CN112946137B - High performance liquid chromatography detection method for anthocyanin in blueberries - Google Patents
High performance liquid chromatography detection method for anthocyanin in blueberries Download PDFInfo
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Abstract
The invention provides a high performance liquid chromatography detection method for anthocyanin in blueberries, and belongs to the technical field of photoelectrode materials. The anthocyanins include delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, pelargonidin-3-O-glucoside, paeoniflorin-3-O-glucoside and malvidin-3-O-glucoside, and the flow rate of HPLC analysis is as follows: 0.8mL/min; detection wavelength: 520nm; and (3) detecting the temperature: 25 ℃; sample introduction volume: 10 mu L of the solution; detection time: 60min; mobile phase: the water phase B is 0.3wt% phosphoric acid water solution, and the organic phase A is acetonitrile; and (4) gradient elution. The method realizes the simultaneous determination of 6 anthocyanin in the blueberry sample, has small standard deviation of peak area and retention time, low detection limit and quantitative limit of 6 anthocyanin, high sensitivity, and good precision, reproducibility and stability.
Description
Technical Field
The invention relates to the technical field of photoelectrode materials, in particular to a high performance liquid chromatography detection method for anthocyanin in blueberries.
Background
Anthocyanidin or anthocyanidin is water soluble natural pigment, and the basic structure mother nucleus is 2-phenyl benzopyran, i.e. anthocyanidin (Flavylium), and its structure is shown in the following formula. There are over 22 and over 500 classes of anthocyanins known in nature, the most common of which are 6 as shown in table 1, cyanidin or hibiscus pigment (Cyanidin), delphinidin or Delphinidin, delphinidin (Delphinidin), peonidin (Peonidin), morning or petuniadin (petuniadin), malvidin (Malvidin) and Pelargonidin (Pelargonidin), respectively. Anthocyanins are generally present in the form of glycosides, and are often glycosidically bonded to one or more of glucose, rhamnose, galactose, arabinose, etc. to form anthocyanins or anthocyanosides (Anthocyanin), the major anthocyanins being anthocyanidin-3-O-glucoside.
TABLE 16 common anthocyanidins
Anthocyanins are a family of compounds capable of presenting red color in flavonoids, and widely exist in cell sap of flower, fruit, stem, leaf and root organs of plants, so that the plant is in different colors from red, purple red to blue. Due to its unique functionality, anthocyanins are used for scavenging free radicals in vivo, proliferating xanthophyll, resisting tumor, resisting cancer, resisting inflammation, inhibiting lipid peroxidation and platelet aggregation, preventing diabetes, reducing weight, protecting vision, etc.
The anthocyanin can be analyzed by ultraviolet spectrophotometry, pH differential method, high performance liquid chromatography, capillary electrophoresis, and chromatography-mass spectrometry. The ultraviolet spectrophotometry and the pH value differential method are mostly used for measuring the total content of anthocyanin, the capillary electrophoresis method and the chromatography-mass spectrometry are mostly used for identifying component types, and the high performance liquid chromatography is mainly used for detecting the content of anthocyanin components. At present, the prior art discloses that 6 basic anthocyanins in grapes and wine are simultaneously measured by using a high performance liquid chromatography (see 6 basic anthocyanins in grapes and wine simultaneously measured by an HPLC method, liu Bing and the like, china brewing, 2017,36 (002): 162-165), but only anthocyanins in grapes and wine containing few kinds of anthocyanins are well separated and analyzed, but blueberries containing many kinds of anthocyanins (containing at least 13 kinds of anthocyanins) are difficult to completely separate the 6 basic anthocyanins, and the analysis result is certainly influenced.
Disclosure of Invention
In view of the above, the invention aims to provide a high performance liquid chromatography detection method for anthocyanin in blueberry. The preparation method provided by the invention can realize simultaneous determination of 6 anthocyanin in the blueberry.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a high performance liquid chromatography detection method of anthocyanin in blueberry, wherein the anthocyanin comprises delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunianin-3-O-glucoside, pelargonidin-3-O-glucoside, paeoniflorin-3-O-glucoside and malvidin-3-O-glucoside, and the method comprises the following steps:
pretreating a blueberry sample to be detected to obtain a blueberry anthocyanin solution to be detected;
performing high performance liquid chromatography analysis on the blueberry anthocyanin solution to be detected to obtain a chromatogram of a sample to be detected, wherein the chromatographic conditions of the high performance liquid chromatography analysis are as follows:
and (3) analyzing the column: a C18 column;
flow rate: 0.8mL/min;
detection wavelength: 520nm;
detecting the temperature: 25 ℃;
sample injection volume: 10 mu L of the solution;
detection time: 60min;
mobile phase: the water phase B is 0.3wt% phosphoric acid water solution, and the organic phase A is acetonitrile;
an elution mode: gradient elution; 0min, a, b =9, 20min, a, b = 10;
and comparing the chromatogram of the sample to be detected with a preset standard chromatogram, and calculating the content of anthocyanin in the blueberry by using an external standard method and a standard equation, wherein the standard equation is an equation with the mass concentration of each anthocyanin as an independent variable and a peak area as a dependent variable.
Preferably, the concentration of 6 anthocyanin in the blueberry anthocyanin solution to be detected is 0-50 mu g/mL independently, the minimum detection limit of 6 anthocyanin is 0.010-0.035 mu g/mL, and the minimum quantification limit is 0.033-0.117 mu g/mL.
