CN107561185B - High performance liquid chromatography detection method for simultaneously determining 11 flavonoids and method for detecting flavonoid content in fruits - Google Patents
High performance liquid chromatography detection method for simultaneously determining 11 flavonoids and method for detecting flavonoid content in fruits Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 68
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 25
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- VEVZSMAEJFVWIL-UHFFFAOYSA-O cyanidin cation Chemical compound [O+]=1C2=CC(O)=CC(O)=C2C=C(O)C=1C1=CC=C(O)C(O)=C1 VEVZSMAEJFVWIL-UHFFFAOYSA-O 0.000 claims abstract description 37
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Abstract
The invention provides a high performance liquid chromatography detection method for simultaneously determining 11 flavonoids, belonging to the field of flavonoid detection, wherein the 11 flavonoids are delphinidin glucoside, delphinidin rutinoside, peony pigment glucoside, cyanidin rutinoside, cyanidin glucoside, rutin, kaempferol, quercetin, myricetin, isorhamnetin and apigenin. According to the invention, through accurate limitation on the chromatographic conditions of the high performance liquid chromatography, different types of flavonoid substances are detected at different retention times, the minimum separation degree of chromatographic peaks is 1.54, and the simultaneous determination of 11 types of flavonoids is realized. The data of the embodiment shows that the method realizes the simultaneous determination of the 11 kinds of flavonoids, is simple and quick to operate, has small standard deviation of peak area and retention time, and has high accuracy.
Description
Technical Field
The invention relates to the technical field of flavonoid content detection, in particular to a high performance liquid chromatography detection method for simultaneously detecting 11 types of flavonoids and a method for detecting the flavonoid content in fruits.
Background
Flavonoids, also called bioflavonoids, are polyphenol compounds which are most abundant in human diet, are widely stored in crops such as fruits and vegetables, and more than four thousand different flavonoids are confirmed at present, wherein the flavonoids with high content in fruits are flavonoids, flavonols and anthocyanin compounds. The flavonoid has strong oxidation resistance, free radical scavenging ability, and important biological activities of regulating cardiovascular system and endocrine system, resisting cancer and resisting aging.
The flavonoid analysis method comprises ultraviolet spectrophotometry, aluminum nitrate colorimetry, high performance liquid chromatography, capillary electrophoresis, chromatography-mass spectrometry, etc. Ultraviolet spectrophotometry and aluminum nitrate colorimetric methods are mostly used for determining the weight content of the flavonoid, capillary electrophoresis and chromatography-mass spectrometry are mostly used for identifying the component types, and the high performance liquid chromatography is mainly used for detecting the content of the flavonoid component. The high performance liquid chromatography has simple operation, high accuracy and good reproducibility, but because the detection conditions and methods of various components of anthocyanin, flavonol and flavonoid are greatly different, the detection is often carried out for many times in batches, and the work is complicated and the amount is large.
Disclosure of Invention
The invention aims to provide a high performance liquid chromatography detection method for simultaneously measuring 11 flavonoids. The detection method provided by the invention has the characteristics of wide measurement range and simple and rapid operation.
The invention provides a high performance liquid chromatography detection method for simultaneously determining 11 flavonoids, wherein the 11 flavonoids are delphinidin glucoside, delphinidin rutinoside, peony pigment glucoside, cyanidin rutinoside, cyanidin glucoside, rutin, kaempferol, quercetin, myricetin, isorhamnetin and apigenin, and the method comprises the following steps of:
(1) preparing a solution to be detected containing the 11 flavonoid standard samples;
(2) performing high performance liquid chromatography analysis on the solution to be detected obtained in the step (1) 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 C-18 column;
mobile phase: the water phase A is 0.3 wt% phosphoric acid water solution; and (3) organic phase B: acetonitrile;
flow rate: 1 mL/min;
and (3) an elution mode: gradient elution for 0min, A: B: 95:5, 20min, A: B: 85:15, 30min, A: B: 75:25, 40min, A: B: 50, 48min, A: B: 95: 5;
detection wavelength: 510nm and 360 nm;
detecting the temperature: 40 ℃;
sample introduction volume: 10 mu L of the solution;
detection time: 50 min;
a detector: a G1314F VWD detector;
(3) and comparing the chromatogram of the sample to be detected with a preset standard chromatogram to obtain the flavonoid species in the sample to be detected.
