CN112557544B - Detection method of carotenoid metabolites of wolfberry - Google Patents
Detection method of carotenoid metabolites of wolfberry Download PDFInfo
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Abstract
The invention discloses a detection method of a wolfberry carotenoid metabolite, and relates to the technical field of detection methods. Taking a proper amount of medlar, preparing a sample solution after pretreatment, and detecting the medlar by using a liquid chromatography; the liquid chromatography conditions are as follows: chromatographic column: YM-C30 column (4.6X105 mm,5 μm); the mobile phase A is methanol/water solution, the mobile phase B is methyl tertiary butyl ether/methanol solution, and gradient elution is adopted, wherein the volume ratio of methanol to water in the methanol water solution is 15:85; the volume ratio of methyl tertiary butyl ether to methanol in the methyl tertiary butyl ether/methanol solution is 25:75. The method has the advantages of rapidness, high efficiency, stability, accuracy and the like, and can rapidly and effectively detect the carotenoid metabolites of the medlar.
Description
Technical Field
The invention relates to the technical field of detection methods, in particular to a detection method of carotenoid metabolites of wolfberry.
Background
Carotenoids are fat-soluble pigments found in plants and microorganisms, most of which are yellow, orange or red. Numerous medical studies have shown that carotenoids have a highly efficient quenching of singlet oxygen and scavenging of free radicals, thereby reducing the damage of free radicals to cytogenetic material and cell membranes. Epidemiological studies have shown that carotenoids play a more important role in protecting human health, for example, in preventing cardiovascular disease and cancer in humans. However, the human body cannot synthesize carotenoids by itself, and fruits and vegetables are important sources of carotenoids for human acquisition.
The medlar is a functional characteristic plant resource with homology of medicine and food, is rich in carotenoid, flavonoid substances, medlar polysaccharide and other bioactive substances, and is widely applied to the health-care food industry. The current method for analyzing carotenoids in medlar mainly comprises high performance liquid chromatography (HPLC, high Performance Liquid Chromatography), which is sensitive, good in repeatability and suitable for batch detection, wherein the selection of mobile phase is a key factor influencing the carotenoid separation effect.
However, the detection methods of carotenoids disclosed in the prior art cannot satisfy simultaneous detection of a plurality of carotenoids. In order to more comprehensively and effectively detect carotenoids in medlar, it is necessary to establish a rapid, simple, accurate and reliable analysis method for simultaneous qualitative and quantitative analysis of index components of various carotenoids in medlar.
Disclosure of Invention
The invention provides a detection method of carotenoid metabolites of medlar aiming at the technical problems in the prior art, and aims to solve the problems in the prior art.
The technical scheme for solving the technical problems is as follows:
a detection method of carotenoid metabolites of fructus Lycii comprises pretreating appropriate amount of fructus Lycii to obtain sample solution, and detecting by liquid chromatography; the liquid chromatography conditions are as follows: chromatographic column: YM-C30 column (4.6X105 mm,5 μm); the mobile phase A is methanol/water solution, the mobile phase B is methyl tertiary butyl ether/methanol solution, and gradient elution is adopted, wherein the volume ratio of methanol to water in the methanol water solution is 15:85; the volume ratio of methyl tertiary butyl ether to methanol in the methyl tertiary butyl ether/methanol solution is 25:75.
The beneficial effects of the invention are as follows: the HPLC method is used for detecting carotenoid metabolites in the medlar, and has the advantages of convenient detection, high detection sensitivity and high sample recovery rate.
On the basis of the technical scheme, the invention can be improved as follows.
In the detection method of the medlar carotenoid metabolite provided by the invention, the gradient elution program is as follows:
in the detection method of the medlar carotenoid metabolite provided by the invention, the detection wavelength is 450nm in the liquid chromatography condition.
In the detection method of the medlar carotenoid metabolite provided by the invention, the column temperature is 30 ℃ in the liquid chromatography condition.
In the detection method of the medlar carotenoid metabolite provided by the invention, the flow rate is 1ml/min in the liquid chromatography condition.