Preferably, the C18 column is SB-C18 with a specification of 5 μm, 4.6X 250mm.
Preferably, the blueberry sample to be tested comprises a solid product, a liquid product or a paste product.
Preferably, the pre-treatment comprises the steps of: mixing the blueberry sample to be detected with an extracting solution, and then carrying out ultrasonic extraction to obtain a filtrate; filtering the filtrate by using an organic microporous filter membrane of 0.22 mu m, wherein the extracting solution is a methanol solution or a hydrochloric acid acidified methanol solution, the volume percentage of methanol in the methanol solution is 0-50%, the hydrochloric acid acidified methanol solution is hydrochloric acid containing 1% by volume, and the mass fraction of the hydrochloric acid is 30-36%.
Preferably, the mass ratio of the blueberry sample to be detected to the extracting solution is 1.
Preferably, when the blueberry sample to be detected is a liquid product, the pretreatment comprises the following steps: filtering the liquid product through a 0.22 mu m organic microporous filter membrane.
Preferably, when the blueberry sample to be detected is fresh blueberry fruit, the pretreatment comprises the following steps:
mixing the fresh blueberry fruits with a compound enzyme, and then carrying out dark enzymolysis to obtain an enzymolysis product, wherein the compound enzyme comprises cellulase and pectinase;
extracting the enzymolysis product by using an extracting agent to obtain filtrate, wherein the extracting agent is a 75wt% ethanol water solution containing citric acid, and the volume percentage of the citric acid in the extracting agent is 0.5-1.5%;
sequentially concentrating the filtrate, extracting with ethyl acetate, mixing the lower layers, concentrating under reduced pressure, passing through D101 macroporous adsorbent resin, eluting with ethanol, and freeze-drying to obtain blueberry anthocyanin extract;
and mixing the blueberry anthocyanin extract with a solvent to obtain the blueberry anthocyanin solution to be detected.
Preferably, the mass ratio of the cellulase to the pectinase in the complex enzyme is 1-4.
Preferably, the mass ratio of the fresh blueberry fruits to the complex enzyme is 100.1-0.5.
The invention provides a high performance liquid chromatography detection method of anthocyanin in blueberry, wherein the anthocyanin comprises delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunianin-3-O-glucoside, pelargonidin-3-O-glucoside, paeoniflorin-3-O-glucoside and malvidin-3-O-glucoside, and the method comprises the following steps: pretreating a blueberry sample to be detected to obtain a blueberry anthocyanin solution to be detected; performing high performance liquid chromatography analysis on the blueberry anthocyanin solution to be detected to obtain a chromatogram of a sample to be detected, wherein the chromatographic conditions of the high performance liquid chromatography analysis are as follows: and (3) analyzing the column: a C18 column; flow rate: 0.8mL/min; detection wavelength: 520nm; detecting the temperature: 25 ℃; sample injection volume: 10 mu L of the solution; detection time: 60min; mobile phase: the water phase B is 0.3wt% phosphoric acid water solution, and the organic phase A is acetonitrile; and (3) an elution mode: gradient elution; 0min, a; and comparing the chromatogram of the sample to be detected with a preset standard chromatogram, and calculating the content of anthocyanin in the blueberry by using an external standard method and a standard equation, wherein the standard equation is an equation with the mass concentration of each anthocyanin as an independent variable and the peak area as a dependent variable.
The method realizes the simultaneous determination of 6 anthocyanins in the blueberry sample, has small standard deviation of peak area and retention time, low detection limit and quantitative limit of the 6 anthocyanins, high sensitivity, high precision, reproducibility and stability, is suitable for the detection of the anthocyanins in various blueberry sample products, is simple and rapid to operate, has high accuracy, and is a good quality control detection method. The invention optimizes the separation condition of the high performance liquid chromatography, simplifies the sample processing method, detects the diversification of samples and achieves the method for simply, quickly, sensitively and accurately detecting the anthocyanin in various products.
Furthermore, the pretreatment method in the detection method of the invention can not degrade anthocyanin at high temperature, thus avoiding the problem that the analysis result is influenced by the reduction of anthocyanin content caused by high temperature, and the detection method of the invention can not treat anthocyanin under the condition of strong acidity and high temperature (100 ℃), thus avoiding the problem that the analysis result is influenced by the degradation of anthocyanin.
Drawings
FIG. 1 is a high performance liquid chromatography chromatogram of 6 anthocyanins in the present invention;
FIG. 2 is a high performance liquid chromatography chromatogram of anthocyanin obtained in example 2 of the invention;
FIG. 3 is a high performance liquid chromatography spectrum of anthocyanin obtained in example 3 of the present invention;
FIG. 4 is a high performance liquid chromatography spectrum of anthocyanin obtained in example 4 of the present invention;
FIG. 5 is a high performance liquid chromatography chromatogram of anthocyanin obtained in example 5 of the present invention
FIG. 6 is a high performance liquid chromatography chromatogram of anthocyanin obtained in example 6 of the present invention;
FIG. 7 is a high performance liquid chromatography spectrum of anthocyanin obtained in example 7 of the present invention;
FIG. 8 is a high performance liquid chromatography chromatogram of anthocyanin obtained in example 8 of the present invention;
in fig. 1 to 8, 1 is delphinidin-3-O-glucoside, 2 is cyanidin-3-O-glucoside, 3 is petunidin-3-O-glucoside, 4 is pelargonidin-3-O-glucoside, 5 is peonidin-3-O-glucoside, and 6 is malvidin-3-O-glucoside.