Preferably, the detection wavelength is 510nm when the detection time is (0,20) min, and the detection wavelength is 360nm when the detection time is [20,50] min.
Preferably, the method further comprises filtering the solution to be detected with a 0.22 μm organic filter membrane before the solution to be detected is subjected to high performance liquid chromatography to obtain a sample solution.
Preferably, the concentration of 11 flavonoids in the mixed standard solution is independently 2.5-500.0 [ mu ] g/mL.
Preferably, the C-18 column is InfinityLab Poroshell120 EC-C18, and the specification of the C-18 column is 4.6X 150 mm.
Preferably, the solvent used for preparing the solution to be tested in the step (1) is a methanol-hydrochloric acid mixed solution, the volume ratio of methanol to hydrochloric acid in the methanol-hydrochloric acid mixed solution is 98:2, and the mass fraction of hydrochloric acid is 30-36%.
The invention also provides a method for detecting flavonoids in fruits, which comprises the following steps:
(1) crushing a fresh fruit sample, performing first light-proof alcohol extraction, and performing solid-liquid separation to obtain a first filtrate and a first filter residue;
(2) repeatedly carrying out light-proof alcohol extraction on the first filter residue obtained in the step (1), and combining the filtrates to obtain a second filtrate;
(3) mixing the first filtrate and the second filtrate, and then sequentially diluting, centrifugally separating and filtering by using an organic filter membrane to obtain a solution to be detected, wherein the aperture of the organic filter membrane is 0.22 mu m;
(4) performing high performance liquid chromatography analysis on the liquid to be detected obtained in the step (3), and calculating by using an external standard method and a standard equation to obtain the content of flavonoids in the fruits, wherein the standard equation is an equation with the mass concentration of each flavonoid as an independent variable and a peak area as a dependent variable;
the chromatographic conditions of the high performance liquid chromatography are as follows:
and (3) analyzing the column: a C-18 column;
mobile phase: the water phase A is 0.3 wt% phosphoric acid water solution; and (3) organic phase B: acetonitrile;
flow rate: 1 mL/min;
and (3) an elution mode: gradient elution for 0min, A: B: 95:5, 20min, A: B: 85:15, 30min, A: B: 75:25, 40min, A: B: 50, 48min, A: B: 95: 5;
detection wavelength: 510nm and 360 nm;
detecting the temperature: 40 ℃;
sample introduction volume: 10 mu L of the solution;
detection time: 50 min;
a detector: G1314F VWD detector.
Preferably, the lixiviant used in the first light-proof alcohol extraction in the step (1) is a methanol-hydrochloric acid mixed solution, the volume ratio of methanol to hydrochloric acid in the methanol-hydrochloric acid mixed solution is 99:1, and the mass fraction of hydrochloric acid is 30-36%.
Preferably, the volume ratio of the mass of the fresh fruit sample to the volume of the leaching agent in the step (1) is 5g: 25-35 mL.
Preferably, the linear range of the standard equation in the step (4) is independently 5-500 [ mu ] g/mL.
The invention provides a high performance liquid chromatography detection method for simultaneously determining 11 flavonoids, wherein the 11 flavonoids are delphinidin glucoside, delphinidin rutinoside, paeoniflorin glucoside, cyanidin rutinoside, cyanidin glucoside, rutin, kaempferol, quercetin, myricetin, isorhamnetin and apigenin, a solution to be detected containing 11 flavonoid standard samples is prepared, a chromatogram of the sample to be detected is obtained, and the chromatographic conditions of high performance liquid chromatography analysis are as follows: and (3) analyzing the column: c-18 column, mobile phase: the water phase A is 0.3 wt% phosphoric acid water solution; and (3) organic phase B: acetonitrile, flow rate: 1mL/min, elution mode: gradient elution, 0min, a: B: 95:5, 20min, a: B: 85:15, 30min, a: B: 75:25, 40min, a: B: 50, 48min, a: B: 95:5, detection wavelength: 510nm, 360nm, detection temperature: 40 ℃, injection volume: 10 μ L, detection time: 50 min; a detector: G1314F VWD detector. The invention detects different flavonoid substances at different retention times through accurately defining the chromatographic conditions of the high performance liquid chromatography, the minimum separation degree of the chromatography is 1.54, and the simultaneous determination of 11 flavonoids can be realized. The data of the embodiment shows that the method realizes the simultaneous determination of the 11 kinds of flavonoids, is simple and quick to operate, has small standard deviation of peak area and retention time, and has high accuracy.