In the detection method of the medlar carotenoid metabolite provided by the invention, the preparation method of the test solution is as follows:
s1, grinding and crushing medlar, then mixing with a proper amount of mobile phase B, and filtering until the extracting solution is colorless;
s2: mixing the extractive solutions, concentrating under reduced pressure, and drying to obtain crude extract;
s3: dissolving the crude extract of S2 with methanol, and adding 30% methanol-KOH with equal volume to saponify for more than 4 hours to obtain saponified solution;
s4: concentrating the saponified solution under reduced pressure to minimum volume, adding appropriate amount of mobile phase B, and extracting until the extract is colorless;
and S5, merging the extracts in the step S4, washing alkali liquor during saponification by using water, and concentrating the upper layer of extract under reduced pressure to the minimum volume to obtain a concentrated carotenoid sample solution.
The invention also provides a detection method of the wolfberry carotenoid metabolite, which comprises the following steps:
(1) Preparation of test sample solution:
s1, grinding and crushing medlar, then mixing with a proper amount of mobile phase B, and filtering until the extracting solution is colorless;
s2: mixing the extractive solutions, concentrating under reduced pressure, and drying to obtain crude extract;
s3: dissolving the crude extract of S2 with methanol, and adding 30% methanol-KOH with equal volume to saponify for more than 4 hours to obtain saponified solution;
s4: concentrating the saponified solution under reduced pressure to minimum volume, adding appropriate amount of mobile phase B, and extracting until the extract is colorless;
s5, merging the extracts in the step S4, washing alkali liquor during saponification with water, and concentrating the upper layer of extract under reduced pressure to the minimum volume to obtain a concentrated carotenoid sample solution;
(2) Preparation of a control solution: taking proper amounts of neoxanthin, cordierite, epoxy zeaxanthin, lutein and beta-carotene reference substances, precisely weighing, adding methanol to prepare a mixed solution containing 10 mug of neoxanthin, 10 mug of cordierite, 10 mug of epoxy zeaxanthin, 10 mug of lutein and 60 mug of beta-carotene per 1 ml;
(3) And (3) detection: taking 10 μl of each of the control and test solutions, and loading into a liquid chromatographic column for detection, wherein the chromatographic conditions are as follows: chromatographic column: YM-C30 column (4.6X105 mm,5 μm); the mobile phase A is a methanol/water solution with the volume ratio of methanol to water being 15:85, the mobile phase B is a methyl tertiary butyl ether/methanol solution with the volume ratio of methyl tertiary butyl ether to methanol being 25:75, the detection wavelength is 450nm, the column temperature is 30 ℃, the flow rate is 1.0ml/min, and the gradient elution condition is as follows:
the beneficial effects of adopting the further scheme are as follows:
1. the method can detect and separate 6 components of neoxanthin, cordierite, epoxy zeaxanthin, lutein and beta-carotene in the medlar, can reflect the inherent quality characteristics of the medlar more comprehensively and truly, and saves time cost and reagent cost;
2. the detection time is reasonable, the peak time of the target substance is short, and the detection efficiency of the sample is greatly improved;
3. the separation degree of each target component is good, the baseline fluctuation is small, the background interference is small, and the components in the sample to be tested can be accurately detected;
4. the detection method has high stability, good repeatability, good linearity and high recovery rate through the invention researches on the repeatability, recovery rate, linearity and the like of the detection method.
Drawings
FIG. 1 high Performance liquid chromatography of sample of example 1
FIG. 2 is a high performance liquid chromatogram of example 2;
FIG. 3 high performance liquid chromatography of example 3 test sample;
FIG. 4 is a high performance liquid chromatogram of example 4;
FIG. 5 high performance liquid chromatography of example 5 test sample;
in the figure: 1. neoxanthin, 2, cordierite, 3, epoxy zeaxanthin, 4, zeaxanthin, 5, lutein, 6, beta-carotene.
Detailed Description
The principles and features of the present invention are described below in connection with specific embodiments, examples of which are provided for illustration only and are not intended to limit the scope of the invention.