Detailed Description
The invention provides a high performance liquid chromatography detection method of anthocyanin in blueberry, wherein the anthocyanin comprises delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunianin-3-O-glucoside, pelargonidin-3-O-glucoside, paeoniflorin-3-O-glucoside and malvidin-3-O-glucoside, and the method comprises the following steps:
pretreating a blueberry sample to be detected to obtain a blueberry anthocyanin solution to be detected;
performing high performance liquid chromatography analysis on the blueberry anthocyanin solution to be detected to obtain a chromatogram of a sample to be detected, wherein the chromatographic conditions of the high performance liquid chromatography analysis are as follows:
and (3) analyzing the column: a C18 column;
flow rate: 0.8mL/min;
detection wavelength: 520nm;
detecting the temperature: 25 ℃;
sample introduction volume: 10 mu L of the solution;
detection time: 60min;
mobile phase: the water phase B is 0.3wt% phosphoric acid water solution, and the organic phase A is acetonitrile;
and (3) an elution mode: gradient elution; 0min, a, b =9, 20min, a, b = 10;
and comparing the chromatogram of the sample to be detected with a preset standard chromatogram, and calculating the content of anthocyanin in the blueberry by using an external standard method and a standard equation, wherein the standard equation is an equation with the mass concentration of each anthocyanin as an independent variable and the peak area as a dependent variable.
The blueberry anthocyanin to-be-detected solution is obtained by pretreating a blueberry sample to be detected.
In the invention, the blueberry sample to be detected preferably comprises a solid product, a liquid product or a sauce-shaped product. The source of the blueberry sample to be detected is not specially limited.
In the present invention, the pretreatment preferably comprises the steps of: mixing the blueberry sample to be detected with an extracting solution, and then carrying out ultrasonic extraction to obtain a filtrate; filtering the filtrate by using a 0.22 mu m organic microporous filter membrane, wherein the extracting solution is a methanol solution or a hydrochloric acid acidification methanol solution, the volume percentage of methanol in the methanol solution is 0-50%, the hydrochloric acid acidification methanol solution is hydrochloric acid containing 1% volume ratio, and the mass fraction of the hydrochloric acid is 30-36%.
In the invention, the mass ratio of the blueberry sample to be detected to the extracting solution is preferably 1.
In the present invention, the number of times of the ultrasonic extraction is preferably 3 times, and more preferably, the ultrasonic extraction is repeated 2 times on the obtained filter residue obtained by the first ultrasonic treatment, and the filtrates are combined. In the invention, the temperature of the ultrasonic extraction is preferably room temperature, and the time is preferably 30min. The power of the ultrasonic extraction is not particularly limited in the present invention.
In the invention, when the blueberry sample to be detected is a liquid product, the pretreatment preferably comprises the following steps: filtering the liquid product through a 0.22 mu m organic microporous filter membrane.
In the invention, when the blueberry sample to be detected is fresh blueberry fruit, the pretreatment preferably comprises the following steps:
mixing the fresh blueberry fruits with a compound enzyme, and then carrying out dark enzymolysis to obtain an enzymolysis product, wherein the compound enzyme comprises cellulase and pectinase;
extracting the enzymolysis product by using an extracting agent to obtain a filtrate, wherein the extracting agent is a 75wt% ethanol water solution containing citric acid, and the volume percentage of the citric acid in the extracting agent is 0.5-1.5%;
sequentially concentrating the filtrate, extracting with ethyl acetate, mixing the lower layers, concentrating under reduced pressure, passing through D101 macroporous adsorbent resin, eluting with ethanol, and freeze-drying to obtain blueberry anthocyanin extract;
and mixing the blueberry anthocyanin extract with a solvent to obtain the blueberry anthocyanin solution to be detected.
The method comprises the steps of mixing fresh blueberries with a complex enzyme, and then carrying out dark enzymolysis to obtain an enzymolysis product, wherein the complex enzyme comprises cellulase and pectinase.
In the present invention, the citric acid content in the extractant is preferably 1% by volume.
In the invention, the mass ratio of the cellulase to the pectinase in the complex enzyme is preferably 1-4, more preferably 2.
In the invention, the mass ratio of the fresh blueberry fruit to the complex enzyme is preferably 100.1-0.5, more preferably 100.
In the present invention, the temperature for protecting from light enzymolysis is preferably 37 ℃, and the time is preferably 4 hours.
After obtaining an enzymolysis product, extracting the enzymolysis product with an extractant to obtain a filtrate, wherein the extractant is a 75wt% ethanol aqueous solution containing citric acid, and the volume percentage of the citric acid in the extractant is 1%.
In the present invention, the mass ratio of the enzymatic hydrolysate to the extractant is preferably 1:10.
in the present invention, the number of times of extraction is preferably 3, and more preferably, the residue obtained from the first extraction is further added with an extraction agent for extraction, and the filtrates are combined. In the present invention, the time for the extraction is independently preferably 6h.
After the filtrate is obtained, sequentially concentrating the filtrate, extracting with ethyl acetate, combining lower layers, concentrating under reduced pressure, passing through D101 macroporous adsorption resin, eluting with ethanol, and freeze-drying to obtain the blueberry anthocyanin extract.
