CN112903835B - Fingerprint spectrum construction method and detection method of momordica grosvenori and red tangerine peel solid beverage - Google Patents
Fingerprint spectrum construction method and detection method of momordica grosvenori and red tangerine peel solid beverage Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention relates to a construction method and a detection method of a fingerprint spectrum of a solid drink of momordica grosvenori and red tangerine peel. The construction method comprises the following steps: preparing a reference solution and a test solution; wherein, the preparation of the reference substance solution comprises the following steps: dissolving chlorogenic acid, naringin and quercitrin with a solvent a; the preparation of the test solution comprises the following steps: dissolving the solid drink of the momordica grosvenori and the exocarpium citri rubrum in a solvent b, and filtering; and detecting the reference substance solution and the test solution by adopting high performance liquid chromatography. The characteristic spectrum of the solid drink of the siraitia grosvenorii and the exocarpium citri rubrum constructed by the invention can comprehensively, accurately, objectively and scientifically reflect the whole chemical components and the functional components of the solid drink of the siraitia grosvenorii and the exocarpium citri rubrum, and the specific identification of the solid drink of the siraitia grosvenorii and the exocarpium citri rubrum can be realized from the inherent quality by adopting the characteristic spectrum to detect the solid drink of the siraitia grosvenorii and the exocarpium citri rubrum, and the method is simple to operate, good in repeatability and good in stability.
Description
Technical Field
The invention relates to the technical field of food quality detection, in particular to a method for constructing a fingerprint spectrum of a solid drink of momordica grosvenori and exocarpium citri rubrum and a detection method.
Background
The momordica grosvenori and red tangerine peel solid beverage is one of diet and nutrition series functional foods specially developed by medical and nutrition combined (Beijing) traditional Chinese medicine research institute aiming at common unbalance states of middle-aged and old sub-healthy people, meets the functional food specification of the community old traditional Chinese medicine health post station, and is one of prepared and marketable products of Guangzhou Zeli medicine science and technology Limited companies. The momordica grosvenori and exocarpium citri rubrum solid beverage mainly comprises momordica grosvenori, exocarpium citri rubrum, folium eriobotryae, rhizoma zingiberis, sweet almond, orange peel, auxiliary materials and other raw materials, contains various effective components such as flavonoids, phenols, sterols, polysaccharides and the like, has the effects of strengthening stomach, promoting qi circulation, relieving cough, reducing phlegm, relieving asthma, clearing heat, moistening lung, relieving sore throat, enhancing lung function, resisting cancer and the like, can supplement energy, protein, carbohydrate and other nutrients required by a human body, and improves the immunity of the human body.
The momordica grosvenori and red tangerine peel solid beverage is a functional food prepared by organically matching various main raw materials, and the components of the beverage are complex. At present, the quality control indexes of the solid drink of the siraitia grosvenorii and the exocarpium citri reticulatae are only limited to the detection of the physical and chemical indexes, the limit of microorganisms and other macroscopic characteristics, a detection method for the chemical components of the solid drink is lacked, the requirement of product quality control cannot be met far away, the increasingly severe requirements of consumers on high-quality products are difficult to meet at present, and therefore the establishment of a method for comprehensively representing the whole components of the solid drink of the siraitia grosvenorii and the exocarpium citri reticulatae is particularly important.
The traditional Chinese medicine fingerprint spectrum is used as a quality control technology, can comprehensively control and reflect the quality of traditional Chinese medicines and related products, and has the characteristics of systematicness and integrity. At present, documents and patent reports for controlling the quality of the momordica grosvenori and red tangerine peel solid beverage by using a fingerprint spectrum are not disclosed.
Disclosure of Invention
Based on the above, the invention mainly aims to provide a construction method and a detection method of a fingerprint of a siraitia grosvenorii and red tangerine peel solid beverage. The fingerprint constructed by the invention can comprehensively, accurately and scientifically reflect the whole chemical components and functional components of the solid drink of the siraitia grosvenorii and the exocarpium citri reticulatae, and the specific identification of the solid drink of the siraitia grosvenorii and the exocarpium citri reticulatae can be realized on the internal quality by adopting the characteristic spectrum to detect the solid drink of the siraitia grosvenorii and the exocarpium citri reticulatae.
The specific technical scheme comprises the following steps:
a construction method of a fingerprint of a solid drink of momordica grosvenori and exocarpium citri rubrum comprises the following steps:
preparing a reference substance solution and a test substance solution; wherein,
the preparation of the reference solution comprises the following steps: dissolving chlorogenic acid, naringin and quercitrin with a solvent a;
the preparation of the test solution comprises the following steps: dissolving the solid drink of the momordica grosvenori and the exocarpium citri rubrum in a solvent b, and filtering;
and detecting the reference substance solution and the test solution by adopting high performance liquid chromatography.