The experimental instrument, medicine and reagent related in the invention are as follows:
LC-2010CHT high performance liquid chromatograph (Shimadzu)
Milli-Q pure water system (America MA)
Methanol is chromatographic pure, and the rest reagents are analytical pure.
Preparation of the solution:
preparing a reference substance solution: taking proper amounts of neoxanthin, cordierite, epoxy zeaxanthin, lutein and beta-carotene reference substances, precisely weighing, adding methanol to prepare a mixed solution containing 10 mug of neoxanthin, 10 mug of cordierite, 10 mug of epoxy zeaxanthin, 10 mug of lutein and 60 mug of beta-carotene per 1 ml;
preparation of test solution:
s1, grinding and crushing medlar, then mixing with a proper amount of mobile phase B, and filtering until the extracting solution is colorless;
s2: mixing the extractive solutions, concentrating under reduced pressure, and drying to obtain crude extract;
s3: dissolving the crude extract of S2 with methanol, and adding 30% methanol-KOH with equal volume to saponify for more than 4 hours to obtain saponified solution;
s4: concentrating the saponified solution under reduced pressure to minimum volume, adding appropriate amount of mobile phase B, and extracting until the extract is colorless;
s5, merging the extracts in the step S4, washing alkali liquor during saponification with water, and concentrating the upper layer of extract under reduced pressure to the minimum volume to obtain a concentrated carotenoid sample solution;
example 1
And (3) detection: taking 10 mu l of each of the reference substance solution and the test substance solution, and injecting into a liquid chromatograph for detection. Wherein, chromatographic conditions: the chromatographic column is YM-C30 chromatographic column (4.6X105 mm,5 μm); mobile phase a is a methanol/water solution with a volume ratio of methanol to water of 15:85, mobile phase B is a methyl tert-butyl ether/methanol solution with a volume ratio of methyl tert-butyl ether to methanol of 25:75, mobile phase a: mobile phase b=16: 84; the detection wavelength is 450nm; the theoretical plate number should be not less than 3000 calculated as lutein peak.
The results of the HPLC detection performed according to the above method are shown in FIG. 1. As can be seen from the results of FIG. 1, beta-carotene could not be detected, and the peaks of neoxanthin and cordierite overlapped, and the separation requirement could not be satisfied.
Example two
And (3) detection: taking 10 mu l of each of the reference substance solution and the test substance solution, and injecting into a liquid chromatograph for detection. Wherein, chromatographic conditions: the chromatographic column is YM-C30 chromatographic column (4.6X105 mm,5 μm); the mobile phase A is a methanol/water solution with the volume ratio of methanol to water being 15:85, and the mobile phase B is a methyl tertiary butyl ether/methanol solution with the volume ratio of methyl tertiary butyl ether to methanol being 25:75; the detection wavelength is 450nm; the theoretical plate number should be not less than 10000 calculated according to lutein peak, and the following gradient elution is adopted.
The results of the HPLC detection performed according to the above method are shown in FIG. 2. As can be seen from the results of FIG. 2, zeaxanthin and lutein cannot be completely separated, the separation requirement cannot be met, and the baseline drift of the detection gradient of beta-carotene is large.
Example III
And (3) detection: taking 10 mu l of each of the reference substance solution and the test substance solution, and injecting into a liquid chromatograph for detection. Wherein, chromatographic conditions: the chromatographic column is YM-C30 chromatographic column (4.6X105 mm,5 μm); the mobile phase A is a methanol/water solution with the volume ratio of methanol to water being 15:85, and the mobile phase B is a methyl tertiary butyl ether/methanol solution with the volume ratio of methyl tertiary butyl ether to methanol being 15:85; the detection wavelength is 450nm; the theoretical plate number should be not less than 10000 calculated according to lutein peak, and the following gradient elution is adopted.
The results of the HPLC detection performed according to the above method are shown in FIG. 3. From the results of FIG. 3, it is clear that the peaks of neoxanthin and cordierite overlap, the separation requirement is not satisfied, and β -carotene cannot be detected.