In addition, the method provided by the invention can simultaneously measure 11 flavonoids, can separate the flavonoids from other components and flavonoids in the fruits, and can realize the measurement of the flavonoids in the fruits through one-time detection.
Drawings
FIG. 1 is a high performance liquid chromatography chromatogram of example 1 of the present invention for simultaneous determination of 11 flavonoids;
FIG. 2 is a high performance liquid chromatography spectrum obtained in example 2 of the present invention;
FIG. 3 is a high performance liquid chromatography spectrum obtained in example 3 of the present invention;
FIG. 4 is a high performance liquid chromatography spectrum obtained in example 4 of the present invention;
FIG. 5 is a high performance liquid chromatography spectrum obtained in example 4 of the present invention;
FIG. 6 is a high performance liquid chromatography spectrum obtained in example 5 of the present invention.
Detailed Description
The invention provides a high performance liquid chromatography detection method for simultaneously determining 11 flavonoids, wherein the 11 flavonoids are delphinidin glucoside, delphinidin rutinoside, peony pigment glucoside, cyanidin rutinoside, cyanidin glucoside, rutin, kaempferol, quercetin, myricetin, isorhamnetin and apigenin, and the method comprises the following steps of:
(1) preparing a solution to be detected containing the 11 flavonoid standard samples;
(2) performing high performance liquid chromatography analysis on the solution to be detected obtained in the step (1) 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 C-18 column;
mobile phase: the water phase A is 0.3 wt% phosphoric acid water solution; and (3) organic phase B: acetonitrile;
flow rate: 1 mL/min;
and (3) an elution mode: gradient elution for 0min, A: B: 95:5, 20min, A: B: 85:15, 30min, A: B: 75:25, 40min, A: B: 50, 48min, A: B: 95: 5;
detection wavelength: 510nm and 360 nm;
detecting the temperature: 40 ℃;
sample introduction volume: 10 mu L of the solution;
detection time: 50 min;
a detector: G1314F VWD detector.
(3) And comparing the chromatogram of the sample to be detected with a preset standard chromatogram to obtain the flavonoid species in the sample to be detected.
In the invention, the 11 flavonoids comprise delphinidin glucoside, delphinidin rutinoside, peochrome glucoside, cyanidin rutinoside and cyanidin glucoside which are anthocyanins, rutin, kaempferol, quercetin, myricetin and isorhamnetin are flavonols, and apigenin is flavone.
The invention prepares a solution to be tested containing the 11 flavonoid standard samples. In the invention, the solvent used for preparing the solution to be tested containing the 11 flavonoid standard samples is preferably a methanol-hydrochloric acid mixed solution, the volume ratio of methanol to hydrochloric acid in the methanol-hydrochloric acid mixed solution is preferably 98:2, and the mass fraction of hydrochloric acid is preferably 30-36%, and more preferably 35%. In the invention, the 11 standard flavonoid samples are preferably high performance liquid chromatography pure products, and the purity of the 11 standard flavonoid samples is more than or equal to 99%.
In the present invention, the concentration of 11 flavonoids in the solution to be tested is preferably 2.5 to 500.0. mu.g/mL, more preferably 5.0 to 100.0. mu.g/mL, and most preferably 50.0. mu.g/mL.
In the present invention, the method further preferably comprises filtering the test solution with a 0.22 μm organic filter membrane before the test solution is subjected to the hplc analysis. In the invention, the filtration can remove impurities in the solution to be detected, and the influence on the detection result of the high performance liquid chromatography is avoided.
