How to Determine Astaxanthin in Health Foods by High Performance Liquid Chromatography

 Astaxanthin is a carotenoid, also known as astaxanthin, lobster shell pigment, astaxanthin exists in living organisms, in shrimp, crab, fish, algae, yeast and bird feathers in high content [1-3]. Astaxanthin is the highest level of carotenoid biosynthesis, astaxanthin has the strongest antioxidant properties, can avoid cell aging [4-6]. Astaxanthin has physiological activities such as anticancer, immunity enhancement, and prevention of cardiovascular diseases [7-9]. Astaxanthin was isolated from the shells of shrimps and crabs in the 1930s, but it was not until the 1980s that its physiological functions attracted widespread attention. Astaxanthin is also commonly used as a feed additive for aquaculture animals, with a dosage of up to 80 mg/kg [10]. 1999, astaxanthin was approved by the FDA as a dietary supplement [11]. The astaxanthin content of Rhodococcus aureus in algae is as high as 5% of the dry weight, which is known as the "concentrate" of natural astaxanthin, and was approved by the Ministry of Health of China in October 2010 as a new resource food [12-13].

 

Astaxanthin is a fat-soluble ketocarotenoid, insoluble in water, soluble in most organic solvents, unstable in acid, alkali, oxygen, high temperature and ultraviolet light conditions, and prone to oxidative degradation. Astaxanthin exists in a variety of forms in organisms, including geometric isomerism (all-trans, 9-cis, 13-cis, etc.), stereoisomerism (levulose, dextrose and endocyclohexane), and esterification (mono- and diester) forms [12]. The structural formula of all-trans astaxanthin is shown in Figure 1.

 

Currently, the commonly used analytical methods for astaxanthin content are high performance liquid chromatography (HPLC) and ultraviolet spectrophotometry [14-15], the samples were pre-treated with astaxanthin esters converted into free astaxanthin, and different methods were used for the determination of astaxanthin. The UV spectrophotometric method is simple, easy to operate, and can fulfill the needs of rapid detection, but there may be other carotenoids in the extract, which may interfere with the determination of astaxanthin content. High-performance liquid chromatography (HPLC) is more complicated, but the chromatographic column can effectively separate astaxanthin and other possible carotenoid impurities, as well as all-trans astaxanthin, 9-cis astaxanthin, 13-cis astaxanthin isomers, to eliminate the interference of impurities.

 

Although astaxanthin is widely used as a raw material for health food, the relevant national departments have not yet promulgated standard methods for the accurate determination of astaxanthin in health food. Currently, the standard methods can be referred to GB/T 31520-2015 "Determination of astaxanthin in red algae by liquid chromatography" [16] and T/CCCMHPIE 1.23-2016 "Plant extracts astaxanthin oil" [17], the pre-treatment of the former method is mainly through the conversion of free astaxanthin esters to free astaxanthin esters by saponification of sodium hydroxide (NaOH), which is the most important method for the determination of astaxanthin esters in health food products. The former method is mainly used to convert astaxanthin esters into the free state by saponification with sodium hydroxide (NaOH), which is a simple and low-cost reagent, but astaxanthin is sensitive to acid and alkali, and may damage astaxanthin; the latter method uses hydrolysis of astaxanthin esters by cholesterol esterase, which is more gentle and less likely to damage astaxanthin than saponification; however, the cholesterol esterase and the internal standard substances used in the experiments are expensive and not easy to be preserved, which increases the variable factors in the experiment and the cost of experiment. However, the cholesterol esterase and the internal standard used in the experiment are expensive and not easy to store, which increases the variable factors and experimental costs.

 

In this study, the method of GB/T 31520-2015 "Determination of astaxanthin in red algae by liquid chromatography"[16] was adopted to determine the astaxanthin content in different traits of health food, focusing on the degradation of astaxanthin by alkali content, in order to reduce the cost of the products and to provide reference for the daily testing of the inspection organization.

 

1 Materials and Methods

1.1 Materials and reagents

Methanol, methyl tert-butyl ether (chromatographic purity, the United States Fisher Company); dichloromethane (chromatographic purity, Tianjin Saifu Century Science and Technology Development Co. Ltd.); 2,6-di-tert-butylated hydroxytoluene (BHT) (chemically pure); all-trans astaxanthin control (97% purity, Toronto Research Chemicals, Canada); 9-cis-astaxanthin control, 13-cis-astaxanthin control (≥90% purity, Sigma-Aldrich Corporation, U.S.A.).