In one embodiment, the mobile phase used for detection comprises: the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid water solution with the phosphoric acid volume concentration of 0.05wt% -0.1 wt%, and the elution mode is gradient elution.
In one embodiment, gradient elution the procedure employed comprises:
the volume percentage of the mobile phase A is increased from 5 percent to 15 percent within 0-20 min;
20-45 min, wherein the volume percentage of the mobile phase A is increased from 15% to 30%;
45-60 min, and the volume percentage of the mobile phase A is increased from 30% to 80%.
In one embodiment, the chromatographic column used for detection is a C18 chromatographic column.
In one embodiment, the wavelength used for detection is 295-305 nm, or/and the device used for detection is Agilent 1100.
In one embodiment, the temperature of the column used for detection is 30-35 ℃, or/and the flow rate used for detection is 0.8-1 mL/min, or/and the sample size used for detection is 5-20 muL.
In one embodiment, the solvent a is methanol, or/and the solvent b is water.
In one embodiment, the fingerprint contains 11 characteristic peaks, and the 11 characteristic peaks include characteristic peaks of chlorogenic acid, naringin and quercetin.
A detection method of a solid drink of momordica grosvenori and exocarpium citri rubrum comprises the following steps:
dissolving the solid drink of the momordica grosvenori and the exocarpium citri rubrum in a solvent b, and filtering to prepare a solution to be detected;
and detecting the solution to be detected by adopting a high performance liquid chromatography, and comparing the obtained detection map of the solution to be detected with the fingerprint map constructed by the construction method.
In one embodiment, the mobile phase used for detection comprises: the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid aqueous solution with the phosphoric acid volume concentration of 0.05wt% -0.1 wt%, and the elution mode is gradient elution.
In one embodiment, gradient elution the procedure employed comprises:
the volume percentage of the mobile phase A is increased from 5 percent to 15 percent within 0-20 min;
20-45 min, wherein the volume percentage of the mobile phase A is increased from 15% to 30%;
45-60 min, and the volume percentage of the mobile phase A is increased from 30% to 80%.
In one embodiment, the chromatographic column used for detection is a C18 chromatographic column.
In one embodiment, the wavelength used for detection is 295nm to 305nm, or/and the device used for detection is Agilent 1100.
In one embodiment, the temperature of the column used for detection is 30-35 ℃, or/and the flow rate used for detection is 0.8-1 mL/min, or/and the sample size used for detection is 5-20 muL.
In one embodiment, the solvent b is water.
Compared with the prior art, the invention has the following beneficial effects:
the method selects chlorogenic acid, naringin and quercitrin as reference substances, constructs the characteristic spectrum of the siraitia grosvenorii red-orange solid beverage containing the characteristic peaks of the chemical components, particularly constructs the characteristic spectrum containing 11 characteristic peaks of the chemical components under the appropriate chromatographic condition, has good chromatographic peak separation degree, can comprehensively, accurately, objectively and scientifically reflect the whole chemical components and functional components of the siraitia grosvenorii red-orange solid beverage, can realize the specificity identification of the siraitia grosvenorii red-orange solid beverage from the inherent quality by adopting the characteristic spectrum to detect the siraitia grosvenorii red-orange solid beverage, and has the advantages of simple operation, good repeatability and good stability.
Drawings
FIG. 1 is an HPLC three-dimensional map of a solid drink of Momordica grosvenori and exocarpium citri rubrum in one embodiment of the present invention;
FIG. 2 is a chromatogram at different wavelengths according to an embodiment of the present invention;
FIG. 3 is a chromatogram of different mobile phases in one embodiment of the invention;
FIG. 4 is a different gradient elution chromatogram in one embodiment of the present invention;
FIG. 5 is a chromatogram of a different column according to an embodiment of the invention;
FIG. 6 is a chromatogram of a different liquid phase system in accordance with an embodiment of the present invention;
FIG. 7 shows a comparison of precision test fingerprints according to an embodiment of the present invention;
FIG. 8 is a comparison of repeatability test fingerprints in accordance with an embodiment of the present invention;
FIG. 9 shows a comparison of stability test fingerprints according to one embodiment of the present invention;
FIG. 10 is a comparison of fingerprints for different batches of products in accordance with an embodiment of the present invention;
FIG. 11 shows a common peak of liquid-phase fingerprint of a Siraitia grosvenorii and exocarpium citri reticulatae solid beverage according to an embodiment of the present invention;
FIG. 12 is a comparison of the spectra of a chlorogenic acid control and a sample in accordance with an embodiment of the present invention;
FIG. 13 is a comparison of the spectra of a naringin control sample and a sample in accordance with an embodiment of the present invention;
FIG. 14 is a comparison of spectra of quercetin control and a sample in accordance with an embodiment of the present invention.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The embodiment of the invention provides a method for constructing a fingerprint of a solid drink of momordica grosvenori and red tangerine peel, which comprises the following steps:
preparing a reference substance solution and a test substance solution; wherein,
the preparation of the reference solution comprises the following steps: dissolving chlorogenic acid, naringin and quercitrin with a solvent a;
the preparation of the test solution comprises the following steps: dissolving the solid drink of the momordica grosvenori and the exocarpium citri rubrum in a solvent b, and filtering;
and detecting the reference substance solution and the test solution by adopting high performance liquid chromatography.
