CN112083057A

CN112083057A - Astaxanthin detection method

Info

Publication number: CN112083057A
Application number: CN202010921219.0A
Authority: CN
Inventors: 甄德帅; 蔡青云; 刘禹森; 刘宏昌
Original assignee: Guizhou Beibeisheng Biotechnology Co ltd
Current assignee: Guizhou Beibeisheng Biotechnology Co ltd
Priority date: 2020-09-04
Filing date: 2020-09-04
Publication date: 2020-12-15
Anticipated expiration: 2040-09-04
Also published as: CN112083057B

Abstract

The invention relates to a method for detecting astaxanthin, which comprises the steps of preparing a standard working solution; weighing a sample to be detected, dissolving the sample in acetone, adding 0.08-0.12g of BHT, transferring the sample to a 50mL volumetric flask after the BHT is dissolved, carrying out constant volume with acetone, and shaking up to obtain a sample solution A; uniformly mixing 2mL of the sample solution A and 3mL of cholesterol esterase solution, and reacting in a thermostat at 37 ℃ for 45-55min to obtain a mixed solution; adding 1g of sodium sulfate decahydrate and 2mL of petroleum ether into the mixed solution, performing ultrasonic dispersion for 40-80s, and centrifuging for layering; then, taking the petroleum ether layer on the upper layer, blowing the petroleum ether layer by argon, adding 3mL of acetone, ultrasonically dissolving for 40-80s, and filtering by using an organic solvent microporous filter membrane with the aperture of 0.22 mu m to obtain a sample solution B; obtaining a blank sample solution A and a blank sample solution B by the same method; and then carrying out MALDI-TOF MS determination, and calculating to obtain the astaxanthin content in the sample by combining the obtained MALDI-TOF MS spectrogram. The detection method of the invention has high sensitivity and good reproducibility.

Description

Astaxanthin detection method

Technical Field

The invention relates to a method for detecting astaxanthin, belonging to the field of quantitative detection.

Background

Astaxanthin is a ketone or carotenoid, has a pink color, is fat-soluble, insoluble in water, and soluble in organic solvents. Also known as astaxanthin. A red carotenoid, whose chemical name is3, 3 ' -dihydroxy-4, 4 ' -diketo-beta, beta ' -carotene, is found in the bodies of aquatic animals such as the shells of river crayfish, oysters and salmon. It can be combined with protein in vivo to form cyan and blue color, especially in feathers of aquatic animals such as shrimp, crab, fish and bird, and has color development effect.

At present, the content of astaxanthin in a sample is detected by a common chromatography, and Chinese patent specification CN107703232A discloses a method for detecting the content of astaxanthin, which comprises the following steps: a) drawing a standard curve; b) extracting; c) saponification; d) detecting the content of astaxanthin by chromatography; and e) calculating the astaxanthin content in the sample to be detected according to the result of the step d), however, the chromatography has the defects of complex operation, long time consumption and the like, and the identity information of the astaxanthin is difficult to accurately determine.

Disclosure of Invention

In view of the shortcomings of the prior art, the present invention aims to provide a novel astaxanthin detection method to obtain stable test results.

The technical scheme of the invention is as follows:

s1, preparing a standard working solution: preparing various standard working solutions with different astaxanthin concentrations by using acetone as a solvent and an astaxanthin standard substance as a solute;

preparing a sample solution B: weighing 0.1-40mg of sample to be detected, heating to 50-52 ℃ in an oil bath, dissolving in acetone, adding 0.08-0.12g of BHT, transferring to a volumetric flask after the BHT is dissolved, fixing the volume with acetone, and shaking up to obtain a sample solution A;