After a solution to be detected is obtained, the high performance liquid chromatography analysis is carried out on the solution to be detected, wherein the chromatographic conditions of the high performance liquid chromatography analysis are as follows:
and (3) analyzing the column: a C-18 column;
mobile phase: the water phase A is 0.3 wt% phosphoric acid water solution; and (3) organic phase B: acetonitrile;
flow rate: 1 mL/min;
and (3) an elution mode: gradient elution for 0min, A: B: 95:5, 20min, A: B: 85:15, 30min, A: B: 75:25, 40min, A: B: 50, 48min, A: B: 95: 5;
detection wavelength: 510nm and 360 nm;
detecting the temperature: 40 ℃;
sample introduction volume: 10 mu L of the solution;
detection time: 50 min;
a detector: G1314F VWD detector.
In the invention, the high performance liquid chromatography detection is carried out by adopting double wavelengths, when the detection time is (0,20) min, the detection wavelength is 510nm, and when the detection time is [20,50] min, the detection wavelength is 360 nm.
In the present invention, the C-18 column is preferably InfinityLab Poroshell120 EC-C18, and the specification of the C-18 column is 4.6X 150 mm.
The invention also provides a method for detecting flavonoids in fruits, which comprises the following steps:
(1) crushing a fresh fruit sample, performing first light-proof alcohol extraction, and performing solid-liquid separation to obtain a first filtrate and a first filter residue;
(2) repeatedly carrying out light-proof alcohol extraction on the first filter residue obtained in the step (1), and combining the filtrates to obtain a second filtrate;
(3) mixing the first filtrate and the second filtrate, and then sequentially diluting, centrifugally separating and filtering by using an organic filter membrane to obtain a solution to be detected, wherein the aperture of the organic filter membrane is 0.22 mu m;
(4) performing high performance liquid chromatography analysis on the liquid to be detected obtained in the step (3), and calculating by using an external standard method and a standard equation to obtain the content of the flavonoid in the fruit, wherein the standard equation is an equation with the mass concentration of 11 flavonoids as an independent variable and a peak area as a dependent variable;
the chromatographic conditions of the high performance liquid chromatography are as follows:
and (3) analyzing the column: a C-18 column;
mobile phase: the water phase A is 0.3 wt% phosphoric acid water solution; and (3) organic phase B: acetonitrile;
flow rate: 1 mL/min;
and (3) an elution mode: gradient elution for 0min, A: B: 95:5, 20min, A: B: 85:15, 30min, A: B: 75:25, 40min, A: B: 50, 48min, A: B: 95: 5;
detection wavelength: 510nm and 360 nm;
detecting the temperature: 40 ℃;
sample introduction volume: 10 mu L of the solution;
detection time: 50 min;
a detector: G1314F VWD detector.
In the present invention, the fresh fruit sample preferably comprises black gem plum peel, blueberry, black grape, dragon fruit or cherry.
According to the method, a fresh fruit sample is crushed and then subjected to first light-proof alcohol extraction to obtain first filtrate and first filter residue. In the present invention, the crushing is preferably carried out in a liquid nitrogen environment, and the grinding in the liquid nitrogen environment does not remove water in the fresh fruit sample. The grinding time is not particularly limited, and the fresh fruit sample can be ground into powder.
After the fresh fruit sample is crushed, the crushed fresh fruit sample is subjected to first light-proof alcohol extraction to obtain first filtrate and first filter residue. In the invention, the leaching agent used in the first light-proof alcohol extraction is preferably a methanol-hydrochloric acid mixed solution, the volume ratio of methanol to hydrochloric acid in the methanol-hydrochloric acid mixed solution is preferably 99:1, and the mass fraction of hydrochloric acid is preferably 30-36%, and more preferably 35%. In the invention, the ratio of the mass of the fresh fruit sample to the volume of the extracting agent is preferably 5g: 25-35 mL, and more preferably 5g:30 mL. In the invention, the first light-proof alcohol extraction time is preferably 6-12 h, and more preferably 8-10 h; the temperature of the first light-proof alcohol extraction is preferably room temperature, and no additional heating or cooling is needed.
