CN112485357B - Method for rapidly detecting carotenoid components in wolfberry medicinal materials - Google Patents

Method for rapidly detecting carotenoid components in wolfberry medicinal materials Download PDF

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CN112485357B
CN112485357B CN202011474325.5A CN202011474325A CN112485357B CN 112485357 B CN112485357 B CN 112485357B CN 202011474325 A CN202011474325 A CN 202011474325A CN 112485357 B CN112485357 B CN 112485357B
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马学琴
陈欢
李艳婷
任如如
李楠
赵小军
王迎利
苏超
郭荣
张万年
牛阳
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Ningxia Medical University
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Abstract

The invention discloses a method for quickly detecting carotenoid components in a wolfberry medicinal material, in particular to a method for qualitatively and quantitatively detecting carotenoid active ingredients in the wolfberry medicinal material, which specifically comprises the steps of adopting a reference substance of the carotenoid component 1 in the medicinal material as a reference substance, and carrying out qualitative and quantitative calculation on other carotenoid components by utilizing relative retention time and relative correction factors between other components to be detected and the reference substance component. Preparing a reference solution, preparing a sample solution, measuring the relative retention time and relative correction factors of each reference, measuring the sample, and calculating the content of components; the method disclosed by the invention can be used for rapidly detecting the active ingredients and the content in the product, uses few reference substances, and can be used for simplifying the detection process, improving the detection efficiency and reducing the detection cost.

Description

Method for rapidly detecting carotenoid components in wolfberry medicinal materials
Technical Field
The invention relates to the technical field of detection of active ingredients in medicinal materials, in particular to a method for quickly detecting carotenoid components in a wolfberry medicinal material, and specifically relates to a method for qualitatively and quantitatively detecting the carotenoid components in the wolfberry medicinal material.
Background
Gou Qi Zi is recorded in Shen nong Ben Cao Jing (Shen nong's herbal), which is listed as the top grade. The pharmacopoeia of the people's republic of China records that wolfberry is derived from dried ripe fruits of Ningxia wolfberry Lycium barbarum L. The main pigment in the medlar is carotenoid, is one of important active ingredients in the medlar, and has the effects of resisting oxidation and tumors, improving the immunologic function of a human body, preventing and inhibiting tumors, preventing atherosclerosis and the like. In addition, the fruit of Lycium barbarum contains free carotenoid such as beta-carotene and zeaxanthin and carotenoid ester such as zeaxanthin dipalmitate and beta-cryptoxanthin palmitate, wherein the content of zeaxanthin dipalmitate is the highest.
Currently, ultraviolet-visible spectrophotometry, a method for removing most of fatty glyceride and free fatty acid in medlar pulp by saponification so as to determine free carotenoid, and a method for directly determining free carotenoid and carotenoid ester are commonly used for determining carotenoid. The ultraviolet-visible spectrophotometry method uses beta-carotene as a reference substance to measure the total amount of the carotenoid, and cannot accurately reflect the composition and the content of each component of the carotenoid in the medlar; although the saponification method can convert carotenoid esters into carotenoids, the saponification conditions and operation are influenced by many aspects, so that the free carotenoids are degraded to a certain extent, and the total content of the free carotenoids is reduced. In addition, the content determination by the existing liquid chromatography or liquid chromatography-mass spectrometry method mostly adopts an external standard method to determine the content of various carotenoid components in the wolfberry medicinal material or the extract, and various reference substances are needed, but the wolfberry carotenoid reference substances are expensive, difficult to supply and unstable, so that the experiment cost is high.
Therefore, the technical problem to be solved by the present invention is to provide a method for rapidly detecting carotenoid components in wolfberry fruit.