uniformly mixing 2mL of the sample solution A and 3mL of cholesterol esterase solution, and reacting in a thermostat at 37 ℃ for 45-55min to obtain a mixed solution; then 0.8-1.2g of sodium sulfate decahydrate and 1.5-2.5mL of petroleum ether are added into the mixed solution, and after ultrasonic dispersion is carried out for 40-80s, centrifugal layering is carried out; then, taking the petroleum ether layer on the upper layer, blowing the petroleum ether layer by argon, adding 3mL of acetone, ultrasonically dissolving for 40-80s, and filtering by using an organic solvent microporous filter membrane to obtain a sample solution B;

preparing a blank sample solution B: dissolving 0.08-0.12g BHT in acetone, transferring to a volumetric flask, performing constant volume with acetone, and shaking up to obtain a blank sample solution A;

uniformly mixing 2mL of the blank sample solution A and 3mL of cholesterol esterase solution, and reacting in a thermostat at 37 ℃ for 45-55min to obtain a mixed solution; then 0.8-1.2g of sodium sulfate decahydrate and 1.5-2.5mL of petroleum ether are added into the mixed solution, and after ultrasonic dispersion is carried out for 40-80s, centrifugal layering is carried out; then, taking the petroleum ether layer on the upper layer, blowing the petroleum ether layer by argon, adding 3mL of acetone, ultrasonically dissolving for 40-80s, and filtering by using an organic solvent microporous filter membrane to obtain a blank sample solution B;

wherein the cholesterol esterase solution is prepared from cholesterol esterase and 0.05mol/L Tris-HCL buffer solution, the pH value of the Tris-HCL buffer solution is 7.0, and the concentration of the cholesterol esterase solution is 4U/mL;

moving equal volumes of HCCA matrix solution and standard working solution to a stainless steel sample target plate for mixing, and performing MALDI-TOF MS determination after natural drying to respectively obtain MALDI-TOF MS spectrograms of the standard working solutions with different astaxanthin concentrations;

moving equal volumes of HCCA matrix solution and sample solution B to a stainless steel sample target plate for mixing, and after natural drying, performing MALDI-TOF MS determination to obtain MALDI-TOF MS spectrogram of the sample solution B;

moving equal volumes of HCCA matrix solution and blank sample solution B to a stainless steel sample target plate for mixing, and after natural drying, carrying out MALDI-TOF MS determination to obtain MALDI-TOF MS spectrogram of the blank sample solution B;

and S2, combining the MALDI-TOF MS spectrogram, and calculating by an external standard method to obtain the astaxanthin content in the sample.

Further, the method for detecting the astaxanthin comprises the following steps:

s1, preparing a standard working solution: preparing at least 2 standard working solutions with different astaxanthin concentrations by using acetone as a solvent and an astaxanthin standard substance as a solute;

preparing a sample solution B: weighing 0.1-40mg of sample to be detected, heating to 50-52 ℃ in an oil bath, dissolving in acetone, adding 0.08-0.12g of BHT, transferring to a 50mL volumetric flask after the BHT is dissolved, fixing the volume with acetone, and shaking up to obtain a sample solution A;

uniformly mixing 2mL of the sample solution A and 3mL of cholesterol esterase solution, and reacting in a thermostat at 37 ℃ for 45-55min to obtain a mixed solution; adding 1g of sodium sulfate decahydrate and 2mL of petroleum ether into the mixed solution, performing ultrasonic dispersion for 40-80s, and centrifuging for layering; then, taking the petroleum ether layer on the upper layer, blowing the petroleum ether layer by argon, adding 3mL of acetone, ultrasonically dissolving for 40-80s, and filtering by using an organic solvent microporous filter membrane with the aperture of 0.22 mu m to obtain a sample solution B;

preparing a blank sample solution B: dissolving 0.08-0.12g BHT in acetone, transferring to a 50mL volumetric flask, performing constant volume with acetone, and shaking up to obtain a blank sample solution A;