After the first light-proof alcohol extraction is completed, the first filtrate and the first filter residue are obtained through solid-liquid separation. The specific manner of the solid-liquid separation is not particularly limited in the present invention, and a solid-liquid separation manner known to those skilled in the art may be adopted, specifically, filtration.
After the first filter residue is obtained, the first filter residue is repeatedly subjected to light-proof alcohol extraction, and the filtrates are combined to obtain a second filtrate. In the present invention, the light-proof alcohol extraction conditions are preferably the same as the first light-proof leaching conditions, and are not described herein again. In the invention, the light-resistant alcohol extraction is preferably performed for 2-5 times, and more preferably for 3-4 times. The invention is beneficial to realizing the complete extraction of flavonoid substances in fresh fruit samples through repeated light-resistant alcohol extraction. The invention combines the filtrates obtained by each lucifugal alcohol extraction to obtain a second filtrate.
After the first filtrate and the second filtrate are obtained, the first filtrate and the second filtrate are mixed and then sequentially diluted, centrifugally separated and filtered by an organic filter membrane to obtain the solution to be detected, wherein the aperture of the organic filter membrane is 0.22 mu m. The first filtrate and the second filtrate are mixed to obtain a mixed solution. The mixing method of the present invention is not particularly limited, and a mixing method known to those skilled in the art may be used.
After the mixed solution is obtained, the mixed solution is diluted to obtain the diluent. In the present invention, the dilution used for the dilution is preferably the same as the leaching agent described above, and will not be described herein. In the present invention, it is preferable that the volume ratio of the diluted solution to the mixed solution is preferably 2 to 5:1, more preferably 2.5 to 3.5: 1, the mixed solution is diluted to be within the linear range of a standard curve, so that the flavonoid content in the fruit can be calculated subsequently.
After the diluent is obtained, the invention centrifugally separates the diluent to obtain a sample solution. The rotating speed and time of the centrifugal separation are not particularly limited, preferably, the rotating speed of the centrifugal separation is 8000-10000 rpm, and more preferably 8500-9000 rpm; the centrifugal separation time is preferably 10-15 min, and more preferably 12 min; the temperature of the centrifugation is preferably 4 ℃. In the present invention, the centrifugation temperature is such as to avoid oxidation of the flavonoid.
After sample liquid is obtained, the invention filters the sample liquid through an organic filter membrane to obtain the liquid to be detected, and the aperture of the organic filter membrane is 0.22 μm. In the invention, the filtration can remove filter residues in the sample liquid, prevent large-particle substances from blocking a chromatographic column and simultaneously avoid influencing the detection result of the high performance liquid chromatography.
After the liquid to be detected is obtained, the high performance liquid chromatography analysis is carried out on the liquid to be detected, the content of the flavonoid in the fruit is calculated by an external standard method and a standard equation, wherein the standard equation takes the mass concentration of the 11 flavonoid as an independent variable and the peak area as a dependent variable. In the present invention, the chromatographic conditions for performing the HPLC analysis are the same as those of the HPLC for simultaneously measuring 11 kinds of flavonoids, and thus, the detailed description thereof is omitted.
In the present invention, the standard equation preferably includes the steps of: and (3) carrying out high performance liquid chromatography analysis on the solution to be detected of the 11 flavonoids with the concentration gradient of 2.5-500.0 mu g/mL by adopting the same method as the detection method of the high performance liquid chromatography for simultaneously detecting the 11 flavonoids, and obtaining a standard equation by taking the peak area as a vertical coordinate, the concentration as a horizontal coordinate and a linear equation of the peak area to the concentration.
In the present invention, the concentration gradient is preferably 2.5, 5.0, 50.0, 100.0, 500.0. mu.g/mL.
The linear range of the standard equation obtained by the invention is independently preferably 5-500 mu g/mL, and the lowest detection limit of the standard equation is 5 mu g/mL.