In the present invention, the concentration is preferably concentration under reduced pressure to half the volume.
In the present invention, the number of the ethyl acetate extractions is preferably 3, and the amount of ethyl acetate used per time is preferably 2 times of the volume of the extract to be extracted.
In the present invention, the specific parameters of the vacuum concentration are not particularly limited, and ethyl acetate can be completely removed.
In the present invention, the purpose of the D101 macroporous adsorption resin is to enrich anthocyanin.
In the invention, the mass concentration of the ethanol solution used for ethanol elution is 30-50%, the flow rate of the elution is preferably 1-3 BV/h, and the dosage of the ethanol solution for each elution is preferably 2-4 BV.
In the present invention, the freeze-drying end point temperature is preferably-30 to-50 ℃, the vacuum degree is preferably 1 to 5Pa, and the thickness of the feed liquid is preferably 10 to 15mm.
After the blueberry anthocyanin extract is obtained, the blueberry anthocyanin extract is mixed with a solvent to obtain the blueberry anthocyanin solution to be detected. In the invention, the solvent is preferably a methanol solution or a hydrochloric acid acidified methanol solution, the volume percentage content of methanol in the methanol solution is 0-50%, the hydrochloric acid acidified methanol solution is hydrochloric acid containing 1% by volume, and the mass fraction of the hydrochloric acid is 30-36%. The specific dosage of the solvent is not specially limited, and the concentration of 6 anthocyanin in the blueberry anthocyanin solution to be detected can be ensured to be 0-50 mu g/mL.
In the invention, the concentration of 6 anthocyanin in the blueberry anthocyanin solution to be detected is preferably 0-50 mu g/mL independently, the minimum detection limit of 6 anthocyanin is preferably 0.010-0.035 mu g/mL, and the minimum quantitative limit is preferably 0.033-0.117 mu g/mL.
After a blueberry anthocyanin solution to be detected is obtained, the blueberry anthocyanin solution to be detected is subjected to high performance liquid chromatography to obtain a chromatogram of a sample to be detected, wherein the chromatographic conditions of the high performance liquid chromatography are as follows:
and (3) analyzing the column: a C18 column;
flow rate: 0.8mL/min;
detection wavelength: 520nm;
and (3) detecting the temperature: 25 ℃;
sample introduction volume: 10 mu L of the solution;
detection time: 60min;
mobile phase: the water phase B is 0.3wt% phosphoric acid water solution, and the organic phase A is acetonitrile;
and (3) an elution mode: gradient elution; 0min, a
In the present invention, the C18 column is preferably SB-C18, and the specification is preferably 5 μm, 4.6X 250mm.
In the present invention, the ratios in the gradient elution all refer to volume ratios.
After obtaining the chromatogram of the sample to be detected, the method compares the chromatogram of the sample to be detected with a preset standard chromatogram, and calculates the content of anthocyanin in the blueberry by an external standard method through a standard equation, wherein the standard equation is an equation with the mass concentration of each anthocyanin as an independent variable and the peak area as a dependent variable.
In the present invention, the predetermined standard spectrum is prepared by a method comprising the steps of:
weighing HPLC chromatographically pure anthocyanin standard substances, dissolving the anthocyanin standard substances with 30wt% of methanol to prepare a series of mixed standard solutions with the concentrations of 0, 2, 5, 15, 25 and 50 mu g/mL, filtering the mixed standard solutions by using a 0.22 mu m organic filter membrane, and performing high performance liquid chromatography analysis, wherein the anthocyanins comprise delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunianin-3-O-glucoside, pelargonidin-3-O-glucoside, peoniflorin-3-O-glucoside and malvidin-3-O-glucoside, and 6 anthocyanins are compared and calibrated, as shown in figure 1, 1 is delphinidin-3-O-glucoside, 2 is cyanidin-3-O-glucoside, 3 is petunianin-3-O-glucoside, 4 is pelargonidin-3-O-glucoside, 5 is peoniflorin-3-O-glucoside, and 6 is malvidin-3-O-glucoside; the chromatographic conditions of the high performance liquid chromatography are consistent with the scheme, and are not described again;
taking the peak area as the ordinate and the concentration as the abscissa, and taking a linear equation of the peak area to the concentration to obtain a standard equation of 6 kinds of anthocyanin.
In order to further illustrate the present invention, the following describes the method for detecting anthocyanin in blueberry by high performance liquid chromatography in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Materials and reagents: the tested blueberries were collected from the blueberry planting base in Majiang county, kaili city, guizhou, and the blueberry varieties were rabbit eye series. The reagent comprises chromatographic pure acetonitrile, methanol, analytically pure formic acid, acetic acid, phosphoric acid, ethanol, citric acid and ethyl acetate
2. The instrument equipment comprises: high performance liquid chromatography (Agilent 1260 liquid chromatograph with 1260 diode array detector, agilent corp.); LGJ-50F vacuum freeze dryer (Beijing Songyuan Huaxing science and technology development Co., ltd.); BEATL-10 ultrapure water preparation instrument (Shandong Tinglan environmental protection science and technology Co., ltd.); in addition, the device also relates to a rotary evaporator, an ultrasonic cleaning instrument, a digital display constant temperature water bath and an ultrafiltration device.
