Chemistry of Natural Compounds, Vol. 49, No. 1, March, 2013 [Russian original No. 1, January–February, 2013]

HIGH-PERFORMANCE THIN LAYER CHROMATOGRAPHY DETERMINATION OF ASTAXANTHIN IN Euphausia superba OIL

Meng Huang and Daicheng Liu*

UDC 543.2

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 (3,3c-dihydroxy-E,Ec-carotene-4,4c-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 E-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 r 1.0% to 85 r 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 4qC [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 (Omax 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%.

Key Laboratory of Animal Resistance, College of Life Science, Shandong Normal University, 88 East Wenhua Road, 250014, Jinan, P. R. China, fax : 86 0531 86185360, e-mail: [email protected]. Published in Khimiya Prirodnykh Soedinenii, No. 1, January–February, 2013, pp. 128–129. Original article submitted September 23, 2011. 0009-3130/13/4901-0145 ”2013 Springer Science+Business Media New York

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TABLE 1. Linear Regression Data for Calibration Curves (n = 5) of Astaxanthin Parameter

Value

Parameter

Value

Linearity range,Pg·spot–1 r2 r SD

0.186 – 0.930 0.9990 r 0.0016

Slope r SD Intercept r SD

47.9693 r 5.80 1309.2935 r 10.50

The RSD of the intra-plates and inter-plates was 0.52 r 0.15 and 1.47 r 0.21, respectively, which indicates that the robustness of this method is acceptable. The LOD (limits of detection) and LOQ (limits of quantification) were respectively 0.046 and 1.000 Pg. Compared with HPLC, the HPTLC method exhibits several advantages in quality and quantity assay. For the sample preparation, complicated pretreatments such as filtration using microfiltration membranes was unnecessary, and the disposable HPTLC plates are not subject to spoilage and contamination like chromatographic columns. Moreover, HPTLC consumes much fewer chemical reagents and produces less environmental pollution. To sum up, this technique was found to be appropriate for routine astaxanthin determination with specificity, precision, accuracy, and robustness. Materials and Reagents. Standard astaxanthin (assigned purity 95%) was purchased from Dr. Ehrenstorfer Co., Germany. All other chemicals and reagents were of analytical grade and were purchased from Tianjin Fuyu Chemical Co., China. The Antarctic krill oil, extracted from Antarctic krill by n-hexane with an extraction ratio of 95 r 2.6%, was received from Shandong Keruier Biological Products Co., China. Instrumentation. A CAMAG (Muttenz, Switzerland) TLC scanner 3 spectrodensitometer with WINCATS 1.4.1 software was used for the densitometric analysis. The chromatographic plates were HPTLC plates [5 u 10 cm2] precoated with silica gel 60 F254 (Qingdao Haiyang Chemical Co., China), with a thickness of 200 Pm. A 10 PL microsyringe was used to spote the samples. Preparation of Sample and Standard Solution. The original sample was prepared as a mixture of Antarctic krill oil (5 g) and acetone (30 mL), which was centrifuged at 10,000 g for 3 min and then divided into a dark red precipitate and a red supernatant. This procedure was repeated at least three times until the supernatant was almost colorless; then all the supernatants were collected and the acetone was removed by a rotavapor at 25qC to give a residue (1.49 g), which was dissolved in 15 mL CH2Cl2 to serve as the original sample. The saponifed sample was prepared with 1 mL original sample and blended with 0.10 g KOH and 9 mL MeOH. The admixture was kept in a freezer at 4qC for 12 h. After that, 20 mL deionized water was added to the admixture. The aqueous layer was located above, the red organic phase was located below, and the supernatant was isolated by a separating funnel after shaking vigorously and after 1 hr cold storage. This step was repeated several times until the underlayer pH value was 7; then the organic solvent was dried by nitrogen and the residue was redissolved in 2 mL CH2Cl2. This is the saponified sample that can be directly analyzed by HPTLC. The standard solution was prepared with an accurately weighed 0.93 mg astaxanthin dissolved in 10 mL MeOH–CH2Cl2 (3:1, v/v) with a concentration of 0.093 mg·mL–1. All the procedures above were conducted in darkness and the samples and standard were stored at –20qC before the subsequent analysis. HPTLC Analysis of Astaxanthin. The densitometric evaluation was performed on silica gel 60 F254 HPTLC plates with n-hexane–acetone (7:2, v/v) as the mobile phase. Standard and samples were applied to the plates as dots with diameters less than 3 mm by a 10 PL microsyringe; the distance between tracks was 8 mm. For the original sample, the application volume of the sample and the standard were both 2 PL. For the saponified sample, the application volume of the sample was 1 PL, and that of the standard solution was 2, 4, 6, 8, and 10 PL. The development distances of the two samples were both 6 cm. The densitometric analysis was performed in the reflectance/absorbance mode at 476 nm; the slit width was 6.00 u 0.90 mm2, the scanning speed was 40 mm·s–1, and the data resolution was 100 Pm·step–1. D2 and W lamps were used. Each scanning was repeated five times. The linear regression calculation was via peak areas.

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