Disclosure of Invention
In view of the above, the invention provides a method for rapidly detecting carotenoid components in a wolfberry medicinal material, which only measures one component by utilizing the inherent functional relationship and proportional relationship of the components to realize the synchronous control of multiple components, can rapidly detect the carotenoid components and the content in the wolfberry medicinal material by adopting 1 component in the wolfberry medicinal material as a reference, uses few reference substances, and can simplify the detection process, improve the detection efficiency and reduce the detection cost.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for rapidly detecting carotenoid components in a wolfberry medicinal material specifically comprises the following steps:
s1 reference solution
Weighing a reference substance, and respectively adding a solvent into the reference substance to prepare a reference substance solution; wherein the reference substance is at least one of lutein, zeaxanthin, beta-carotene, beta-cryptoxanthin palmitate and zeaxanthin dipalmitate;
s2 sample solution preparation
Weighing a sample to be detected, and adding a solvent into the sample to be detected to obtain a sample solution;
s3 liquid chromatography detection
Respectively carrying out liquid chromatography detection on the reference substance solution and the sample solution, respectively obtaining the retention time and the peak area of each reference substance including the reference substance and the reference substance by taking the zeaxanthin as the reference substance, and simultaneously reading the retention time and the peak area of each chromatographic peak of the component to be detected and the retention time and the peak area of the chromatographic peak of the reference substance in the detection result of the sample solution;
s4 determining relative retention time and relative correction factor of each control
Taking zeaxanthin as a reference substance, respectively calculating the ratio of the chromatographic peak retention time of each reference substance to the chromatographic peak retention time of the reference substance to obtain the relative retention time of the reference substance, respectively calculating the concentration and peak area ratio of each reference substance to obtain the correction factor of each reference substance, and then calculating the ratio of the correction factor of each reference substance to the correction factor of the reference substance to obtain the relative correction factor of each reference substance;
calculation of attribution and content of S5 component chromatographic peak
Determining the attribution of each component to be detected in the detection chromatographic peak of the sample solution according to the relative retention time; and respectively calculating the contents of lutein, zeaxanthin, beta-carotene, beta-cryptoxanthin palmitate and zeaxanthin dipalmitate in the sample according to the relative correction factor, the peak area of the chromatographic peak of each component to be detected, the concentration of the reference substance and the peak area of the chromatographic peak of the reference substance.
S6 calculating the relative deviation between the results of the measurement by the method and the external standard method
The content of each component to be measured calculated by the method through the relative correction factor is compared with the content of each component to be measured by the external standard method, and the relative deviation of the measurement result of the method and the measurement result of the external standard method is calculated so as to determine the accuracy of the result of the method.
Preferably, the solvent in step S1 and step S2 is the same, and the solvent is at least one of methanol, ethanol, and water.
More preferably, the step S1 is specifically: adding solvent into the reference substances respectively to obtain reference substance solutions with concentration of 0.1-1 mg/mL.
More preferably, the step S2 is specifically: precisely weighing a sample to be measured, adding 10-50 times of solvent, performing reflux, ultrasonic treatment or cold soaking treatment, and filtering to obtain a sample solution.
Preferably, in step S3, the chromatographic conditions of the liquid chromatography detection are: the packing material is a chromatographic column of C30, the inner diameter of the chromatographic column is 4.6mm, the length of the chromatographic column is 250mm, the grain diameter of the packing material is 5 mu m, the elution conditions are eluent A-eluent B, and gradient elution is carried out: 70-50% of eluent A and 30-50% of eluent B in 0-20 min; 50% of eluent A and 50% of eluent B in 20-48 min; 50-70% of eluent A and 50-30% of eluent B in 48-50 min; 70% of eluent A and 30% of eluent B in 50-55 min; the flow rate is 1 mL/min; sample introduction amount: 20 mu l of the mixture; column temperature: 20 ℃; the detection wavelength is 450 nm; the eluent A is methanol: acetonitrile: a water (81:14:5) solution, and the eluent B is dichloromethane.
Preferably, in step S4, the calculation formula of the relative retention time R is:
R＝Rx/Rs；
in the formula, RxAnd RsThe retention time of the component to be measured and the retention time of the reference substance are respectively.