In order to further illustrate the present invention, the following examples are provided to describe the HPLC method for simultaneously measuring 11 kinds of flavonoids and the method for measuring the flavonoid content in fruits according to the present invention in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
Respectively weighing delphinidin glucoside, delphinidin rutinoside, peony pigment glucoside, cyanidin rutinoside, cyanidin glucoside, rutin and kaempferol in HPLC chromatogram purity (purity is more than or equal to 99 percent)Quercetin, myricetin, isorhamnetin, apigenin, and methanol-hydrochloric acid (V)Methanol:VHydrochloric acid98:2) were dissolved to prepare mixed standard solutions having concentrations of 2.5, 5.0, 50.0, 100.0, and 500.0 μ g/mL, and the solutions were filtered through a 0.22 μm organic filter and analyzed by high performance liquid chromatography.
And (3) carrying out high performance liquid chromatography analysis on the mixed standard solution, wherein the chromatographic conditions are as follows:
and (3) analyzing the column: c-18 column, InfinityLab Poroshell120 EC-C18, 4.6X 150 mm;
mobile phase: the water phase A is 0.3 wt% phosphoric acid water solution; and (3) organic phase B: acetonitrile;
flow rate: 1 mL/min;
and (3) an elution mode: gradient elution for 0min, A: B: 95:5, 20min, A: B: 85:15, 30min, A: B: 75:25, 40min, A: B: 50, 48min, A: B: 95: 5;
detection wavelength: 510nm and 360nm, wherein when the detection time is (0,20) min, the detection wavelength is 510nm, and when the detection time is [20,50] min, the detection wavelength is 360 nm;
detecting the temperature: 40 ℃;
sample introduction volume: 10 mu L of the solution;
detection time: 50 min;
a detector: G1314F VWD detector.
The HPLC detection spectrum of the mixed standard solution with a concentration of 2.5 μ g/mL is shown in FIG. 1, and it can be seen from FIG. 1 that the detection method provided by the present invention realizes the simultaneous determination of 11 kinds of flavones, in FIG. 1, 1 is delphinidin glucoside, 2 is delphinidin pigment rutinoside, 3 is cyanidin glucoside, 4 is cyanidin rutinoside, 5 is paeoniflorin glucoside, 6 is rutin, 7 is myricetin, 8 is quercetin, 9 is apigenin, 10 is isorhamnetin, and 11 is kaempferol.
The peak area is used as a vertical coordinate, the concentration is used as a horizontal coordinate, the peak area is used for making a linear equation to the concentration to obtain a standard equation of 11 kinds of flavone, the result is shown in table 1, and the standard deviation of the peak area and the retention time is smaller as can be seen from table 1.
TABLE 111 Standard equation, correlation coefficient, Linear Range, and relative Standard deviation results for Flavonoids
Wherein in the regression equation, y represents the peak area and x represents the concentration of flavonoid (. mu.g/mL).
Example 2
Taking 5g of black plum peel, grinding with liquid nitrogen, and adding 25ml methanol-hydrochloric acid solution (V)Methanol:VHydrochloric acid99:1) extracting for 12h in dark and filtering; filtering residue, and adding 25ml methanol-hydrochloric acid solution (V)Methanol:VHydrochloric acid99:1) repeatedly leaching for 2 times; all filtrates were collected with methanol-hydrochloric acid solution (V)Methanol:VHydrochloric acid99:1) adding the volume to 100ml to obtain a sample solution, centrifuging the sample solution at 8000rpm and 4 ℃ for 10min, and filtering by using a 0.22-micron organic filter membrane to obtain a solution to be detected for later use.
The detection conditions of the high performance liquid chromatography which are completely the same as those of the detection conditions of the high performance liquid chromatography in the embodiment 1 are adopted to detect the liquid to be detected, and the obtained high performance liquid chromatography spectrogram is shown in fig. 2, wherein 1 in fig. 2 is delphinidin glucoside, 2 is delphinidin rutinoside, 3 is cyanidin glucoside, 4 is cyanidin rutinoside, 5 is rutin, 6 is myricetin, and 7 is kaempferol.