uniformly mixing 2mL of the blank sample solution A and 3mL of cholesterol esterase solution, and reacting in a thermostat at 37 ℃ for 45-55min to obtain a mixed solution; adding 1g of sodium sulfate decahydrate and 2mL of petroleum ether into the mixed solution, performing ultrasonic dispersion for 40-80s, and centrifuging for layering; then, taking the petroleum ether layer on the upper layer, blowing the petroleum ether layer by argon, adding 3mL of acetone, ultrasonically dissolving for 40-80s, and filtering by using an organic solvent microporous filter membrane with the aperture of 0.22 mu m to obtain a blank sample solution B;

wherein the cholesterol esterase solution is prepared by cholesterol esterase and 0.05mol/LTris-HCL buffer solution, the pH value of the Tris-HCL buffer solution is 7.0, and the concentration of the cholesterol esterase solution is 4U/mL (U: enzyme activity unit);

moving 0.5 mu L HCCA matrix solution and 0.5 mu L standard working solution to a stainless steel sample target plate for mixing, naturally drying, and then performing MALDI-TOF MS determination to respectively obtain MALDI-TOF MS spectrograms of the standard working solutions with different astaxanthin concentrations;

moving 0.5 mu L of LHCCA matrix solution and 0.5 mu L of sample solution B to a stainless steel sample target plate for mixing, and after natural drying, carrying out MALDI-TOF MS determination to obtain a MALDI-TOF MS spectrogram of the sample solution B;

moving 0.5 mu of LHCCA matrix solution and 0.5 mu of blank sample solution B to a stainless steel sample target plate for mixing, and performing MALDI-TOF MS determination after natural drying to obtain an MALDI-TOF MS spectrogram of the blank sample solution B;

Optionally, the concentration of astaxanthin in the standard working solution is 0.05-0.5 mg/mL.

Further, in S1, about 3 to 7mg of astaxanthin standard was put into a 10mL centrifuge tube and dissolved with acetone to obtain a standard working solution.

In this application, acetone is used as the solvent for astaxanthin throughout the process. The astaxanthin extraction sample contained free astaxanthin, astaxanthin monoester and astaxanthin diester. BHT can be used as antioxidant to prevent oxidative decomposition of free astaxanthin. The sterol esterase can carry out enzymolysis on the astaxanthin monoester and the astaxanthin diester to convert the astaxanthin monoester and the astaxanthin diester into free astaxanthin. Petroleum ether was used to extract free astaxanthin. Anhydrous sodium sulfate was used for water removal. The HCCA can react with the astaxanthin in the sample solution to form cocrystal, which is beneficial to improving the detection sensitivity.

Further, in S1, the volumetric flask is a brown volumetric flask, which can prevent the visible light decomposition of the free astaxanthin and ensure the accuracy of the test result.

Further, in S1, centrifugation was performed at 5000-.

Further, the MALDI-TOF MS mass spectrometry test conditions were as follows: the point target mode is a dry point method, the frequency is 2000Hz, the shooting times are 500 times, the laser wavelength is 355nm, the laser intensity is 90 percent, and the mode is RP 700-.

Further, the method also comprises the step of measuring the content of free astaxanthin in the sample solution A, namely moving 0.5 mu L of LHCCA matrix solution and 0.5 mu L of sample solution A to mix with a stainless steel sample target plate, and performing MALDI-TOF MS measurement after natural drying to obtain a MALDI-TOF MS spectrogram of the sample solution A; and then combining a related MALDI-TOF MS spectrogram, and calculating by an external standard method to obtain the astaxanthin content in the sample.