And, a relative correction factor fx/sThe calculation formula of (2) is as follows:
Figure BDA0002834561900000031
f isx/sIs the relative correction factor of each component, said fxIs the correction factor of the component to be measured, said fsIs a reference correction factor, said CxIs the concentration of the reference solution of the component to be measured, CsAs concentration of reference control solution, said AsIs the peak area of chromatographic peak of reference substance control solution, AxIs the peak area of chromatographic peak of the reference solution of the component to be measured.
Preferably, in the step S5, the content W of the component to be measuredxThe calculation formula of (2) is as follows:
Figure BDA0002834561900000041
the W isxIs the content of the component to be measured, CxIs the concentration of the component to be measured in the sample solution, V is the volume of the sample solution, m is the sample weighing amount of the sample, AxIs the peak area of chromatographic peak of each component to be measured, CsAs concentration of reference control solution, said fx/sIs a relative correction factor, said AsIs the peak area of the reference substance chromatographic peak in the sample solution.
Preferably, in step S6, the calculation formula of the relative deviation (statistical ethoddiference) SMD is:
Figure BDA0002834561900000042
the SMD is a relative deviation; the W isExternal standard methodFor the content of the component to be measured by the external standard method, WThis methodIs the content of the component to be measured by the method of the present invention.
According to the technical scheme, compared with the prior art, the invention discloses a qualitative and quantitative detection method for carotenoid components in a wolfberry medicinal material, and the method has the following beneficial effects:
(1) according to the method, a reference object is selected, and a relative correction factor of a component to be detected and the reference object is obtained through peak area and concentration calculation;
(2) according to the invention, by establishing the relative correction factor, the content of 5 components of lutein, zeaxanthin, beta-carotene, beta-cryptoxanthin palmitate and zeaxanthin dipalmitate can be simultaneously determined according to the measurement result of the reference substance in the process of determining the content of the reference substance of the medlar;
(3) the method has the advantages of no significant difference between the content measurement result and the external standard method commonly used for pharmaceutical analysis, simplicity, convenience, high accuracy, great saving of experiment cost and suitability for popularization and application.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a HPLC chromatogram of a xanthophyll control solution;
FIG. 2 is an HPLC chromatogram of a zeaxanthin control solution;
FIG. 3 is an HPLC chromatogram of a beta-carotene control solution;
FIG. 4 is an HPLC chromatogram of a beta-cryptoxanthin palmitate control solution;
FIG. 5 is an HPLC chromatogram of a zeaxanthin dipalmitate control solution;
FIG. 6 is a HPLC chromatogram of a mixed control solution;
FIG. 7 is an HPLC chromatogram of a sample solution of fructus Lycii.
In the figure: 1-lutein; 2-zeaxanthin; 3-beta-carotene; 4- β -cryptoxanthin palmitate; 5-zeaxanthin dipalmitate.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A method for rapidly detecting carotenoid components in a wolfberry medicinal material specifically comprises the following steps:
s1 reference solution
Respectively weighing reference substances, adding solvent to obtain reference substance solution, wherein the reference substances comprise lutein, zeaxanthin, beta-carotene, beta-cryptoxanthin palmitate and zeaxanthin dipalmitate.
S2 sample solution preparation
And (3) precisely weighing a sample to be measured, carrying out reflux, ultrasonic treatment or cold soaking treatment, and filtering to obtain a clear sample solution.
S3 liquid chromatography detection
And respectively carrying out liquid chromatography detection on the reference substance solution and the sample solution, respectively taking the zeaxanthin as a reference substance, respectively obtaining the retention time and the peak area of each reference substance including the reference substance, and simultaneously reading the retention time and the peak area of each chromatographic peak of the component to be detected in the detection result of the sample solution and the retention time and the peak area of the chromatographic peak of the reference substance.