Example IV
And (3) detection: taking 10 mu l of each of the reference substance solution and the test substance solution, and injecting into a liquid chromatograph for detection. Wherein, chromatographic conditions: the chromatographic column is YM-C30 chromatographic column (4.6X105 mm,5 μm); the mobile phase A is a methanol/water solution with the volume ratio of methanol to water being 15:85, and the mobile phase B is a methyl tertiary butyl ether/methanol solution with the volume ratio of methyl tertiary butyl ether to methanol being 15:85; the detection wavelength is 450nm; the theoretical plate number should be not less than 10000 calculated according to lutein peak, and the following gradient elution is adopted.
The results of the HPLC detection performed in the above-described manner are shown in FIG. 4. From the results of FIG. 4, it is clear that the peaks of neoxanthin and cordierite overlap, the separation requirement is not satisfied, and β -carotene cannot be detected.
Example five
And (3) detection: taking 10 mu l of each of the reference substance solution and the test substance solution, and injecting into a liquid chromatograph for detection. Wherein, chromatographic conditions: the chromatographic column is YM-C30 chromatographic column (4.6X105 mm,5 μm); the mobile phase A is a methanol/water solution with the volume ratio of methanol to water being 15:85, and the mobile phase B is a methyl tertiary butyl ether/methanol solution with the volume ratio of methyl tertiary butyl ether to methanol being 25:75; the detection wavelength is 450nm; the theoretical plate number should be not less than 10000 calculated according to lutein peak, and the following gradient elution is adopted.
The results of the HPLC detection performed in the above-described manner are shown in FIG. 5. As can be seen from the results of FIG. 5, the separation degree of the 6 components is greater than 1.5, the detection effect is achieved, the gradient detection baseline of each component is the most stable, the interference is the least, and the detection effect is the best.
And (3) experimental verification:
linear relationship:
taking a mixed reference solution of neoxanthin, cordierite, epoxy zeaxanthin, lutein and beta-carotene (wherein the concentration of neoxanthin is 0.22mg/ml, the concentration of the cordierite is 0.28mg/ml, the concentration of the epoxy zeaxanthin is 0.75mg/ml, the concentration of the zeaxanthin is 0.64mg/ml, the concentration of the lutein is 0.58mg/ml and the concentration of the beta-carotene is 0.68 mg/ml), precisely measuring 0.5ml, 1ml, 2.5ml, 5ml and 10ml into a volumetric flask by a pipette, adding methanol to dilute to a scale, and shaking uniformly. According to the above chromatographic conditions, 10. Mu.l of each was aspirated and injected into a liquid chromatograph to measure the peak area. And (3) carrying out linear regression by taking the sample injection amounts (mug) of the neoxanthin, the cordierite, the epoxy zeaxanthin, the lutein and the beta-carotene as an abscissa and taking the peak areas (A) of the neoxanthin, the cordierite, the epoxy zeaxanthin, the lutein and the beta-carotene as an ordinate to respectively obtain standard curves of the neoxanthin, the cordierite, the epoxy zeaxanthin, the lutein and the beta-carotene. The results show that the components have good linear relation in the corresponding concentration range, and the linear range, regression equation and correlation coefficient are shown in the following table.
Linear relationship of carotenoid components in Chinese wolfberry
Repeatability:
taking the same batch of medlar samples, precisely weighing, preparing 6 parts of test solution in parallel according to the method under the test solution preparation item, carrying out sample injection measurement according to the chromatographic conditions, recording a chromatogram, and calculating RSD (reactive species decomposition) of neoxanthin, cordierite, epoxy zeaxanthin, lutein and beta-carotene content to be 1.2%, 1.3%, 0.8%, 1.1%, 0.9% and 0.9% respectively, wherein the RSD is less than 2.0% and the repeatability of the method is good.
Stability:
preparing a sample solution according to a sample preparation method; and respectively placing for 0, 2, 4, 8, 12 and 24 hours, injecting 10 μl of sample, and injecting into a liquid chromatograph to respectively determine peak areas of the neoxanthin, the cordierite, the epoxy-zeaxanthin, the lutein and the beta-carotene of the sample solution. RSD is calculated. The RSD of the contents of neoxanthin, cordierite, epoxy zeaxanthin, lutein and beta-carotene are calculated to be 1.6%, 1.4%, 1.8%, 1.9%, 1.7% and 1.3% respectively, which are less than 2.0%, so that the method is good in stability.