The health food samples used in the experiment were samples of different brands and dosage forms, among which 1-7 were soft capsules and 8-12 were tablets, which were purchased and collected from the market. Sample 1 was taken as a representative of the oil sample, and sample 8 was taken as a representative of the powder sample for analytical and methodological studies.

 

1.2 Instruments and equipment

Waters 2695 HPLC (with variable wavelength UV detector and Empower2 chromatography workstation, Waters Corporation, USA); electronic analytical balance [Sartorius Scientific Instruments (Beijing) Co. Ltd.); Vortex-Genie vortex mixer (Scientific industries, USA); MD200-2 nitrogen blower (Hangzhou Aosheng Instrument Co., Ltd.); ultrapure water machine (Shanghai Lefeng Bio-technology Co., Ltd.).

 

1.3 Experimental Methods

1.3.1 Sample pre-treatment

Weigh the sample accurately (accurate to 0.1 mg) in a volumetric flask, add dichloromethane-methanol mixture (1:3, V:V) solution, ultrasonic 5 min to make the sample fully dissolved and dispersed, cooled to room temperature, and then add dichloromethane-methanol (1:3, V:V) solution was determined to the gradient, and then mixed well. Dilute according to different samples, and reserve. Then, measure 5.00 mL of the backup solution into a 10 mL cuvette, add 0.70 mL of 0.1 mol/L sodium hydroxide-methanol solution, vortex to mix, seal with nitrogen, and react in a refrigerator at 4 ℃ for 12-15 h. Add 0.40 mL of 2% phosphoric acid-methanol solution to neutralize the residual alkali in the reaction solution, mix well, and then volume up to 5 mL under nitrogen purge, mix well, and use a 0.45 μm filter. The solution was then filtered through a 0.45 μm membrane to obtain the HPLC test solution.

 

1.3.2 Chromatographic conditions

Chromatographic column: C30 column (4.6 mm×250 mm, 5 μm), mobile phase: methanol + methyl tert-butyl ether + 1% phosphoric acid gradient elution, the elution ratio is shown in Table 1. The flow rate was 1.0 mL/min, the detection wavelength was 474 nm, and the temperature of the column was 25 ℃.

 

1.3.3 Linearity and minimum detection limits

All-trans astaxanthin was used as the basis for quantification of all isomers. Weigh the appropriate amount of all-trans astaxanthin control, dissolve in acetone to make a standard stock solution. Dilute different concentrations of astaxanthin standard solution with acetone before measurement.

 

1.3.4 Determination under chromatographic conditions and preparation of a standard curve.

A sample without astaxanthin was selected as the blank matrix, and the sample was processed according to the method of 1.3.1, and the basic noise value was obtained after 10 injections. On this basis, different concentrations of all-trans astaxanthin standard solutions were added to the samples, and the limits of detection (LOD) and the limits of quantification (LOQ) of astaxanthin were determined at the signal-to-noise ratios of S/N=3 and S/N=10, respectively.

 

1.3.5 Effect of alkali content on the stability of astaxanthin

Samples 1 and 8 were weighed and treated according to the method of 1.3.1, but different volumes of NaOH solution were added, and the astaxanthin content was determined by HPLC. The effect of alkali on the stability of astaxanthin was investigated by observing the change of sample content.

 

1.3.6 Precision

Six parallel (n=6) samples of Sample 1 and Sample 8 were weighed on the same day and subjected to the method precision test. The samples were processed according to the method 1.3.1 and the results were analyzed by HPLC.

 

1.3.7 Recovery rate

For each group of recovery experiments, 6 samples 1 and 8 were weighed into volumetric flasks, 3 of which were added into the standard stock solution of all-trans astaxanthin according to 0.5, 1 and 1.5 times of the content of the samples, and the other 3 were used as the blank control of each level, and the results were analyzed by HPLC according to the operation of sample processing in accordance with 1.3.1. The results were analyzed by HPLC. The experiment was repeated 3 times, and the average recoveries of the three levels were calculated (n=3).

 

1.3.8 Daytime Repeatability Experiments

Three samples (n=3) of Sample 1 and Sample 8 were weighed in parallel on each day of the 3-day period for inter-day reproducibility, and the samples were processed according to the method of 1.3.1, and the results were analyzed by HPLC.