The chromatographic conditions of the liquid chromatographic detection are as follows: the packing material is a chromatographic column of C30, the inner diameter of the chromatographic column is 4.6mm, the length of the chromatographic column is 250mm, the grain diameter of the packing material is 5 mu m, the elution conditions are eluent A-eluent B, and gradient elution is carried out: 70-50% of eluent A and 30-50% of eluent B in 0-20 min; 50% of eluent A and 50% of eluent B in 20-48 min; 50-70% of eluent A and 50-30% of eluent B in 48-50 min; 70% of eluent A and 30% of eluent B in 50-55 min; the flow rate is 1 ml/min; sample introduction amount: 20 mu l of the mixture; column temperature: 20 ℃; the detection wavelength is 450 nm; the eluent A is methanol: acetonitrile: a water (81:14:5) solution, and the eluent B is dichloromethane.
S4 determining relative retention time and relative correction factor of each control
Taking zeaxanthin as a reference substance, and respectively calculating the ratio of the retention time of the chromatographic peak of each reference substance to the retention time of the chromatographic peak of the reference substance to obtain the relative retention time of the reference substance; respectively calculating the concentration and peak area ratio of each reference substance to obtain the correction factor of each reference substance, and then calculating the ratio of the correction factor of each reference substance to the correction factor of the reference substance to obtain the relative correction factor of each reference substance;
the relative retention time R is calculated as:
R＝Rx/Rs；
the R isxAnd RsThe retention time of the component to be measured and the retention time of the reference substance are respectively.
Relative correction factor fx/sThe calculation formula of (2) is:
Figure BDA0002834561900000061
f isx/sIs the relative correction factor of each component, said fxIs the correction factor of the component to be measured, said fsIs a reference correction factor, said CxIs the concentration of the reference solution of the component to be measured, CsAs concentration of reference control solution, said AsIs the peak area of chromatographic peak of reference substance control solution, AxIs the peak area of chromatographic peak of the reference solution of the component to be measured.
S5 ingredient chromatographic peak attribution and content calculation
Determining the attribution of each component to be detected in the detection chromatographic peak of the sample solution according to the relative retention time; calculating the lutein, the lutein-A-B-C in the sample respectively from the relative correction factor, the peak area of the chromatographic peak of each component to be detected, the concentration of the reference substance solution and the peak area of the chromatographic peak of the reference substance,The content W of zeaxanthin, beta-carotene, beta-cryptoxanthin palmitate and zeaxanthin dipalmitatex；
The calculation formula adopted is as follows:
Figure BDA0002834561900000062
the W isxIs the content of the component to be measured, CxIs the concentration of the component to be measured in the sample solution, V is the volume of the sample solution, m is the sample weighing amount of the sample, AxIs the peak area of chromatographic peak of each component to be measured, CsAs concentration of the reference stock solution, said fx/sIs a relative correction factor, said AsIs the peak area of the reference substance chromatographic peak in the sample solution.
S6 calculation of relative deviation between measurement results of original method and external standard method
The content of each component to be measured calculated by the method through the relative correction factor is compared with the content of each component to be measured by the external standard method, and the relative deviation (standard method) SMD of the measurement results of the method and the external standard method is calculated to determine the accuracy of the result of the method.
The calculation formula adopted is as follows:
Figure BDA0002834561900000071
the SMD is a relative deviation; the W isExternal standard methodFor the content of the component to be measured by the external standard method, WThis methodThe content of the component to be measured is determined by the method. Generally regarded as SMD<The two were not significantly different at 5%.
In order to further optimize the technical scheme, the solvent comprises at least one of methanol, ethanol or water.
The technical solution disclosed and protected by the present invention will be further described with reference to the following specific embodiments.
