High-performance thin layer chromatography determination of astaxanthin in Euphausia superba oil

Abstract

The Antarctic krill (Euphausia superba) is a kind of marine zooplankton belonging among Crustacea [1]. It is the principal prey of many marine predators by reason of its gigantic population [2]. The study of Antarctic krill has become a hot research field in the past few decades. It is not only the key organism in the southern ocean but also the biggest fishery resource in the world. With its high content of astaxanthin as a valuable natural product [2, 3], it urgently awaits development and utilization. The ketocarotenoid astaxanthin (3,3 -dihydroxy, -carotene-4,4 -dione) and astaxanthin esters are the principal components of Antarctic krill pigment [4], and astaxanthin is one of most important carotenoids by virtue of its biologic functions as a vitamin A precursor and as a much more efficient antioxidant than -carotene and vitamin E [5]. It is also a natural coloring (in fish muscles, bird feathers, yolks, etc.), supports the immune system, has an anti-tumor effect, and protects from ultraviolet radiation [6–8]. Overall, astaxanthin can play an important role in healthcare and cosmetic manufacture, with enormous commercial and industrial prospects. As a general rule, high-performance liquid chromatography (HPLC) is preferred in astaxanthin assay due to its wide use all around the world [9], whereas the HPTLC technique with high sensitivity and resolution can also function excellently in the separation and purification of bioactive compounds from natural materials. In short, we aim to establish and validate an HPTLC method that is adapted to astaxanthin determination in Antarctic krill oil and that can be applied to other materials that contain astaxanthin. The astaxanthin in Antarctic krill is mostly in the form of astaxanthin esters for its molecular stabilization [10]. Some are diesters and others are monoesters, attributed to the two hydroxyls that can combine with different fatty acids at both ends of the astaxanthin molecule [11]. While the concentration of free astaxanthin was amall, all of it and other impurities are fat soluble and can be dissolved in low-polarity reagents; thus, n-hexane–acetone (7:2, v/v) was chosen to be the mobile phase after repeated attempts, combined with the high-performance silica gel 60 F254, which can accomplish the HPTLC separation within 10 min. The Rf value of the free astaxanthin was 0.32. It was necessary to apply the saponification procedure to release the astaxanthin combined with fatty acids because the concentration of free astaxanthin was from 5 1.0% to 85 5.0%, so that the qualification and quantification of astaxanthin can be carried out more successfully, as well as the further purification, since there was no standard for astaxanthin esters. KOH was essential in saponification, but it can result in significant degradation of astaxanthin, especially when the reaction temperature was high. There was almost no degradation of astaxanthin at 4 C [12]; therefore it was selected as the saponification temperature, and the maximum concentration of free astaxanthin can reach 89.7466%. The linear regression equation of astaxanthin after saponification is Y = 1309.2935 + 47.9693X (where Y is the response and X is the mass of astaxanthin). The correlation coefficient was found to be 0.9990, the RSD (relative standard deviation) was 2.56%, and the astaxanthin in the saponification sample was 0.7045 mg·mL–1; more data are shown in Table 1. The three main components in the original sample were separated without interference from other impurities, and the spectral evaluation of the samples showed the same maximum absorption ( max 476 nm) as the standard; therefore, the specificity of this method is verified. The RSD of intraday precision was 1.68% and the value of the interday precision was 5.07%. Both values were determined when the plates were stored in the dark. The accuracy was clarified as percent recovery, and the average recovery of astaxanthin was 98.53%.