Further, the HCCA matrix solution is formed by mixing HCCA and TA30, wherein the concentration of HCCA is 10 mg/mL; TA30 was prepared by mixing acetonitrile and aqueous TFA at a 30:70 volume ratio, with the concentration of aqueous TFA being 0.1 wt%.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely applied to the fields of biomolecule detection, bacterium identification and the like due to the advantages of high sensitivity, high flux, high analysis speed and the like. However, since MALDI-TOF MS is relatively expensive in the market at present, the usage rate has a certain limit. The applicant researches and discovers that astaxanthin and an organic matrix (HCCA, DHB, SA, HPA and the like) generate cocrystallization during the preparation process of a MALDI-TOF MS sample, wherein discrete hot spots are generated when astaxanthin and the matrix such as DHB, SA, HPA and the like are cocrystallized, and the phenomenon reduces the laser ionization efficiency and is not favorable for the reproducibility of detection results. The applicant found through repeated experiments that the HCCA matrix and astaxanthin have good co-crystallization ability and stable test results can be obtained. Specifically, as can be seen from fig. 5 and 6, the comparison of the HCCA matrix with DHB, SA and HPA matrices shows that the HCCA matrix can be better and uniformly dispersed on the target plate over the entire target, increasing the sufficient contact area with the astaxanthin molecule, forming a large co-crystal area, and facilitating the improvement of the reproducibility of the detection result.

The detection method has the advantages of small detection sample consumption (micro-upgrading), short detection time (femtosecond level) and the like. The method is particularly suitable for determining and confirming the anthocyanin in the astaxanthin oil obtained by extracting and refining the shrimp shells serving as the raw materials.

Drawings

FIG. 1 is a MALDI-TOF MS spectrum of free astaxanthin in sample solution A in example 1.

FIG. 2 is a MALDI-TOF MS spectrum of total astaxanthin in sample solution B in example 1.

FIG. 3(a) is a MALDI-TOF MS spectrum of a white sample solution in example 1, and FIG. 3(b) is an enlarged view of a part of the m/z region in FIG. 3 (a).

FIG. 4 is a MALDI-TOF MS spectrum of 0.5mg/mL standard working solution.

FIG. 5 is an optical picture of a cocrystal of HCCA and astaxanthin, wherein the marked positions of the white circles are random points which have been detected and the unmarked positions are regions to be detected.

FIG. 6 is an optical photograph of four matrices, HCCA, DHB, SA and HPA.

FIG. 7 is a MALDI-TOF MS spectrum of 0.05mg/mL standard working solution.

Detailed Description

The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. For convenience of description, the words "upper", "lower", "left" and "right" in the following description are used only to indicate the correspondence between the upper, lower, left and right directions of the drawings themselves, and do not limit the structure.

Example 1

In this example, all reagents were analytically pure except for the special indication, and water was ultrapure water (18.2 M.OMEGA.cm).

Acetone: and (4) carrying out chromatographic purification.

Anhydrous sodium sulfate: analytically pure, CAS: 7757-82-6.

Sodium sulfate decahydrate.

Petroleum ether.

Tris (hydroxymethyl) aminomethane: and (4) chemical purity.

Hydrochloric acid.

2, 6-di-tert-butyl-p-cresol (BHT): and (4) chemical purity.

Cholesterol esterase (CAS: 9026-00-0), storage temperature-20 ℃, Aladdin or other equivalents.

Preparation of 0.05mol/l tris-HCl (PH 7.0) buffer: trishydroxymethyl aminomethane (0.606 g) was weighed out accurately, dissolved in 75mL of deionized water, adjusted to pH 7.0 with 1mol/L hydrochloric acid, and diluted to 100mL with water.

Preparation of cholesterol esterase solution: accurately weighing a proper amount of cholesterol esterase into a 25mL volumetric flask, dissolving the cholesterol esterase in 0.05mol/L Tris-HCL (PH 7.0) buffer solution to prepare an enzyme solution with the concentration of 4 enzyme activity units/mL, and preparing the enzyme solution at present.

An astaxanthin standard substance: astaxanthin, CAS:472-61-7, purity: HPLC 98% or more, was stored at 2-8 ℃ in the dark.

Acetonitrile: acetonitrile, chromatographic purity, CAS: 75-05-8.

Trifluoroacetic acid: TFA, chromatographically pure, CAS: 76-05-1.

TA 30: 30/70(v/v) Acetonitriles (TFA 0.1wt% aqueous solution).