3. Preparation of blueberry anthocyanin extract:
adding 0.3wt% of complex enzyme into fresh blueberry fruits, wherein the complex enzyme consists of cellulase and pectinase according to the mass ratio of 2; then adding 5 times of 75wt% ethanol containing 1% (volume percentage) of citric acid, extracting for 6 hours, and filtering to obtain filtrate and filter residue; adding 5 times volume of 75wt% ethanol containing 1% (volume percentage) citric acid into the filter residue, extracting for 6 hours, and repeating for 2 times; combining the three filtrates, concentrating under reduced pressure to half volume, adding 2 times volume of ethyl acetate, and extracting for 3 times (the amount of ethyl acetate is 2 times of the volume of the extraction agent each time); combining the lower layers, concentrating under reduced pressure, performing anthocyanin enrichment through D101 macroporous adsorption resin, eluting with 30wt% ethanol at a rate of 2BV/h for 3BV, and freeze-drying (the freezing end temperature is-40 ℃, the vacuum degree is 1Pa, and the thickness of the feed liquid is 11 mm.) to obtain the blueberry anthocyanin extract.
4. High performance liquid chromatography separation of blueberry anthocyanin extract
Chromatographic column screening study
The experiment used Shim-pack VP-ODS (4.6 mm. Times.150 mm,
5μm)、Agilent ZORBAX SB-C18(4.6×250mm，
5 μm) and Infinity Lab Poroshell 120 EC-C18 (4.6 x 150mm,
5 mu m) of the blueberry anthocyanin extract are separated by three different chromatographic columnsThe results are shown in Table 2. As can be seen from Table 2, the difference of the chromatographic columns has a certain influence on the separation effect of blueberry anthocyanin, and the blueberry anthocyanin can be separated by all three chromatographic columns, so that the Agilent ZORBAX SB-C18 chromatographic column has the best separation effect, and is preferably the Agilent ZORBAX SB-C18 chromatographic column.
Table 2 results of separation of blueberry anthocyanidin extract by different chromatographic columns
Mobile phase screening study
And respectively carrying out mobile phase screening on the A-phase methanol, the acetonitrile, the B-phase formic acid water, the acetic acid water and the phosphoric acid water, carrying out gradient elution, and inspecting the separation effect of chromatographic peaks. Table 3 shows the separation of the blueberry anthocyanidin extract in different mobile phases. As can be seen from Table 3, the different proportions of the mobile phase have a great influence on the separation effect of blueberry anthocyanin. The methanol-water system can not well separate blueberry anthocyanin at the same time, the acetonitrile-water has a good separation effect on the blueberry anthocyanin, and the experiment prefers that 0.3wt% of phosphoric acid water-acetonitrile is used as a mobile phase.
TABLE 3 separation of blueberry anthocyanidin extracts in different mobile phases
Screening study of elution conditions
The study was carried out with acetonitrile (solution A) and 0.3wt% phosphoric acid (solution B) from 90% to 80% in 0 to 100 minutes. Other elution conditions were: the sample injection volume is 10 mu L, the flow rate is 0.8mL/min, the column temperature is 25 ℃, and the monitoring wavelength is 520nm. The initial gradient composition was then adjusted to 5vol% of solvent A and 95vol% of solvent B. Finally, the initial gradient composition was further adjusted to 9vol% of a and 91vol% of solvent B to obtain a separation spectrum as shown in fig. 1, and by comparison, the elution gradient obtained by the optimization screening was as shown in table 4.
TABLE 4 different gradient elution modes of HPLC
Investigation of flow Rate
Through comparison of three flow rates of 0.8mL/min,1.0mL/min and 1.5mL/min, the flow rate has no great influence on the peak separation effect and retention time of anthocyanin, but the slower the flow rate, the more perfect the peak shape of anthocyanin, and the too fast the flow rate, which can cause unstable baseline. In conclusion, 0.8mL/min was selected as the flow rate for the preparation of blueberry anthocyanin.
Investigation of separation temperature
The temperature affects the physical properties of the solvent, such as density, viscosity, etc. Generally, as the column temperature increases, the peak-to-peak time decreases, but the peak symmetry decreases. The blueberry anthocyanin monomers are similar in structure and polarity and sensitive to temperature. Higher temperatures can affect the separation effect and degrade anthocyanin. The experiment was carried out at 20 deg.C, 25 deg.C and 30 deg.C, and the separation temperature was determined to be 25 deg.C based on the experimental results.
Investigation of sample size
Different injection concentrations will affect the peak shape and the time to peak of the sample. 5mg/mL blueberry anthocyanin extract is injected with 8 muL, 10 muL and 15 muL respectively. With the increase of the sample amount, the solvent effect is prominent, the peak emergence time is shortened, and the peak symmetry is reduced. By comparison, a sample size of 10 μ L was selected.
Determination of detection wavelength
The maximum absorption wavelength of anthocyanin is about 520nm, and the absorption conditions of 515nm, 520nm and 530nm are experimentally observed. The result shows that the peak shape and the absorption at 520nm are both good, the symmetry factor is about 1, and 520nm is selected as the detection wavelength.
The optimized chromatographic conditions are as follows: agilent ZORBAX SB-C18 column (5 μm)
4.6 × 250 mm), flow rate: 0.8mL/min; detection wavelength: 520nm; detecting the temperature: 25 ℃; sample introduction volume: 10 muL; detection time: and (5) performing treatment for 60min.
Mobile phase: the aqueous phase B was 0.3wt% phosphoric acid in water and the organic phase A was acetonitrile.