In one example, the mobile phase used for detection includes: the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid water solution with the phosphoric acid volume concentration of 0.05wt% -0.1 wt%, and the elution mode is gradient elution.
In one example, the gradient elution employed comprises:
the volume percentage of the mobile phase A is increased from 5 percent to 15 percent within 0-20 min;
20-45 min, wherein the volume percentage of the mobile phase A is increased from 15% to 30%;
45-60 min, and the volume percentage of the mobile phase A is increased from 30% to 80%.
In one example, the column used for detection is a C18 column.
In one example, the wavelength used for detection is 295nm to 305nm, or/and the device used for detection is Agilent 1100.
In one example, the temperature of the column used for detection is 28-32 ℃, or/and the flow rate used for detection is 0.8-1.2 mL/min, or/and the sample size used for detection is 5-20 μ L.
In one example, the solvent a is methanol, or/and the solvent b is water.
In one example, the fingerprint contains 11 characteristic peaks, wherein the 11 characteristic peaks comprise characteristic peaks of chlorogenic acid, naringin and quercetin.
The embodiment of the invention provides a method for detecting a solid drink of momordica grosvenori and red tangerine peel, which comprises the following steps:
dissolving the solid drink of the momordica grosvenori and the exocarpium citri rubrum in a solvent b, and filtering to prepare a solution to be detected;
and detecting the solution to be detected by adopting a high performance liquid chromatography, and comparing the obtained detection map of the solution to be detected with the fingerprint map constructed by the construction method.
In one example, the mobile phase used for detection includes: the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid water solution with the phosphoric acid volume concentration of 0.05wt% -0.1 wt%, and the elution mode is gradient elution.
In one example, gradient elution the procedure employed comprises:
the volume percentage of the mobile phase A is increased from 5 percent to 15 percent within 0-20 min;
20-45 min, wherein the volume percentage of the mobile phase A is increased from 15% to 30%;
45-60 min, and the volume percentage of the mobile phase A is increased from 30% to 80%.
In one example, the column used for detection is a C18 column.
In one example, the wavelength used for detection is 295nm to 305nm, or/and the device used for detection is Agilent 1100.
In one example, the temperature of the column used for detection is 28-32 ℃, or/and the flow rate used for detection is 0.8-1.2 mL/min, or/and the sample size used for detection is 5-20 μ L.
In one example, the solvent b is water.
The method is characterized in that single component is quantitatively detected as a quality control index of the solid drink of the siraitia grosvenorii and the exocarpium citri reticulatae according to the conventional method, and the information of various components in the product is difficult to monitor comprehensively; for the momordica grosvenori and red tangerine solid beverage with complex components, multiple types of components which are qualitative and quantitative are difficult to separate simultaneously under the conventional chromatographic condition, and the momordica grosvenori and red tangerine solid beverage fingerprint detection method has the characteristics of strong specificity and good stability, and can be used for comprehensively, accurately and quickly detecting the quality of the product.
The following drugs involved in the embodiments of the present invention are commercially available: acetonitrile (chromatographically pure, Dima, USA), methanol (chromatographically pure, Dima, USA), phosphoric acid (analytically pure, Chengdu city Kelong chemical reagent factory), chlorogenic acid as reference (Beijing extract purification remote chemical technology research institute, lot No. 13241-233), naringin (Beijing extract purification remote chemical technology research institute, lot No. 10236-47-2), and quercetin (China pharmaceutical biologicals institute, lot No. 111538-200504).
The solid drink containing the momordica grosvenori and the exocarpium citri rubrum is Runkeshuang.
Example 1
The embodiment of the invention provides a method for constructing a liquid-phase fingerprint of a siraitia grosvenorii and red tangerine peel solid beverage, which comprises the following steps:
preparing a test solution: taking 1g of the momordica grosvenori exocarpium citri solid beverage, adding water to dissolve the momordica grosvenori exocarpium citri solid beverage and fixing the volume to 15mL, filtering the mixture by using a 0.45 mu m filter membrane, and taking a proper amount of a subsequent filtrate for later use.