Sample recovery rate:
taking 6 parts of medlar samples, accurately weighing and placing the medlar samples with the content of about 0.1g into a conical bottle with a plug, respectively adding proper amounts of neoxanthin, cordierite, epoxy zeaxanthin, lutein and beta-carotene reference substances, preparing a sample-adding recovered sample solution according to the preparation method of the sample solution, carrying out sample injection measurement according to the chromatographic conditions, recording a chromatogram, and calculating the sample-adding recovery rate of the neoxanthin, the cordierite, the epoxy zeaxanthin, the lutein and the beta-carotene, wherein the results are shown in the following table.
Recovery measurement (n=6)
The above table shows that the sample recovery rate of each component in the sample is between 95.0% and 105.0%, the RSD% is less than 3.0%, and the test result shows that the sample recovery rate is good.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (1)
1. A detection method of a medlar carotenoid metabolite is characterized in that the detected medlar carotenoid metabolite is neoxanthin, cordierite, epoxy zeaxanthin, zeaxanthin and lutein, and the detection method is as follows:
(1) Preparation of test solution:
s1, grinding and crushing medlar, then mixing with a proper amount of mobile phase B, and filtering until the extracting solution is colorless;
s2: mixing the extractive solutions, concentrating under reduced pressure, and drying to obtain crude extract;
s3: dissolving the crude extract of S2 with methanol, and adding 30% methanol-KOH with equal volume to saponify for more than 4 hours to obtain saponified solution;
s4: concentrating the saponified solution under reduced pressure to minimum volume, adding appropriate amount of mobile phase B, and extracting until the extract is colorless;
s5, merging the extracts in the step S4, washing alkali liquor during saponification with water, and concentrating the upper layer of extract under reduced pressure to the minimum volume to obtain a concentrated carotenoid sample solution;
(2) Preparation of a control solution: taking proper amounts of neoxanthin, cordierite, epoxy zeaxanthin, zeaxanthin and lutein reference substances, precisely weighing, adding methanol to prepare a mixed solution containing 10 mug of neoxanthin, 10 mug of cordierite, 10 mug of epoxy zeaxanthin, 10 mug of zeaxanthin and 10 mug of lutein per 1 ml;
(3) And (3) detection: taking 10 μl of each of the control and test solutions, and loading into a liquid chromatographic column for detection, wherein the chromatographic conditions are as follows: chromatographic column: YM-C30 column 4.6X105 mm,5 μm; the mobile phase A is a methanol/water solution with the volume ratio of methanol to water being 15:85, the mobile phase B is a methyl tertiary butyl ether/methanol solution with the volume ratio of methyl tertiary butyl ether to methanol being 25:75, the detection wavelength is 450nm, the column temperature is 30 ℃, the flow rate is 1.0ml/min, and the gradient elution condition is as follows:
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| CN104749293A (en) * | 2015-04-21 | 2015-07-01 | 江苏省农业科学院 | Method for efficiently extracting carotenoids in yellow peach fruits and determining carotenoids in yellow peach fruits by liquid phase |
| CN108120788A (en) * | 2016-11-29 | 2018-06-05 | 康迎春 | HPLC analysis methods on five Carotenoids of detection |
| CN107561171B (en) * | 2017-07-06 | 2020-02-21 | 北京林业大学 | Efficient extraction and determination method for carotenoid in Chinese rose petals |
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| CN105717209A (en) * | 2016-01-29 | 2016-06-29 | 甘肃农业大学 | Method for extracting carotenoid components in pepper leaves and quantitatively detecting content of carotenoid components |
| CN110412153A (en) * | 2019-06-26 | 2019-11-05 | 西南大学 | A method of carotenoid metabolin is marked based on LC-MS/MS technology screening honeysuckle yellow petal |
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