The content of different kinds of flavonoids in the pericarp of prunus saphire was calculated according to the standard equation provided in example 1, and the results are shown in table 2, and it can be seen from fig. 2 and table 2 that example 2 realizes the determination of flavonoids in the pericarp of prunus saphire by one-time detection.
TABLE 2 Flavonoids content (μ g/g-FW) in the Black Gem plum peel
Among them, FW is the fresh weight (g) of fresh fruit, and it should be noted that in the analysis of the results in the above examples, only the content of flavonoids which can be detected is given, and the content is less than 5. mu.g/g.FW or is not detected and is not shown in the table.
Example 3
The same procedure as in example 2 was followed except that the pericarp of the black gem plum was replaced with cherry (pericarp and pulp). The obtained high performance liquid chromatography chromatogram is shown in FIG. 3, wherein 1 in FIG. 3 is cyanidin glucoside, 2 is cyanidin rutinoside, and 3 is rutin.
The contents of different kinds of flavonoids in the pericarp of prunus humilis bunge were calculated according to the standard equation provided in example 1, and the results are shown in table 3, and as can be seen from fig. 3 and table 3, example 3 achieved the determination of flavonoids in cherries by one-time detection.
TABLE 3 flavonoid content in cherry fruits (μ g/g-FW)
Among them, FW is the fresh weight (g) of fresh fruit, and it should be noted that in the analysis of the results in the above examples, only the content of flavonoids which can be detected is given, and the content is less than 5. mu.g/g.FW or is not detected and is not shown in the table.
Example 4
The same procedure as in example 2 was followed except that the black gem plum peel was replaced with blueberries (peel and pulp). The obtained high performance liquid chromatography chromatogram is shown in FIG. 4, wherein 1 in FIG. 4 is delphinidin glucoside, 2 is delphinidin rutinoside, 3 is cyanidin glucoside, 4 is cyanidin rutinoside, 5 is rutin, and 6 is myricetin.
The content of different types of flavonoids in the pericarp of the prunus saphire is calculated according to the standard equation provided in example 1, the result is shown in table 4, and as can be seen from fig. 4 and table 4, example 4 realizes the determination of the flavonoids in the blueberries through one-time detection.
TABLE 4 flavonoid content in blueberry fruit (μ g/g-FW)
Among them, FW is the fresh weight (g) of fresh fruit, and it should be noted that in the analysis of the results in the above examples, only the content of flavonoids which can be detected is given, and the content is less than 5. mu.g/g.FW or is not detected and is not shown in the table.
Example 5
The same procedure as in example 2 was followed except that the pericarp of the prunus sapiensis was replaced with dragon fruit (pulp). The obtained high performance liquid chromatography chromatogram is shown in FIG. 5, wherein 1 in FIG. 5 is delphinidin rutinoside, 2 is cyanidin rutinoside, 3 is peoniflorin glucoside, 4 is rutin, 5 is quercetin, 6 is isorhamnetin, and 7 is kaempferol.
The content of different kinds of flavonoids in the pericarp of prunus humilis bunge was calculated according to the standard equation provided in example 1, and the result is shown in table 5, and as can be seen from fig. 5 and table 5, example 5 realizes the determination of flavonoids in pitaya through one-time detection.
TABLE 5 flavonoid content in Pitaya pulp (μ g/g-FW)
Among them, FW is the fresh weight (g) of fresh fruit, and it should be noted that in the analysis of the results in the above examples, only the content of flavonoids which can be detected is given, and the content is less than 5. mu.g/g.FW or is not detected and is not shown in the table.
Example 6
The same procedure as in example 2 was followed except that the black gem plum peel was replaced with black grape (peel and pulp). The obtained high performance liquid chromatography chromatogram is shown in FIG. 6, wherein 1 in FIG. 6 is delphinidin glucoside, 2 is cyanidin rutinoside, 3 is rutin, and 4 is kaempferol.
The content of different kinds of flavonoids in the pericarp of prunus sapiensis was calculated according to the standard equation provided in example 1, and the results are shown in table 6, and it can be seen from fig. 6 and table 6 that example 6 realizes the determination of flavonoids in black grape through one-time detection.