An elution mode: gradient elution is shown in Table 5, where A is acetonitrile and B is 0.3wt% phosphoric acid solution.
TABLE 5 optimized gradient elution mode for HPLC
Under the optimized chromatographic condition, HPLC-chromatographically pure delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, pelargonidin-3-O-glucoside, peoniflorin-3-O-glucoside and malvidin-3-O-glucoside standard substances are respectively weighed, dissolved by 30wt% of methanol to prepare mixed standard solutions with the concentrations of 0, 2, 5, 15, 25 and 50 mu g/mL, filtered by a 0.22 mu m organic filter membrane and subjected to high performance liquid chromatography analysis, and 6 corresponding anthocyanins in blueberry anthocyanins are compared and calibrated, as shown in figure 1, 1 in figure 1 is the delphinidin-3-O-glucoside, 2 is the cyanidin-3-O-glucoside, 3 is the petunidin-3-O-glucoside, 4 is the pelargonidin-3-O-glucoside, 5 is the peoniflorin-3-O-glucoside, and 6 is the malvidin-3-O-glucoside.
The peak area is used as the ordinate, the concentration is used as the abscissa, the peak area is used for making a linear equation to the concentration, 6 standard equations are obtained, the result is shown in table 6, as can be seen from table 6, the standard deviation of the peak area and the retention time is small, and the detection method provided by the invention has high accuracy.
TABLE 6 Retention time, regression equation, correlation coefficient, linear range, and relative standard deviation of the various anthocyanins
In the regression equation, Y represents the peak area, and C represents the concentration of anthocyanin μ g/mL.
Instrumental detection limits for 6 anthocyanins were calculated at a 3-fold signal-to-noise ratio (S/N = 3) under optimized HPLC assay conditions, and instrumental quantitation limits for 6 anthocyanins were obtained under S/N =10 conditions. As can be seen from Table 7, the minimum detection limit of 6 anthocyanin is 0.010-0.035 μ g/mL, and the minimum quantification limit is 0.033-0.117 μ g/mL), which indicates that the method has higher sensitivity for detecting anthocyanin.
TABLE 7 detection Limit test results
Example 2
Dissolving blueberry anthocyanin powder 10mg in 30wt% methanol solution, diluting to a constant volume of 10mL to obtain a sample solution, and filtering the sample solution with an organic filter membrane of 0.22 mu m to obtain a solution to be detected for later use.
The detection of the solution to be detected was carried out under the same conditions as those of example 1, and the obtained hplc chromatogram 2 was obtained, where 1 in fig. 2 was delphinidin-3-O-glucoside, 2 was cyanidin-3-O-glucoside, 3 was petunidin-3-O-glucoside, 4 was pelargonidin-3-O-glucoside, 5 was peoniflorin-3-O-glucoside, and 6 was malvidin-3-O-glucoside.
The content of 6 major anthocyanins in the blueberry anthocyanin powder is calculated according to the standard equation provided in example 1, and the results are shown in table 8, and as can be seen from fig. 2 and table 8, example 2 realizes the determination of 6 major anthocyanins in the blueberry anthocyanin powder through one detection, the anthocyanin in blueberries has the highest content of malvidin-3-O-glucoside, but the content of cyanidin-3-O-glucoside commonly used in the literature is relatively low.
TABLE 8 blueberry anthocyanidin powder with 6 major anthocyanins (μ g/mL)
Example 3
Filtering a proper amount of blueberry red wine with a 0.22 mu m organic filter membrane to obtain a liquid to be detected for later use.
The same conditions as those in example 1 were used to perform a sample injection of 60 μ L of the solution to be tested, and a high performance liquid chromatography chromatogram 3 was obtained, in fig. 3, 1 is delphinidin-3-O-glucoside, 2 is cyanidin-3-O-glucoside, 3 is petunidin-3-O-glucoside, 4 is pelargonidin-3-O-glucoside, 5 is peoniflorin-3-O-glucoside, and 6 is malvidin-3-O-glucoside.
The content of 6 major anthocyanins in the blueberry red wine is calculated according to the standard equation provided in example 1, the result is shown in table 3, and as can be seen from fig. 3 and table 9, example 3 realizes the determination of 5 major anthocyanins in the blueberry red wine through one detection. As shown in Table 3, the blueberry wine has the highest malvidin-3-O-glucoside content, but the conventional cyanidin-3-O-glucoside content in the literature is relatively low.
TABLE 9 blueberry Red wine 6 major anthocyanins content (μ g/mL)
Note: ND indicates not detected, the same applies below.
Example 4
Filtering a proper amount of grape wine with a 0.22 mu m organic filter membrane to obtain a solution to be detected for later use.
By adopting the same high performance liquid chromatography detection conditions as those in example 1, 60 μ L of the solution to be detected was injected and detected to obtain a high performance liquid chromatography spectrogram 4, wherein 1 in fig. 4 is delphinidin-3-O-glucoside, 2 is cyanidin-3-O-glucoside, 3 is petunidin-3-O-glucoside, 5 is paeoniflorin-3-O-glucoside, and 6 is malvidin-3-O-glucoside.
The content of 6 major anthocyanins in wine was calculated according to the standard equation provided in example 1, and the results are shown in table 10, and it can be seen from fig. 4 and table 10 that example 4 achieved the determination of 5 major anthocyanins in wine by one test.