HCCA matrix: saturated HCCA solution (10mg/mL) in TA 30.

Instruments and devices:

an analytical balance.

An ultrasonic machine.

A centrifuge: at 6000 rpm.

Microporous filter membrane: 0.22 μm, organic phase.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS): a 355nm laser source; and an RP mode.

A constant temperature box at 37 ℃.

Liquid-transfering gun

Preparing a sample to be detected: enzymolysis of shrimp shell → solid-liquid separation of enzymolysis liquid, liquid removal and retention → supercritical carbon dioxide extraction or solvent extraction and purification → sample.

Generally, the samples should be stored in dry, ventilated and clean places, and stored at low temperature and in the dark.

The method for measuring astaxanthin comprises the following steps:

1.1 preparation of Standard working solutions

Accurately weighing 5mg astaxanthin standard substance into a 10mL centrifuge tube by using an analytical balance, dissolving with acetone, and shaking up to obtain 0.5mg/mL standard solution. It is used as it is. Other low concentration standard solutions are obtained by diluting based on 5mg/mL standard solutions, for example, 0.1mL of 0.5mg/mL standard solution is added with acetone to make the volume to 1mL, to obtain 0.05mg/mL standard solution, and the other concentration configurations are analogized in sequence.

2.1 measurement of free astaxanthin in sample solutions

Weighing 30mg of a sample (sample) to be tested in a centrifuge tube, placing the centrifuge tube in an oil bath at 50-52 ℃, preserving heat for 30min, dissolving the sample (sample) with acetone, adding 0.1g of BHT, transferring the dissolved sample to a 50mL brown volumetric flask, fixing the volume with acetone, and shaking up to obtain a sample solution A for later use.

2.2 determination of Total astaxanthin in sample solutions

Accurately sucking 2mL of the sample solution A and 3mL of the cholesterol esterase solution by using a pipette gun respectively, adding the sample solution A and the cholesterol esterase solution into a 10mL centrifuge tube, and inverting and uniformly mixing. The centrifuge tubes were placed in a 37 ℃ incubator for 50min, where they were mixed back-to-back every 10 min. After the reaction, 1g of sodium sulfate decahydrate and 2mL of petroleum ether were added to the centrifuge tube in this order and dispersed by ultrasound for 1min, and then placed in a centrifuge for 2min at 6000 rpm. Subsequently, the petroleum ether layer (located at the upper layer) in the centrifuge tube was pipetted into a new 10mL centrifuge tube (to which 1g anhydrous sodium sulfate was added in advance) taking care not to pipette the middle emulsion layer. Blowing dry petroleum ether by using argon gas at room temperature, adding 3mL of acetone, ultrasonically dissolving for 1min, filtering by using a 0.22 mu m organic phase microporous filter membrane, and obtaining a filtered solution which is a sample solution B and is used for determining the total astaxanthin in the sample.

2.3 matrix-assisted laser Desorption ionization Mass Spectrometry (MALDI-TOF MS) determination

Precisely transferring 0.5. mu.L HCCA matrix solution and 0.5. mu.L test solution (i.e. standard working solution, sample solution A, sample solution B, each of which is independently measured under the same conditions) to a stainless steel sample target plate (Bruker Daltonics), mixing, and naturally drying to obtain the final product. And data acquisition is carried out by adopting a reflection mode, and data processing is carried out by adopting Flex analysis3.4 software. The MS signal was obtained as an average of 15 laser shots taken from each target at random.

MALDI-TOF MS mass spectrometry test conditions:

(1) matrix: HCCA;

(2) target plate: group steel;

(3) point target mode: dry point method;

(4) frequency: 2000 Hz;

(5) the shooting times are as follows: 500, a step of;

(6) laser wavelength and intensity: 355nm, 90%;

(7) mode (2): RP 700-.

2.4 blank experiment

Repeating steps 2.1-2.2 except for the weighed sample to obtain a blank sample solution, and then processing according to step 2.3.