Preparation of control solutions: accurately weighing appropriate amount of chlorogenic acid, naringin and quercetin reference substance in 10mL volumetric flasks respectively, and metering volume with methanol to obtain chlorogenic acid reference substance solution, naringin reference substance solution and quercetin reference substance solution.
The specific chromatographic condition optimization process is as follows:
(1) determination of detection wavelength:
the detection wavelength is subjected to three-dimensional spectrogram analysis through DAD detector detection, as shown in figure 1, spectrograms under wavelengths of 220nm, 290nm, 300nm, 310nm and 3200nm are selected and analyzed under the condition of avoiding interference of the overall peak profile due to too strong absorption, as shown in figure 2, the spectrograms under 300nm are preferably good in separation degree, stable in baseline, rich in component types and high in content, and therefore the 300nm wavelength is selected for scanning in the later stage experiment of the embodiment of the invention.
(2) Selection of mobile phase system:
methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid aqueous solution and acetonitrile-0.1% phosphoric acid aqueous solution are taken as mobile phase systems, and the results of sample injection analysis under the same liquid phase condition show that the chromatogram obtained by the acetonitrile-0.1% phosphoric acid aqueous solution has rich information content and stable baseline, so the later experiment of the invention takes the acetonitrile-0.1% phosphoric acid aqueous solution as the mobile phase, as shown in figure 3.
(3) Optimization of elution gradient:
gradient optimization was performed according to the following 3 experiments using acetonitrile (a) -0.1% phosphoric acid in water (B) as mobile phase:
gradient one: 0min to 5min, 5 percent of A; 5-20 min, 5-15% A; 20 min-25 min, 15% -25% A; 25-50 min, 25-30% A; 50-60 min, 30-50% A.
And (2) gradient II: 0min to 20min, 5 percent to 15 percent of A; 20 min-45 min, 15% -30% A; 45-60 min, 30-80% A.
Gradient three: 0min to 20min, 10 percent to 15 percent of A; 20 min-50 min, 15% -30% A; 50-60 min, 25-80% A.
After each spectrogram analysis, as shown in fig. 4, the spectrogram obtained under the gradient two-condition has the most types and contents of components, stable baseline and good separation degree, so that the gradient condition is selected for sample analysis in the later stage experiment of the invention.
(4) Selection of chromatographic column:
according to the existing chromatographic column, sample injection is carried out under the same liquid phase conditionAnd (3) analyzing, wherein the chromatogram obtained by using an Inertsl ODS-3(4.6mm multiplied by 250mm, 5 mu m) chromatographic column has rich information and stable baseline, so that the later experiment of the invention adopts an Inertsl ODS-3 chromatographic column for sample injection analysis (shown in figure 5). In FIG. 5, the ODS column is specifically Inertsll ODS-3(4.6 mm. times.250 mm, 5 μm), C 18 The column is concretely Diamonsil 5 mu m C 18 (2)(250×4.6mm)
(5) Optimization of liquid phase equipment:
according to the existing experimental equipment, the spectrum information obtained by Agilent 1100 is abundant relative to Shimadzu-GL in the same liquid phase sample injection analysis, so that the Agilent 1100 liquid phase equipment is used for sample injection analysis in the later period of the experiment (shown in figure 6).
(5) Determination of chromatographic conditions:
by combining the test results, the chromatographic conditions for establishing the liquid-phase fingerprint of the siraitia grosvenorii and red tangerine peel solid beverage are as follows:
an Inertsll ODS-3(4.6 mm. times.250 mm, 5 μm) column;
the mobile phase is acetonitrile (A) -0.1 percent phosphoric acid water solution (B);
a gradient elution procedure was used, see table 1;
TABLE 1 conditions for elution of fingerprint mobile phase of Momordica grosvenori and exocarpium citri rubrum solid beverage
| Time (minutes) | Mobile phase A (%) |
| 0min～ |
5→15 |
| 20min～45min | 15→30 |
| 45min～60min | 30→80 |
The detection wavelength is 300 nm;
the column temperature is 30 ℃;
the flow rate is 1 mL/min;
the amount of the sample was 10. mu.L.
(6) Methodology investigation: precision, stability, repeatability tests
1) And (3) precision test:
taking the same newly prepared test solution, preparing a sample according to the test solution preparation method, determining a chromatogram under the finally optimized chromatographic condition, carrying out continuous sample introduction for 6 times, and inspecting the RSD of each common peak relative retention time and relative peak area.