 

2 Results and analysis

2.1 Chromatographic separation and determination of astaxanthin

According to the HPLC chromatographic conditions, all-trans astaxanthin, 9-cis-astaxanthin, and 13-cis-astaxanthin were well separated, and the chromatographic peaks of the three astaxanthin isomer standards are shown in Figure 2. The chromatographic peaks of astaxanthin esters and the three isomers of astaxanthin after extraction and saponification of oil sample No.1 are shown in Figs. 3 and 4, respectively. It can be seen from the chromatograms that on the one hand, astaxanthin esters can be saponified into astaxanthin, and on the other hand, the astaxanthin isomers can be well separated, and there is no interference of impurity peaks within the detection wavelengths. Among them, all-trans astaxanthin accounted for the major part of all astaxanthin isomers and was well separated, so it was chosen as the main basis for quantitative calculation in this study.

 

The linearity of all-trans astaxanthin was tested, and the results showed that the concentration of all-trans astaxanthin was in the range of 0.25~12.70 μg/mL, and the linearity was good, and the regression equation was Y=103023.6X+2015, r=1.0000.

The limit of detection (LOD) was 1.09 ng/mL at S/N=3 and the limit of quantification (LOQ) was 3.64 ng/mL at S/N=10 for all-reversed astaxanthin.

 

2.2 Effect of alkalinity on the stability of astaxanthin

The free astaxanthin after saponification is unstable under alkaline conditions, so the amount of alkali used in the saponification process is a key factor in the pretreatment process. Therefore, the amount of alkali used in the saponification process is a key factor in the pretreatment process. If the amount of alkali is too little, the saponification will be incomplete, and if it is too much, the free astaxanthin will be destroyed, therefore, the amount of sodium hydroxide was further tested in this study. The results of this study are shown in Table 2. When the NaOH concentration was lower than 21.9 mmol/L, the astaxanthin content was stable, and when the NaOH concentration was higher than 35.94 mmol/L, the astaxanthin content was stable. When NaOH concentration was lower than 21.9 mmol/L, the content of astaxanthin was stable, when NaOH concentration was higher than 35.94 mmol/L, the content of astaxanthin decreased, and when NaOH concentration reached 73.7 mmol/L, the content of astaxanthin was lowest.

A higher NaOH final concentration of 21.9 mmol/L was used for the recovery of all-trans astaxanthin, and the recovery reached 95.7% (see Table 3), indicating that the astaxanthin content was stable when the NaOH concentration was lower than 21.9 mmol/L. The recoveries of all-trans astaxanthin were as follows.

 

2.3 Method Precision Results

The precision of the method was tested with the oil sample No.1 and the powder sample No.8 at the same time, and the results are shown in Table 4, which indicate that the precision of this method for the determination of astaxanthin is good and the method is reliable.

 

2.4 Results of Method Recovery Determination

The recoveries of all-trans astaxanthin were determined by three levels of spiked recoveries of all-trans astaxanthin in samples 1 and 8, and the results are shown in Table 5. The recoveries of all-trans astaxanthin were stable without any loss of content.

 

2.5 Day-to-day reproducibility results

The results are shown in Table 6. The measured levels of astaxanthin were stable and the precision was good in different days, indicating that the method is reproducible from day to day.

 

2.6 Determination of astaxanthin content in samples

According to the above pre-treatment and chromatographic conditions, the astaxanthin content in 12 health food products was determined by saponification with NaOH concentration of 12.3 mmol/L. The samples 1-7 were in soft capsule form, and the samples 8-12 were in solid dosage form. The results are shown in Table 7, which indicate that the precision of astaxanthin determination in different samples is relatively high and the values are reliable, and the method can be applied to the determination of astaxanthin in health foods.

 

3 CONCLUSIONS

There is no standard method for the determination of astaxanthin in health foods in China. In this paper, a method for the determination of astaxanthin in health foods was developed, which consisted of extraction with dichloromethane-methanol mixture, saponification with NaOH methanol, and separation by high performance liquid chromatography (HPLC). The separation was performed on a C30 column (4.6 mm×250 mm, 5 μm) at 25 ℃ with a gradient elution of methanol+methyl tert-butyl ether+1% phosphoric acid in the mobile phase at a flow rate of 1.0 mL/min. The detection wavelength was 474 nm. A methodological study was carried out for the determination of astaxanthin in two kinds of dosage forms of oil and solid, which showed that the recoveries of astaxanthin in the samples were high, the precision was good, and the measurement results were accurate. The method can be applied to the determination of astaxanthin in health food products.

 

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