3 calculation of results

And (3) quantifying by adopting an external standard method, and calculating the content of astaxanthin in the sample according to the formula (1):

in the calculation formula:

wt. the weight fraction of astaxanthin in the sample to be tested;

c, astaxanthin concentration (mg/mL) obtained by standard curve and tested MALDI-TOF MS mass spectrum peak intensity;

v volume of sample solution after preparation (free astaxanthin: 50 mL; total astaxanthin: 65mL (2.6X 25));

m mass (30mg) weighed during the test of the sample solution.

Table 1 primary reference mass spectral data

Detection object	[M+H]⁺	[M+Na]⁺
			Astaxanthin	597.5	619.6

Firstly, sequentially dissolving a sample in acetone, adding BHT (butylated hydroxytoluene) to prevent oxidation of free astaxanthin, then fixing the volume by using a 50mL brown volumetric flask, and detecting the content of the free astaxanthin in the sample by using a MALDI-TOF MS (matrix-assisted laser Desorption-time of flight mass spectrometry). Detecting that the mass fraction of the free astaxanthin is 0.513 percent,

then, taking the 2mL sample solution B, adding cholesterol esterase to decompose the astaxanthin esterase into all free astaxanthin, uniformly mixing at a constant temperature, finally extracting by using petroleum ether, adding anhydrous sodium sulfate into the extracted petroleum ether layer to remove redundant moisture, blowing argon to blow the petroleum ether, dissolving by using acetone, filtering by using a 0.22 mu m organic phase microporous filter membrane, and obtaining a filtered solution which is used for determining the total astaxanthin, wherein the mass fraction of the total astaxanthin detected by the sample is as follows: 2.863 percent.

Through MALDI-TOF MS test of astaxanthin with different standard concentrations c, the mass spectrum peak intensity y of corresponding astaxanthin is obtained, which is in a linear relation in a smaller concentration range, and finally the formula shown in the table 2 is obtained:

TABLE 2 calculation formula of astaxanthin concentration c

The foregoing examples are set forth to illustrate the present invention more clearly and are not to be construed as limiting the scope of the invention, which is defined in the appended claims to which the invention pertains, as modified in all equivalent forms, by those skilled in the art after reading the present invention.

Claims

1. The method for detecting astaxanthin is characterized by comprising the following steps:

2. The method according to claim 1, wherein about 3 to 7mg of astaxanthin standard is taken in a 10mL centrifuge tube and dissolved in acetone to obtain a standard working solution in S1.

3. The method according to claim 1, wherein in S1, the volumetric flask is a brown volumetric flask.

4. The detection method according to claim 1, wherein in S1, the centrifugation is performed at 6000rpm and 5000-.

5. The detection method according to any one of claims 1 to 4, wherein the MALDI-TOF MS mass spectrometry conditions are as follows: the point target mode is a dry point method, the frequency is 2000Hz, the shooting times are 500 times, the laser wavelength is 355nm, the laser intensity is 90 percent, and the mode is RP 700-.

6. The detection method according to any one of claims 1 to 4, further comprising a step of measuring the content of free astaxanthin in the sample solution A, wherein 0.5. mu.L of HCCA matrix solution and 0.5. mu.L of sample solution A are transferred to a stainless steel sample target plate and mixed, and after natural drying, MALDI-TOF MS measurement is performed to obtain a MALDI-TOF MS spectrum of the sample solution A; and then combining a related MALDI-TOF MS spectrogram, and calculating by an external standard method to obtain the astaxanthin content in the sample.

7. The assay of any one of claims 1-4, wherein the HCCA matrix solution is a mixture of HCCA and TA30, wherein the concentration of HCCA is 10 mg/mL; TA30 was prepared by mixing acetonitrile and aqueous TFA at a 30:70 volume ratio, with the concentration of aqueous TFA being 0.1 wt%.