As a result, the RSD of the relative retention time is less than or equal to 1.5 percent (shown in Table 2), the RSD of the relative peak area is less than or equal to 5.0 percent (shown in Table 3), and the similarity of each chromatogram is more than 0.99 (shown in Table 4), which indicates that the precision of the instrument is good and meets the technical requirements of the fingerprint, and is shown in FIG. 7.
TABLE 2 relative retention time of each common peak in precision test
| Numbering | S1 | S2 | S3 | S4 | S5 | S6 | RSD/％ |
| 1 | 0.264 | 0.265 | 0.264 | 0.265 | 0.265 | 0.264 | 0.103 |
| 2 | 0.326 | 0.327 | 0.324 | 0.324 | 0.324 | 0.324 | 0.353 |
| 3 | 0.399 | 0.399 | 0.399 | 0.399 | 0.399 | 0.399 | 0.088 |
| 4 | 0.475 | 0.488 | 0.487 | 0.488 | 0.488 | 0.487 | 1.071 |
| 5 | 0.511 | 0.512 | 0.511 | 0.511 | 0.512 | 0.511 | 0.049 |
| 6 | 0.552 | 0.553 | 0.552 | 0.552 | 0.552 | 0.552 | 0.044 |
| 7 | 0.570 | 0.570 | 0.570 | 0.570 | 0.570 | 0.570 | 0.035 |
| 8 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.000 |
| 9 | 1.027 | 1.027 | 1.027 | 1.027 | 1.026 | 1.026 | 0.027 |
| 10 | 1.139 | 1.139 | 1.139 | 1.139 | 1.139 | 1.139 | 0.015 |
| 11 | 1.392 | 1.391 | 1.390 | 1.390 | 1.391 | 1.390 | 0.037 |
TABLE 3 relative peak area of each common peak in precision test
TABLE 4 evaluation of precision map similarity
| Numbering | S1 | S2 | S3 | S4 | S5 | S6 | R |
| S1 | 1.000 | 0.996 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S2 | 0.996 | 1.000 | 0.996 | 0.996 | 0.996 | 0.996 | 0.997 |
| S3 | 1.000 | 0.996 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S4 | 1.000 | 0.996 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S5 | 1.000 | 0.996 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S6 | 1.000 | 0.997 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
2) And (3) repeatability test:
taking 6 parts of momordica grosvenori and exocarpium citri rubrum solid beverage, preparing a sample according to a preparation method of a test solution, determining a chromatogram under a final chromatographic condition, and inspecting the RSD of the relative retention time and the relative peak area of each common peak.
As a result, the RSD of the relative retention time was not more than 1.0% (shown in Table 5), the RSD of the relative peak area was not more than 5.0% (shown in Table 6), and the similarity of each chromatogram was not less than 0.99 (shown in Table 7), indicating good reproducibility and meeting the technical requirements of the fingerprint, as shown in FIG. 8.
TABLE 5 repeatability test relative retention time of each common peak
TABLE 6 relative peak area of each common peak in the repeatability test
| Numbering | S1 | S2 | S3 | S4 | S5 | S6 | RSD/％ |
| 1 | 0.039 | 0.038 | 0.042 | 0.039 | 0.040 | 0.040 | 3.520 |
| 2 | 0.078 | 0.083 | 0.084 | 0.077 | 0.077 | 0.078 | 4.041 |
| 3 | 0.057 | 0.064 | 0.064 | 0.064 | 0.062 | 0.061 | 4.534 |
| 4 | 0.034 | 0.035 | 0.033 | 0.037 | 0.037 | 0.036 | 4.156 |
| 5 | 0.037 | 0.038 | 0.036 | 0.037 | 0.038 | 0.036 | 2.714 |
| 6 | 0.100 | 0.102 | 0.103 | 0.104 | 0.108 | 0.104 | 2.476 |
| 7 | 0.050 | 0.049 | 0.050 | 0.052 | 0.053 | 0.052 | 3.108 |
| 8 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.000 |
| 9 | 0.042 | 0.043 | 0.041 | 0.042 | 0.039 | 0.041 | 2.792 |
| 10 | 0.067 | 0.066 | 0.067 | 0.067 | 0.066 | 0.066 | 0.352 |
| 11 | 0.026 | 0.026 | 0.026 | 0.026 | 0.026 | 0.026 | 1.015 |
TABLE 7 repeatability map similarity evaluation
| Numbering | S1 | S2 | S3 | S4 | S5 | S6 | R |
| S1 | 1.000 | 0.999 | 0.999 | 1.000 | 1.000 | 0.999 | 1.000 |
| S2 | 0.999 | 1.000 | 1.000 | 0.999 | 0.999 | 1.000 | 1.000 |
| S3 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S4 | 1.000 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S5 | 1.000 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
| S6 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 |
3) And (3) stability test:
taking a fructus momordicae and exocarpium citri rubrum solid beverage product, preparing a sample according to a preparation method of a test solution, determining chromatograms at 0h, 2h, 4h, 6h, 8h and 24h under final chromatographic conditions, and inspecting the RSD of the relative retention time and the relative peak area of each common peak.