TABLE 10 content of 6 major anthocyanins in wine (μ g/mL)
Example 5
Filtering a proper amount of blueberry essence oral liquid with a 0.22 mu m organic filter membrane to obtain a liquid to be detected for later use.
The same conditions as those of the example 1 were adopted, and 30 μ L of the solution to be detected was injected and detected to obtain a high performance liquid chromatography spectrogram 4, in fig. 4, 1 is delphinidin-3-O-glucoside, 2 is cyanidin-3-O-glucoside, 3 is petunidin-3-O-glucoside, 4 is pelargonidin-3-O-glucoside, 5 is peoniflorin-3-O-glucoside, and 6 is malvidin-3-O-glucoside.
The content of 6 major anthocyanins in the blueberry essence oral liquid is calculated according to the standard equation provided in example 1, the result is shown in table 11, and as can be seen from fig. 5 and table 11, example 5 realizes the determination of 6 major anthocyanins in the blueberry essence oral liquid through one-time detection. As shown in Table 11, the blueberry essence oral liquid contains the highest content of malvidin-3-O-glucoside, but contains the lower content of cyanidin-3-O-glucoside, which is commonly used in the literature.
TABLE 11 blueberry essence oral liquid in 6 kinds of main anthocyanin content (mug/mL)
Example 6
Taking 5g of blueberry jam, stirring for 2h at room temperature by using 15mL of methanol solution containing 1% hydrochloric acid, filtering, diluting to 50mL by using 30% methanol to obtain a sample solution, centrifuging the sample solution at 4 ℃ and 5000rpm for 10min, and filtering by using a 0.22-micron organic filter membrane to obtain a solution to be detected for later use.
A high performance liquid chromatography spectrogram 6 is obtained by injecting 10 mu L of a liquid to be detected under the same high performance liquid chromatography detection conditions as those in example 1, wherein 1 in the figure 6 is delphinidin-3-O-glucoside, 2 is cyanidin-3-O-glucoside, 3 is petunidin-3-O-glucoside, 5 is paeoniflorin-3-O-glucoside, and 6 is malvidin-3-O-glucoside.
The content of 6 major anthocyanins in the blueberry jam is calculated according to the standard equation provided in example 1, the result is shown in table 12, and as can be seen from fig. 6 and table 12, example 5 realizes the determination of 5 major anthocyanins in the blueberry jam through one detection. As shown in Table 12, the blueberry jam contains the highest content of anthocyanidin, malvidin-3-O-glucoside, but contains the lower content of cyanidin-3-O-glucoside, which is commonly used in the literature.
TABLE 12 blueberry jam with 6 major anthocyanins (μ g/mL)
Example 7
Taking 5g of strawberry jam, stirring for 2h at room temperature by using 15mL of methanol solution containing 1% hydrochloric acid, filtering, fixing the volume to 50mL by using methanol to obtain a sample solution, centrifuging the sample solution at 4 ℃ and 5000rpm for 10min, and filtering by using a 0.22-micron organic filter membrane to obtain a solution to be detected for later use.
And (2) injecting 10 mu L of a solution to be detected to detect by adopting the same high performance liquid chromatography detection conditions as the conditions in the example 1 to obtain a high performance liquid chromatography spectrogram 7, wherein 2 in the chart 7 is cyanidin-3-O-glucoside, and 4 is pelargonidin-3-O-glucoside.
The content of 6 major anthocyanins in the blueberry jam is calculated according to the standard equation provided in example 1, the result is shown in table 13, and as can be seen from fig. 7 and table 13, example 5 realizes the determination of 2 major anthocyanins in the blueberry jam through one-time detection.
TABLE 13 strawberry jam with 6 major anthocyanins (μ g/mL)
Example 8
Filtering a proper amount of blueberry juice with a 0.22 mu m organic filter membrane to obtain a liquid to be detected for later use.
The high performance liquid chromatography detection conditions completely the same as those in example 1 were adopted, and 80. Mu.L of the solution to be detected was injected and detected to obtain a high performance liquid chromatography spectrogram 8, in FIG. 8, 2 is cyanidin-3-O-glucoside, 3 is petuniain-3-O-glucoside, and 5 is peoniflorin-3-O-glucoside.
The content of 6 major anthocyanins in blueberry juice was calculated according to the standard equation provided in example 1, and the results are shown in table 14, and it can be seen from fig. 8 and table 14 that example 3 realizes the determination of 3 major anthocyanins in blueberry juice by one-time detection.
TABLE 14 major anthocyanin content (μ g/mL) in blueberry juice
Comparative example 1
The method for detecting 6 anthocyanin in blueberry is detected according to the method disclosed in the HPLC method for simultaneously detecting 6 basic anthocyanin in grape and wine (Liu Bing et al, chinese brewing, 2017,36 (002): 162-165), and comprises the following steps:
preparing a sample to be tested: weighing blueberry anthocyanin extract 10mg into a 100mL conical flask, adding 50mL of absolute ethyl alcohol/hydrochloric acid/water (2. Taking out the sample, cooling to room temperature, filtering with 0.22 μm microporous membrane, and testing. The method for pretreating the blueberry red wine sample is the same as the method for pretreating the blueberry anthocyanin extract.