Example 1
The embodiment of the invention discloses a method for quickly detecting carotenoid components in a wolfberry medicinal material, which specifically comprises the following steps:
s1 reference solution
Taking lutein, zeaxanthin, beta-carotene, beta-cryptoxanthin palmitate and zeaxanthin dipalmitate reference substances respectively, precisely weighing, adding methanol, and preparing into lutein reference substance solution, zeaxanthin reference substance solution, beta-carotene reference substance solution, beta-cryptoxanthin palmitate reference substance solution and zeaxanthin dipalmitate reference substance solution respectively, to obtain 5 reference substance solutions with concentrations of 1 mg/ml.
S2 sample solution preparation
Taking at least one of fructus Lycii material or fructus Lycii extract as sample, precisely weighing 0.01g sample, adding 10ml methanol into the sample, ultrasonic extracting for 30min, cooling, adding methanol to supplement the weight loss, and filtering to obtain fructus Lycii material sample solution.
S3 liquid chromatography detection
Respectively carrying out liquid chromatography detection on the obtained reference substance solution and the sample solution, wherein the chromatographic conditions are as follows: the packing material is a chromatographic column of C30, the inner diameter of the chromatographic column is 4.6mm, the length of the chromatographic column is 250mm, the grain diameter of the packing material is 5 mu m, the elution conditions are eluent A-eluent B, and gradient elution is carried out: 70-50% of eluent A and 30-50% of eluent B in 0-20 min; 50% of eluent A and 50% of eluent B in 20-48 min; 50-70% of eluent A and 50-30% of eluent B in 48-50 min; 70% of eluent A and 30% of eluent B in 50-55 min; the flow rate is 1 ml/min; sample introduction amount: 20 mu l of the mixture; column temperature: 20 ℃; the detection wavelength is 450 nm; the eluent A is methanol: acetonitrile: a water (81:14:5) solution, and the eluent B is dichloromethane. The results for each of the control solutions and the sample solutions are shown in FIGS. 1-7.
S4 determining relative retention time and relative correction factor of each control
Taking zeaxanthin as a reference substance and the retention time of the zeaxanthin as a reference retention time, and calculating the relative correction factors of the 5 components to be detected and the reference substance by adopting the following formula. According to the formula of the calculation of the relative retention time R as
R＝Rx/Rs
In the formula RxAnd RsThe retention time of the component to be measured and the retention time of the reference substance are respectively.
And calculating the ratio of the retention time of the reference substance chromatographic peak to the retention time of the reference substance zeaxanthin chromatographic peak to obtain the relative retention time of the reference substance, thereby determining the attribution of the chromatographic peaks of 5 components to be detected, namely lutein, zeaxanthin, beta-carotene, beta-cryptoxanthin palmitate and zeaxanthin dipalmitate in the chromatographic peaks of the sample solution, and further obtaining the peak areas of the chromatographic peaks of the components to be detected, wherein the relative retention time of each component to be detected and the zeaxanthin reference peak is shown in the following table 1.
Table 1 relative corrected retention time of each component with zeaxanthin as reference (n-5)
Figure BDA0002834561900000081
As shown in table 1, the ratio of the retention time of the lutein to the zeaxanthin chromatographic peak is 0.91, the ratio of the zeaxanthin chromatographic peak to the zeaxanthin chromatographic peak is 1.00, the ratio of the beta-carotene chromatographic peak to the zeaxanthin chromatographic peak is 2.44, the ratio of the beta-cryptoxanthin palmitate chromatographic peak to the zeaxanthin chromatographic peak is 3.92, and the ratio of the zeaxanthin dipalmitate chromatographic peak to the zeaxanthin chromatographic peak is 6.34. When the content of the above 5 components in the subsequent wolfberry medicinal materials is measured, the components of each chromatographic peak can be determined according to the ratio of the retention time of the chromatographic peak.
Taking zeaxanthin as a reference substance, and calculating by adopting the following formula to obtain relative correction factors of other 5 components to be detected and the reference substance.