As a result, the RSD of the relative retention time is less than or equal to 1.0 percent (shown in Table 8), the RSD of the relative peak area is less than or equal to 5.0 percent (shown in Table 9), and the similarity of each chromatogram is more than 0.99 (shown in Table 10), which indicates that the sample stability is good and meets the technical requirements of the fingerprint, as shown in FIG. 9.
TABLE 8 stability test relative retention time of each common peak
| Numbering | S1 | S2 | S3 | S4 | S5 | S6 | RSD/％ |
| 1 | 0.269 | 0.266 | 0.266 | 0.265 | 0.265 | 0.265 | 0.580 |
| 2 | 0.332 | 0.327 | 0.327 | 0.326 | 0.326 | 0.326 | 0.677 |
| 3 | 0.405 | 0.399 | 0.399 | 0.398 | 0.398 | 0.398 | 0.684 |
| 4 | 0.480 | 0.487 | 0.488 | 0.487 | 0.487 | 0.486 | 0.558 |
| 5 | 0.517 | 0.510 | 0.511 | 0.503 | 0.510 | 0.510 | 0.873 |
| 6 | 0.559 | 0.552 | 0.552 | 0.551 | 0.552 | 0.552 | 0.532 |
| 7 | 0.577 | 0.569 | 0.569 | 0.569 | 0.569 | 0.569 | 0.564 |
| 8 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.000 |
| 9 | 1.027 | 1.026 | 1.026 | 1.026 | 1.026 | 1.026 | 0.038 |
| 10 | 1.140 | 1.140 | 1.141 | 1.141 | 1.142 | 1.142 | 0.075 |
| 11 | 1.389 | 1.391 | 1.391 | 1.391 | 1.391 | 1.390 | 0.058 |
TABLE 9 relative peak area of each common peak in stability test
TABLE 10 evaluation of stability Profile similarity
| Numbering | S1 | S2 | S3 | S4 | S5 | S6 | R |
| S1 | 1.000 | 0.999 | 0.999 | 1.000 | 1.000 | 0.999 | 1.000 |
| S2 | 0.999 | 1.000 | 1.000 | 0.999 | 0.999 | 1.000 | 1.000 |
| S3 | 0.999 | 1.000 | 1.000 | 0.999 | 0.999 | 1.000 | 1.000 |
| S4 | 1.000 | 0.999 | 0.999 | 1.000 | 1.000 | 0.999 | 1.000 |
| S5 | 1.000 | 0.999 | 0.999 | 1.000 | 1.000 | 0.999 | 1.000 |
| S6 | 0.999 | 1.000 | 1.000 | 0.999 | 0.999 | 1.000 | 1.000 |
(7) Establishment of fingerprint spectrum of momordica grosvenori and red tangerine peel solid beverage and identification of common peaks
Fingerprint spectra of different batches of fructus momordicae and exocarpium citri grandis solid beverage samples are measured under the condition of final chromatography, and the result is shown in figure 10. By using an average method, the time window width is 0.1S, and S4 is used as a reference map, so as to generate a Runkeshuang product common mode control map (figure 11). The different batches of the Runkeshuang product are different from each other due to the difference of the factors such as raw materials, equipment, time and the like, and the fingerprint spectrums of the Runkeshuang product are slightly different, but the commonness of the Runkeshuang product is not changed.
And (3) analyzing by software of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), selecting chromatographic peaks with better separation degree, wherein HPLC chromatograms of different batches of Runkshuang samples have 11 common peaks. The reference substance chromatogram analysis confirms that the peak 6 is chlorogenic acid, the peak 8 is naringin and the peak 9 is quercitrin, as shown in fig. 12, fig. 13 and fig. 14, wherein the peak type and the separation degree of the peak 8 are better, so the peak is selected as the reference peak. The relative retention time and relative peak area of the peaks common to each batch of samples were calculated as shown in tables 11 and 12. The result shows that the RSD of the relative retention time is less than or equal to 1.0 percent, the RSD of the relative peak area is less than or equal to 5.0 percent, all common peaks are stable, the fingerprint characteristics are realized, and the index component group of the momordica grosvenori and tangerine peel solid beverage product can be preliminarily drawn up. The similarity of each spectrogram is more than 0.99 (shown in table 13), and meets the technical requirements of the fingerprint.
