retinol, retinyl palmitate and β-carotene were present in the shellfish and there were seasonal variations of vitamin A substances in different marine economic shellfish.