Relative correction factor fx/sThe calculation formula of (2) is:
Figure BDA0002834561900000091
fx/sis the relative correction factor of each component, fxIs the correction factor of the component to be measured, fsIs a reference correction factor, CxIs the concentration of the component to be measured in the control solution, CsConcentration of reference control solution, AsAs the peak area of the chromatographic peak of the reference control solution, AxIs the peak area of chromatographic peak of the reference solution of the component to be measured.
The final calculated relative correction factors for lutein, zeaxanthin, beta-carotene, beta-cryptoxanthin palmitate and zeaxanthin dipalmitate are shown in table 2 below.
Table 2 relative correction factor for each component using zeaxanthin as a reference (n-5)
Figure BDA0002834561900000092
From the results in Table 2 above, it can be seen that the relative correction factor f of lutein isLutein/zeaxanthinIs 1.37, relative correction factor f for zeaxanthinZeaxanthin/zeaxanthinIs a relative correction factor f for 1.00, beta-caroteneBeta-carotene/zeaxanthinIs a relative correction factor f for 1.11, beta-cryptoxanthin palmitateBeta-cryptoxanthin palmitate/zeaxanthinIs 1.41, relative correction factor f for zeaxanthin dipalmitateZeaxanthin dipalmitate/zeaxanthinIs 1.16.
Calculation of attribution and content of S5 component chromatographic peak
Determining the attribution of each component to be detected in the detection chromatographic peak of the sample solution according to the relative retention time; respectively calculating the contents W of lutein, zeaxanthin, beta-carotene, beta-cryptoxanthin palmitate and zeaxanthin dipalmitate in the sample according to the relative correction factor, the peak area of the chromatographic peak of each component to be detected, the concentration of the reference substance stock solution and the peak area of the chromatographic peak of the reference substancex；
Wherein the formula is
Figure BDA0002834561900000093
WxIs the content of the component to be measured, CxThe concentration V of the component to be measured in the sample solution is the volume of the sample solution, m is the sample weighing amount, AxIs the peak area of chromatographic peak of each component to be measured, CsConcentration of stock solution as reference, fx/sIs a relative correction factor, AsIs the peak area of the reference substance chromatographic peak in the sample solution.
The contents of 4 ingredients in the wolfberry fruit by the method of the present invention in example 1 are shown in table 3 below.
TABLE 3 comparison of the results of the simultaneous determination of 4 carotenoid components in fructus Lycii by the external standard method and the present method (μ g/g)
Figure BDA0002834561900000101
Note: lutein was not detected in the collected lycium barbarum samples; q1-10 refers to fructus Lycii material of different batches.
As can be seen from the results shown in Table 3, the results obtained by comparing the results of the method for determining the content of 4 components in each wolfberry fruit medicinal material with the results obtained by the external standard method commonly used in pharmaceutical analysis are close to each other, which indicates that the results obtained by the method are reliable.
S6 calculation of relative deviation between measurement results of original method and external standard method
The content of each component to be measured calculated by the method through the relative correction factor is compared with the content of each component to be measured by the external standard method, and the relative deviation (standard method) SMD of the measurement results of the method and the external standard method is calculated to determine the accuracy of the result of the method.
Wherein the formula is
Figure BDA0002834561900000102
The SMD is a relative deviation; w isExternal standard methodFor the content of the component to be measured by the external standard method, WThis methodThe content of the component to be measured is determined by the method.
TABLE 4 relative deviation of the content of 4 carotenoid components in Lycium barbarum determined by the external standard method and the method
Figure BDA0002834561900000103
Figure BDA0002834561900000111
As can be seen from the test results in Table 4, the relative deviation of the content of the 4 components in each wolfberry fruit measured by the method is less than 1% compared with the result obtained by the external standard method commonly used in pharmaceutical analysis, which indicates that the result obtained by the method has no significant difference from the result obtained by the external standard method, i.e., the result obtained by the method is reliable.