TABLE 11 relative retention time of common peaks for different batches of Lo Han Guo orange solid beverage samples
| Numbering | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | RSD/％ |
| 1 | 0.266 | 0.266 | 0.265 | 0.266 | 0.265 | 0.265 | 0.265 | 0.265 | 0.265 | 0.125 |
| 2 | 0.326 | 0.327 | 0.326 | 0.327 | 0.326 | 0.326 | 0.326 | 0.326 | 0.326 | 0.145 |
| 3 | 0.398 | 0.399 | 0.398 | 0.399 | 0.398 | 0.400 | 0.398 | 0.398 | 0.398 | 0.189 |
| 4 | 0.474 | 0.487 | 0.487 | 0.487 | 0.486 | 0.486 | 0.486 | 0.486 | 0.486 | 0.849 |
| 5 | 0.510 | 0.510 | 0.511 | 0.511 | 0.510 | 0.510 | 0.510 | 0.510 | 0.510 | 0.090 |
| 6 | 0.551 | 0.552 | 0.552 | 0.553 | 0.552 | 0.551 | 0.551 | 0.552 | 0.551 | 0.083 |
| 7 | 0.568 | 0.569 | 0.569 | 0.570 | 0.569 | 0.568 | 0.568 | 0.569 | 0.568 | 0.079 |
| 8 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.000 |
| 9 | 1.026 | 1.026 | 1.026 | 1.026 | 1.026 | 1.026 | 1.026 | 1.026 | 1.026 | 0.022 |
| 10 | 1.143 | 1.141 | 1.140 | 1.140 | 1.141 | 1.141 | 1.141 | 1.141 | 1.141 | 0.066 |
| 11 | 1.393 | 1.392 | 1.391 | 1.391 | 1.392 | 1.391 | 1.391 | 1.391 | 1.391 | 0.055 |
TABLE 12 relative peak area of each common peak of different batches of fructus Siraitiae Grosvenorii exocarpium Citri rubrum solid beverage samples
TABLE 13 evaluation of fingerprint similarity of different batches of Lo Han Guo orange solid beverage samples
| Numbering | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | R |
| S1 | 1.000 | 0.999 | 0.999 | 0.999 | 0.999 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 |
| S2 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.999 | 0.999 | 0.999 | 1.000 |
| S3 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.999 | 0.999 | 0.999 | 1.000 |
| S4 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.999 | 0.999 | 0.999 | 1.000 |
| S5 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.999 | 0.999 | 0.999 | 1.000 |
| S6 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.999 | 0.999 | 0.999 | 1.000 |
| S7 | 1.000 | 0.999 | 0.999 | 0.999 | 0.999 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 |
| S8 | 1.000 | 0.999 | 0.999 | 0.999 | 0.999 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 |
| S9 | 1.000 | 0.999 | 0.999 | 0.999 | 0.999 | 0.999 | 1.000 | 1.000 | 1.000 | 1.000 |
Example 2
First, experimental material
Equipment: agilent 1100 high performance liquid chromatograph, Inertsll ODS-3(4.6 mm. times.250 mm, 5 μm) column, model AUW220D electronic balance (Shimadzu, Japan).
Reagent: acetonitrile (chromatographically pure, Dima, USA), methanol (chromatographically pure, Dima, USA), phosphoric acid (analytically pure, Chengdu city Kelong chemical reagent factory), chlorogenic acid as reference (Beijing extract purification remote chemical technology research institute, lot No. 13241-233), naringin (Beijing extract purification remote chemical technology research institute, lot No. 10236-47-2), quercetin (China pharmaceutical biologicals institute, lot No. 111538-200504), and primary water.
Sample preparation: the solid beverage comprises 20200401 of fructus momordicae and tangerine, 20204002 and 20200403 of solid beverage of fructus momordicae and tangerine, 20200501 of solid beverage of fructus momordicae and tangerine, 20200502 of solid beverage of fructus momordicae and tangerine, 20200503 of solid beverage of fructus momordicae and tangerine, 20200601 of solid beverage of fructus momordicae and tangerine, 20200602 of solid beverage of fructus momordicae and tangerine, and 20200603 of solid beverage of fructus momordicae and tangerine.
Second, experimental procedures and results
1. Preparing a test solution: precisely measuring 1.0g of sample, placing in a 15ml volumetric flask, adding water for dissolving and fixing volume, filtering with a 0.45 mu m filter membrane, and taking a proper amount of subsequent filtrate to obtain the product.
2. Preparation of a reference solution: accurately weighing appropriate amount of naringin, chlorogenic acid and quercetin reference substance in 10mL volumetric flask, and diluting to constant volume with methanol to obtain naringin reference substance solution, chlorogenic acid reference substance solution and quercetin reference substance solution.
