Separation and detection method for astaxanthin in haematococcus pluvialis extract

Abstract

The invention relates to a separation and detection method for astaxanthin in a haematococcus pluvialis extract. The separation and detection method comprises the following steps of 1 extraction of carotenoid in the haematococcus pluvialis extract; 2 enzymolysis of the carotenoid; 3 astaxanthin separation and detection through a normal-phase high performance liquid analysis method, wherein the liquid-phase on-device conditions comprise the detection wavelength is 474 nm, a chromatographic column is Luna 3micro Silica(2), the chromatographic column temperature ranges from 20 DEG C to 25 DEG C, the flow speed is 1-1.2 ml/min, a flow phase is prepared from n-hexane and acetone according to the volume ratio of 75%:25%-90%:10%, and isocratic elution is conducted. According to the separation and detection method, the pretreatment efficiency is high, the intersolubility between the extracting reagent acetone and the astaxanthin is good, the enzymolysis time is shorter than the saponification time, and the efficiency is high; the high performance liquid chromatography on-device conditions are good; isocratic elution is achieved, a base line is easier to stabilize, the peak shape is good, the separation degree is high, and more isomers can be separated out; the peak flowing time is short, and the method is not prone to be influenced by outside light and heat and more suitable for large-scale detection.

Description

technical field [0001] The invention belongs to the field of chemical analysis, in particular to a separation and detection method for astaxanthin in Haematococcus pluvialis extract. Background technique [0002] Astaxanthin is an oxidized carotenoid, natural sources include Haematococcus pluvialis, yeast fungi, shrimp shells and crab shells, astaxanthin is also a ketone carotenoid, and has the same Long conjugated double bonds, with unsaturated ketone and hydroxyl groups at the end, hydroxyl and ketone groups constitute α-hydroxy ketones, and natural products mostly exist in the form of esters. [0003] Astaxanthin is beneficial in anti-oxidation, anti-inflammation, improving immunity, improving vision, lowering blood lipids and cholesterol, etc. It can effectively scavenge free radicals, inhibit the production of free radicals, and avoid cell damage. Natural astaxanthin is also a It is a super antioxidant with unique advantages in the carotenoid family. It quenches single...

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that the content of total carotenoids is measured in this wavelength range, resulting in high detection results

The disadvantage of this method is that compared with enzymatic hydrolysis, saponification has complicated operation and low conversion efficiency

The pretreatment of this method is: dissolving and extracting the sample with acetonitrile and n-hexane, using the reversed-phase system on the machine, making a working curve with the external standard method, and calculating the content of astaxanthin. The disadvantage of this method is: without enzymatic hydrolysis, As a result, the content of astaxanthin in the test sample is relatively low, and the machine conditions of the reversed-phase system are used, and the peak time is relatively long

[0012] Chen Jinming et al. used reversed-phase high-performance liquid chromatography to detect the astaxanthin content of Haematococcus pluvialis powder. The pretreatment of this method is: dissolving and extracting samples with methanol and dichloromethane, operating conditions on the reversed-phase system, and making work by external standard method Curve, and calculate the content of astaxanthin, the shortcomings of this method are: the content of astaxanthin in the test sample is low due to the lack of enzymatic hydrolysis, and the computer conditions of the reversed-phase system cannot be used to separate the different planes. conformation

[0013] In the Chinese patent CN 103630626 A, a C18 column is used, and the mobile phase is dichloromethane, methanol, acetonitrile, water and other reversed-phase machine conditions, and gradient elution requires high machine conditions, and the baseline is not easy to stabilize. Enzymatic hydrolysis, resulting in low calculated astaxanthin content, failing to separate the plane isomers

[0014] In the comprehensive analysis of the existing astaxanthin detection methods, the spectrophotometric method also includes other carotenoid substances in the maximum absorption wavelength range, which will cause high detection results. The thin-layer chromatography method is relatively cumbersome to operate. Most of the detection methods are based on high-performance liquid phase methods, and the extraction reagents are mostly organic reagents, such as acetonitrile, methanol, acetone, chloroform, etc. The pretreatment step needs to convert esterified astaxanthin into free astaxanthin. It is carried out by saponification, the saponification time is long, and the conversion efficiency is not high; some do not adopt the step of converting esterified astaxanthin into free astaxanthin, which will cause the detection content of astaxanthin to decrease

Most of the machine conditions use acetonitrile (water), methanol and other highly polar solutions as eluents. The miscibility between hydrophilic solvents and astaxanthin is not high, and the extraction rate is not high. Gradient elution is often used during elution, which has high requirements on the machine conditions, and the baseline is not easy to stabilize, which will have a certain impact on the quantification. The elution time of the target peak is usually 15-30 minutes, and the peak time is relatively short. Long, not conducive to batch detection content, the peak resolution is not high, can not separate the various plane isomers of astaxanthin, the plane isomers include: trans astaxanthin (trans astaxanthin), double cis Astaxanthin #1 (Di-cis astaxanthin #1), Di-cis astaxanthin #2 (Di-cis astaxanthin #2), 9-cis astaxanthin (9-cis astaxanthin), 13-cis astaxanthin 13-cis astaxanthin, 15-cis astaxanthin (15-cis astaxanthin)

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