The invention provides a method for rapidly qualitatively and quantitatively detecting 5 carotenoid components in a wolfberry medicinal material, which selects zeaxanthin which has stable properties, is cheap and is easy to obtain as a reference substance, and calculates a relative correction factor according to the comparison between the peak area and the concentration of each component and the peak area and the concentration of the reference substance zeaxanthin; once the relative correction factor is established, when the content of the carotenoid components in the subsequent wolfberry medicinal materials is measured, only 1 zeaxanthin reference substance is needed, the content of 5 components such as lutein, zeaxanthin, beta-carotene, beta-cryptoxanthin palmitate and zeaxanthin dipalmitate can be simultaneously measured, and the content measurement result has no significant difference from the external standard method commonly used for pharmaceutical analysis. The method is simple to operate, low in cost, fast and accurate.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (8)

1. A method for rapidly detecting carotenoid components in wolfberry medicinal materials is characterized in that 1 kind of zeaxanthin control substances contained in the wolfberry medicinal materials are adopted, and the content of 5 carotenoid components such as lutein, zeaxanthin, beta-carotene, beta-cryptoxanthin palmitate and zeaxanthin dipalmitate in the wolfberry is simultaneously measured and calculated by adopting a liquid chromatography; wherein the relative retention time and relative correction factor of lutein and zeaxanthin are 0.86-0.96 and 1.30-1.44, respectively; the relative retention time and relative correction factor of zeaxanthin and zeaxanthin are 1.00, respectively; the relative retention time and relative correction factor of beta-carotene and zeaxanthin are respectively 2.32-2.56 and 1.05-1.17; the relative retention time and relative correction factor of the beta-cryptoxanthin palmitate and the zeaxanthin are respectively 3.72-4.12 and 1.34-1.48; the relative retention time and relative correction factor of zeaxanthin dipalmitate and zeaxanthin are respectively 6.02-6.66, 1.10-1.22;
wherein, the chromatographic conditions of the liquid chromatographic detection are as follows: the chromatographic column is C30, the inner diameter of the chromatographic column is 4.6mm, the length of the chromatographic column is 250mm, the particle size of the filler is 5 mu m, the elution conditions are eluent A-eluent B, and gradient elution is carried out: 70-50% of eluent A and 30-50% of eluent B in 0-20 min; 50% of eluent A and 50% of eluent B in 20-48 min; 50-70% of eluent A and 50-30% of eluent B in 48-50 min; 70% of eluent A and 30% of eluent B in 50-55 min; the flow rate is 1 mL/min; sample introduction amount: 20 mu l of the mixture; column temperature: 20 ℃; the detection wavelength is 450 nm; the eluent A is 81:14:5 methanol: acetonitrile: the eluent B is dichloromethane; the extraction solvent of the sample to be detected is at least one of methanol and ethanol.
2. The method of claim 1, wherein the relative retention time and relative calibration factor of lutein and zeaxanthin are 0.91 and 1.37, respectively; the relative retention time and relative correction factors of zeaxanthin and zeaxanthin are 1.00 and 1.00, respectively; the relative retention time and relative correction factor of beta-carotene and zeaxanthin were 2.44, 1.11, respectively; the relative retention time and relative correction factor of β -cryptoxanthin palmitate with zeaxanthin were 3.929, 1.41, respectively; relative retention time and relative correction factor for zeaxanthin dipalmitate and zeaxanthin were 6.34, 1.16, respectively.