3. Liquid chromatography conditions: agilent 1100 high performance liquid chromatograph, Inertsll ODS-3(4.6 mm. times.250 mm, 5 μm) chromatographic column, sample amount of 10 μ L, flow rate of 1.0mL/min, column temperature of 30 ℃, mobile phase: acetonitrile-0.1% aqueous phosphoric acid, elution gradient as shown in table 1 above.
4. And (3) sample determination: respectively and precisely sucking 10uL of the test solution and the reference solution, and injecting into a high performance liquid chromatograph for determination to obtain the fingerprint.
5. And (3) data analysis: and (3) calculating according to 2012 edition of Chinese pharmacopoeia committee 'traditional Chinese medicine chromatogram fingerprint similarity evaluation software' to obtain similarity values of each batch. The chromatogram similarity of a plurality of batches of fructus momordicae and tangerine solid beverage (20200401-20200603) produced in 4-2020 and 6-2020 is over 0.99, the RSD of the relative retention time is less than or equal to 1.0 percent, and the RSD of the relative peak area is less than or equal to 5.0 percent, so that the method meets the internal control standard of enterprises and can provide reference for quality evaluation and quality control of fructus momordicae and tangerine solid beverage products.
The embodiment of the invention optimizes chromatographic conditions such as liquid phase equipment, chromatographic columns, a mobile phase system, wavelength, mobile phase elution gradient and the like, examines a methodology, establishes a common mode, evaluates similarity of a plurality of batches of products, and establishes a fingerprint spectrum; the established fingerprints are used for analyzing the product spectrograms of a plurality of batches of fructus momordicae and exocarpium citri reticulatae solid beverages, the similarity of the product spectrograms is analyzed by using traditional Chinese medicine chromatogram fingerprint similarity evaluation software (2012 edition), the product quality is objectively, comprehensively and scientifically evaluated and controlled, the product quality can be comprehensively, accurately and rapidly detected and monitored, the quality control of the product with high quality is achieved, and the large-scale production and application of companies are met.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (5)
1. A construction method of a fingerprint of a solid drink of momordica grosvenori and exocarpium citri rubrum is characterized by comprising the following steps:
preparing a reference solution and a test solution; wherein,
the preparation of the reference solution comprises the following steps: dissolving chlorogenic acid, naringin and quercitrin with a solvent a;
the preparation of the test solution comprises the following steps: dissolving the solid drink of the momordica grosvenori and the exocarpium citri rubrum in a solvent b, and filtering;
detecting the reference substance solution and the test solution by adopting high performance liquid chromatography;
the chromatographic column used for detection is a C18 chromatographic column;
the mobile phase adopted for detection comprises: the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid aqueous solution with the phosphoric acid volume concentration of 0.05wt% -0.1 wt%, and the elution mode is gradient elution;
gradient elution the procedure employed included:
the volume percentage of the mobile phase A is increased from 5 percent to 15 percent within 0-20 min;
20-45 min, wherein the volume percentage of the mobile phase A is increased from 15% to 30%;
45-60 min, wherein the volume percentage of the mobile phase A is increased from 30% to 80%;
the solvent b is water.
2. The method for constructing the fingerprint of the siraitia grosvenorii and exocarpium citri reticulatae solid beverage according to claim 1, wherein the wavelength used for detection is 295-305 nm, or/and
the equipment used for detection was Agilent 1100.
3. The method for constructing the fingerprint of the siraitia grosvenorii and exocarpium citri reticulatae solid beverage according to claim 1 or 2, wherein the column temperature adopted for detection is 30-35 ℃, or/and
the flow rate adopted by the detection is 0.8mL/min to 1mL/min, or/and
the sample injection amount adopted for detection is 8-10 mu L; or/and
the solvent a is methanol, or/and
the fingerprint spectrum comprises 11 characteristic peaks, wherein the 11 characteristic peaks comprise characteristic peaks of chlorogenic acid, naringin and quercetin.
4. A detection method of a solid drink of momordica grosvenori and red tangerine is characterized by comprising the following steps:
dissolving the solid drink of the momordica grosvenori and the exocarpium citri rubrum in a solvent b, and filtering to prepare a solution to be detected;
detecting the solution to be detected by adopting the conditions of the high performance liquid chromatography in the construction method of any one of claims 1 to 3, and comparing the obtained detection spectrum of the solution to be detected with the fingerprint spectrum constructed by the construction method of any one of claims 1 to 3.
5. The method for detecting the solid drink of momordica grosvenori and red tangerine peel according to claim 4,
the solvent b is water.
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| CN108606220A (en) * | 2018-03-22 | 2018-10-02 | 广东医科大学 | A kind of anti-fog haze solid beverage of Exocarpium Citri Rubrum Sugarless type and preparation method thereof |
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