TABLE 6 Flavonoids content in the Black grape fruit (. mu.g/g-FW)
Among them, FW is the fresh weight (g) of fresh fruit, and it should be noted that in the analysis of the results in the above examples, only the content of flavonoids which can be detected is given, and the content is less than 5. mu.g/g.FW or is not detected and is not shown in the table.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. 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 (9)
1. A high performance liquid chromatography detection method for simultaneously detecting 11 flavonoids, wherein the 11 flavonoids are delphinidin glucoside, delphinidin rutinoside, paeoniflorin glucoside, cyanidin rutinoside, cyanidin glucoside, rutin, kaempferol, quercetin, myricetin, isorhamnetin and apigenin, comprises the following steps:
(1) preparing a solution to be detected containing the 11 flavonoid standard samples;
(2) performing high performance liquid chromatography analysis on the solution to be detected obtained in the step (1) 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;
mobile phase: the water phase A is 0.3 wt% phosphoric acid water solution; and (3) organic phase B: acetonitrile;
flow rate: 1 mL/min;
and (3) an elution mode: gradient elution for 0min, A: B: 95:5, 20min, A: B: 85:15, 30min, A: B: 75:25, 40min, A: B: 50, 48min, A: B: 95: 5;
detecting the temperature: 40 ℃;
sample introduction volume: 10 mu L of the solution;
detection time: 50 min; when the detection time is (0,20) min, the detection wavelength is 510nm, and when the detection time is [20,50] min, the detection wavelength is 360 nm;
a detector: a G1314F VWD detector;
(3) and comparing the chromatogram of the sample to be detected with a preset standard chromatogram to obtain the flavonoid species in the sample to be detected.
2. The detection method according to claim 1, wherein the step of filtering the solution to be detected with a 0.22 μm organic filter membrane to obtain the sample solution is further included before the step of performing the HPLC analysis.
3. The method according to claim 1 or 2, wherein the concentration of 11 flavonoids in the solution to be tested is independently 2.5 to 500.0 μ g/mL.
4. The detection method according to claim 1, wherein the C18 column is InfinityLab Poroshell120 EC-C18, and the specification of the C-18 column is 4.6 x 150 mm.
5. The detection method according to claim 1, wherein a solvent used for preparing the solution to be detected in the step (1) is a methanol-hydrochloric acid mixed solution, a volume ratio of methanol to hydrochloric acid in the methanol-hydrochloric acid mixed solution is 98:2, and a mass fraction of the hydrochloric acid is 30-36%.
6. A method for detecting flavonoids in fruits comprises the following steps:
(1) crushing a fresh fruit sample, performing first light-proof alcohol extraction, and performing solid-liquid separation to obtain a first filtrate and a first filter residue;
(2) repeatedly carrying out light-proof alcohol extraction on the first filter residue obtained in the step (1), and combining the filtrates to obtain a second filtrate;
(3) mixing the first filtrate and the second filtrate, and then sequentially diluting, centrifugally separating and filtering by using an organic filter membrane to obtain a solution to be detected, wherein the aperture of the organic filter membrane is 0.22 mu m;
(4) performing high performance liquid chromatography analysis on the liquid to be detected obtained in the step (3), and calculating by using an external standard method and a standard equation to obtain the content of flavonoids in the fruits, wherein the standard equation is an equation with the mass concentration of each flavonoid as an independent variable and a peak area as a dependent variable;
the chromatographic conditions of the HPLC analysis are those in the HPLC detection method for simultaneously detecting 11 kinds of flavonoids according to claim 1.
7. The detection method according to claim 6, wherein a leaching agent used in the first light-proof alcohol extraction in the step (1) is a methanol-hydrochloric acid mixed solution, the volume ratio of methanol to hydrochloric acid in the methanol-hydrochloric acid mixed solution is 99:1, and the mass fraction of hydrochloric acid is 30-36%.
8. The detection method as claimed in claim 7, wherein the volume ratio of the mass of the fresh fruit sample to the volume of the leaching agent in the step (1) is 5g: 25-35 mL.
9. The detection method according to claim 6, wherein the linear range of the standard equation in the step (4) is independently 5 to 500 μ g/mL.
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