3. The method for rapidly detecting carotenoid components in wolfberry fruit as claimed in claim 1, comprising the following steps:
s1 reference solution
Weighing a reference substance, and respectively adding a solvent into the reference substance to prepare a reference substance solution; wherein the reference substance is lutein, zeaxanthin, beta-carotene, beta-cryptoxanthin palmitate, and zeaxanthin dipalmitate;
s2 sample solution preparation
Weighing a sample to be detected, and adding a solvent into the sample to be detected to obtain a sample solution;
s3 liquid chromatography detection
Respectively carrying out liquid chromatography detection on the reference substance solution and the sample solution, respectively obtaining the retention time and the peak area of each reference substance including the reference substance and the reference substance by taking the zeaxanthin as the reference substance, and simultaneously reading the retention time and the peak area of each chromatographic peak of the component to be detected and the retention time and the peak area of the chromatographic peak of the reference substance in the detection result of the sample solution;
s4 determining relative retention time and relative correction factor of each control
Taking zeaxanthin as a reference substance, respectively calculating the ratio of the chromatographic peak retention time of each reference substance to the chromatographic peak retention time of the reference substance to obtain the relative retention time of the reference substance, respectively calculating the concentration and peak area ratio of each reference substance to obtain the correction factor of each reference substance, and then calculating the ratio of the correction factor of each reference substance to the correction factor of the reference substance to obtain the relative correction factor of each reference substance;
calculation of attribution and content of S5 component chromatographic peak
Determining the attribution of each component to be detected in the detection chromatographic peak of the sample solution according to the relative retention time; respectively calculating the contents of lutein, zeaxanthin, beta-carotene, beta-cryptoxanthin palmitate and zeaxanthin dipalmitate in the sample according to the relative correction factor, the peak area of the chromatographic peak of each component to be detected, the concentration of the reference substance and the peak area of the chromatographic peak of the reference substance;
s6 calculating the relative deviation between the results of the measurement by the method and the external standard method
The content of each component to be measured calculated by the method through the relative correction factor is compared with the content of each component to be measured by the external standard method, and the relative deviation of the measurement result of the method and the measurement result of the external standard method is calculated so as to determine the accuracy of the result of the method.
4. The method for quantitatively detecting carotenoid components in wolfberry, as claimed in claim 3, wherein the step S1 specifically comprises: adding solvent into the reference substances respectively to obtain reference substance solutions with concentration of 0.1-1 mg/mL.
5. The method for rapidly detecting carotenoid components in wolfberry, as claimed in claim 3, wherein the step S2 specifically comprises: precisely weighing a sample to be measured, adding 10-50 times of solvent, performing reflux, ultrasonic treatment or cold soaking treatment, and filtering to obtain a sample solution.
6. The method of claim 3, wherein in step S4, the calculation formula of the relative retention time R is as follows:
R＝Rx/Rs；
in the formula, RxAnd RsRespectively the retention time of the component to be detected and the retention time of the reference substance;
and, a relative correction factor fx/sThe calculation formula of (2) is as follows:
Figure FDA0003560598050000031
f isx/sIs the relative correction factor of each component, said fxIs the correction factor of the component to be measured, said fsIs a reference correction factor, said CxIs the concentration of the reference solution of the component to be measured, CsAs concentration of reference control solution, said AsIs the peak area of chromatographic peak of reference substance control solution, AxIs the peak area of chromatographic peak of the reference solution of the component to be measured.
7. The method for rapidly detecting carotenoid components in wolfberry fruit as claimed in claim 3, wherein in step S5, the content W of the component to be detectedxThe calculation formula of (2) is as follows:
Figure FDA0003560598050000032
the W isxIs the content of the component to be measured, CxIs the concentration of the component to be measured in the sample solution, V is the volume of the sample solution, m is the sample weighing amount of the sample, AxIs the peak area of chromatographic peak of each component to be measured, CsAs concentration of the reference stock solution, said fx/sIs a relative correction factor, said AsIs the peak area of the reference substance chromatographic peak in the sample solution.
8. The method as claimed in claim 1, wherein in step S6, the calculation formula of the relative deviation SMD is as follows:
Figure FDA0003560598050000033
the SMD is relative deviation; the W isExternal standard methodFor the content of the component to be measured by the external standard method, WThis methodThe content of the component to be measured is measured by adopting the method; and when SMD<When the concentration is 5%, the difference is not significant.
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