The conditions of the high performance liquid chromatography are as follows: agilent-ZORBAXSB-C18 column (4.6 mm. Times.250mm, 5 μm); column temperature: 40 ℃; detection wavelength: 525nm; mobile phase: a, acetonitrile; b0.1 wt% phosphoric acid aqueous solution; flow rate: 0.8mL/min, gradient elution; sample introduction volume: 5 μ L, elution program: 0-25 min:10% -25% A, 25-25.1 min:25% to 10% of A. As shown in table 15, the method can only detect 3 anthocyanins in the blueberry product, and the detected contents are all low, and the analysis causes the degradation of anthocyanins due to too high sample pretreatment temperature and detection temperature, and the other causes is that the 6 anthocyanins cannot be separated well by the separation condition.
TABLE 15 anthocyanin content of blueberry products (μ g/mL)
Comparative example 2
According to the patent of CN107561185A, a high performance liquid chromatography detection method for simultaneously detecting flavonoid and a method for detecting the flavonoid content in fruits are disclosed for detection:
preparing a sample to be tested: weighing blueberry anthocyanin extract 5mg, dissolving in 20mL of methanol/hydrochloric acid (98, 2, V/V) mixed solution, filtering by a 0.22 mu m microporous filter membrane, and then testing. The method for pretreating the blueberry red wine sample is the same as the method for pretreating the blueberry anthocyanin extract.
The conditions of the high performance liquid chromatography are as follows: infinityLab Poroshell 120 EC-C18 column (4.6 mm. Times.150mm, 5 μm); column temperature: at 40 ℃; detection wavelength: 510nm; mobile phase: a, acetonitrile; b0.3 wt% phosphoric acid aqueous solution; flow rate: 1mL/min, gradient elution; sample introduction volume: 10 μ L, elution program: 0-20 min: 5-15% of A, 20-30 min:15% -25% A, 30-40 min: 25-50% of A, 40-48 min:50% to 5% by weight of A. As shown in Table 16, it can be seen that only 3 anthocyanins in the blueberry product can be detected by the method, and the detected contents are all low, and the analysis causes anthocyanin degradation due to high acidity of sample pretreatment and detection temperature, and the other causes is that the 6 anthocyanins cannot be well separated by the separation condition.
TABLE 16 anthocyanin content of blueberry products (μ g/mL)
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be construed as the protection scope of the present invention.
Claims (5)
1. A high performance liquid chromatography detection method for anthocyanin in blueberry comprises delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunianin-3-O-glucoside, pelargonidin-3-O-glucoside, peoniflorin-3-O-glucoside and malvidin-3-O-glucoside, and is characterized by comprising the following steps:
pretreating a blueberry sample to be detected to obtain a blueberry anthocyanin solution to be detected;
performing high performance liquid chromatography analysis on the blueberry anthocyanin solution to be detected to obtain a chromatogram of a sample to be detected, wherein the chromatographic conditions of the high performance liquid chromatography analysis are as follows:
and (3) analyzing the column: a C18 column;
flow rate: 0.8mL/min;
detection wavelength: 520nm;
and (3) detecting the temperature: 25 ℃;
sample introduction volume: 10 mu L of the solution;
detection time: 60min;
mobile phase: the water phase B is 0.3wt% phosphoric acid water solution, and the organic phase A is acetonitrile;
and (3) an elution mode: gradient elution; 0min, a, b =9, 20min, a, b = 10;
comparing the chromatogram of the sample to be detected with a preset standard chromatogram, and calculating by using an external standard method and a standard equation to obtain the content of anthocyanin in the blueberry, wherein the standard equation is an equation with the mass concentration of each anthocyanin as an independent variable and a peak area as a dependent variable;
when the blueberry sample to be detected is fresh blueberry fruit, the pretreatment comprises the following steps:
mixing the fresh blueberry fruits with a compound enzyme, and then carrying out dark enzymolysis to obtain an enzymolysis product, wherein the compound enzyme comprises cellulase and pectinase;
extracting the enzymolysis product by using an extracting agent to obtain a filtrate, wherein the extracting agent is a 75wt% ethanol water solution containing citric acid, and the volume percentage of the citric acid in the extracting agent is 0.5-1.5%;
sequentially concentrating the filtrate, extracting with ethyl acetate, mixing the lower layers, concentrating under reduced pressure, passing through D101 macroporous adsorbent resin, eluting with ethanol, and freeze-drying to obtain blueberry anthocyanin extract;
mixing the blueberry anthocyanin extract with a solvent to obtain a solution to be detected of the blueberry anthocyanin;
when the blueberry sample to be detected is blueberry anthocyanin powder, the pretreatment comprises the following steps: dissolving blueberry anthocyanin powder in 30wt% methanol solution;
when the blueberry sample to be detected is a liquid product, the pretreatment comprises the following steps: filtering the liquid product through a 0.22 mu m organic microporous filter membrane.
2. The detection method as claimed in claim 1, wherein the concentration of 6 anthocyanins in the blueberry anthocyanin solution to be detected is 0-50 μ g/mL independently, the minimum detection limit of 6 anthocyanins is 0.010-0.035 μ g/mL, and the minimum quantification limit is 0.033-0.117 μ g/mL.
3. The detection method according to claim 1, wherein the C18 column is SB-C18, and has a size of 5 μm and 4.6X 250mm.
4. The detection method according to claim 1, wherein the mass ratio of the cellulase to the pectinase in the complex enzyme is 1-4.
5. The detection method as claimed in claim 1, wherein the mass ratio of the fresh blueberry fruits to the complex enzyme is 100: 0.1-0.5.
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