CN115015452B - Method for measuring content of allantoin and adenosine in Chinese yam by adopting one-measurement-multiple-evaluation method - Google Patents
Method for measuring content of allantoin and adenosine in Chinese yam by adopting one-measurement-multiple-evaluation method Download PDFInfo
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- POJWUDADGALRAB-UHFFFAOYSA-N allantoin Chemical compound NC(=O)NC1NC(=O)NC1=O POJWUDADGALRAB-UHFFFAOYSA-N 0.000 title claims abstract description 86
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 title claims abstract description 84
- POJWUDADGALRAB-PVQJCKRUSA-N Allantoin Natural products NC(=O)N[C@@H]1NC(=O)NC1=O POJWUDADGALRAB-PVQJCKRUSA-N 0.000 title claims abstract description 43
- 229960000458 allantoin Drugs 0.000 title claims abstract description 43
- 239000002126 C01EB10 - Adenosine Substances 0.000 title claims abstract description 42
- 229960005305 adenosine Drugs 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 24
- 235000002722 Dioscorea batatas Nutrition 0.000 title claims abstract description 16
- 235000006536 Dioscorea esculenta Nutrition 0.000 title claims abstract description 16
- 240000001811 Dioscorea oppositifolia Species 0.000 title claims abstract description 16
- 235000003416 Dioscorea oppositifolia Nutrition 0.000 title claims abstract description 16
- 238000011156 evaluation Methods 0.000 title claims abstract description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 24
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- 238000001914 filtration Methods 0.000 claims abstract description 6
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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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
- G01N30/86—Signal analysis
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
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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
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
The application discloses a method for measuring the contents of allantoin and adenosine in Chinese yam by adopting a one-measurement-multiple-evaluation method, which comprises the following steps: (1) preparation of a sample solution: pulverizing rhizoma Dioscoreae into powder, sieving, precisely weighing, adding methanol water solution, sealing, ultrasonic extracting, shaking, centrifuging part of the extractive solution, centrifuging, collecting supernatant, and filtering; (2) preparation of a control solution: taking appropriate amounts of adenosine and allantoin reference substances, precisely weighing, adding appropriate amount of methanol for dissolving, and preparing into mixed reference solution; (3) Precisely sucking the reference solution and the sample solution respectively, injecting into a liquid chromatograph, and calculating the contents of allantoin and adenosine in the sample according to the peak area by an external standard method. The method provided by the application is convenient and quick, low in cost, strong in specificity, high in sensitivity and convenient to popularize and apply, can provide effective reference for quality evaluation of the Chinese yam decoction pieces, and can be used for inspection and quality control of raw materials for producing Chinese patent medicines and traditional Chinese medicine formula particles.
Description
Technical Field
The application relates to a method for measuring the contents of allantoin and adenosine in Chinese yam by adopting a one-measurement-multiple-evaluation method, and belongs to the technical field of detection of Chinese medicinal components.
Background
The Chinese yam has the effects of strengthening spleen and tonifying stomach, promoting the production of body fluid and tonifying lung, and the like, is a common traditional Chinese medicine, and is also widely accepted health food. At present, the places of origin of the Chinese yam are numerous, and items such as characters, identification, inspection, extract and the like are only carried in the first part of Chinese pharmacopoeia of 2020 edition, and the quality evaluation of the Chinese yam decoction pieces is not facilitated due to the lack of content measurement items. Allantoin and adenosine are main active ingredients contained in yam, and the allantoin has the effects of promoting cell growth and accelerating wound healing, and has good therapeutic effects on digestive tract ulcer, inflammation, diabetes, osteomyelitis and other diseases. After entering the human body, the adenosine can participate in the energy metabolism of myocardial cells, can dilate coronary vessels, increase blood flow and has obvious protection effect on cardiovascular systems.
The high performance liquid chromatography is an accurate and efficient detection method for the content of the effective components in the Chinese yam at present, but most reports in the literature are that different chromatographic conditions are adopted for separate detection, so that the time is consumed, and more resources are wasted. There is also a method for simultaneously detecting adenosine and allantoin in yam by using a liquid chromatography-mass spectrometry technology in China, but the equipment is expensive, and the method is not favorable for popularization and application.
Disclosure of Invention
In order to solve the problems, the method for measuring the content of allantoin and adenosine in the Chinese yam by adopting a one-measurement-multiple-evaluation method is convenient, quick, low in cost, strong in specificity, high in sensitivity and convenient to popularize and apply, provides effective reference for quality evaluation of Chinese yam decoction pieces, and can be used for inspection and quality control of raw materials for producing Chinese patent medicines and traditional Chinese medicine formula particles.
According to one aspect of the present application, there is provided a method for determining contents of allantoin and adenosine in yam by a multiple evaluation method, comprising the steps of:
(1) Preparation of test solution: pulverizing rhizoma Dioscoreae into powder, sieving, precisely weighing, adding methanol water solution, sealing, ultrasonic extracting, shaking, centrifuging part of the extractive solution, centrifuging, collecting supernatant, and filtering;
(2) Preparation of a control solution: taking appropriate amounts of adenosine and allantoin reference substances, precisely weighing, adding appropriate amount of methanol for dissolving, and preparing into mixed reference solution;
(3) Precisely sucking the reference solution and the sample solution respectively, injecting into a liquid chromatograph, and calculating the contents of allantoin and adenosine in the sample according to the peak area by an external standard method.
Optionally, the step (1) is to take the Chinese yam slices, crush the powder, pass through a No. 5 sieve, precisely weigh 2g of the sieved powder, put the powder into a measuring flask, add 10% methanol aqueous solution by volume fraction, seal the powder by a sealing film, ultrasonically extract the powder for 50-70min, shake the powder uniformly, take part of the extract to a centrifuge tube, centrifuge the extract, and suck the supernatant to filter the supernatant by a microporous filter membrane to obtain the Chinese yam slices.
Optionally, the centrifugal speed in the step (1) is 4000-6000r/min, the centrifugal time is 8-12min, and the aperture of the microporous filter membrane is 0.45um.
Preferably, the centrifugal speed in the step (1) is 5000r/min, and the centrifugal time is 10min.
Optionally, step (2) is to take a proper amount of adenosine and allantoin reference substances, precisely weigh, add 10% methanol aqueous solution for dissolution, and prepare a mixed reference solution containing 80ug of adenosine and 200ug of allantoin per 1 ml.
Alternatively, the chromatographic column conditions are: the column was cosmosil his (250 mm. Times.4.6 mm,5 um) and the column temperature was 25 ℃.
Optionally, gradient elution is carried out by taking water as a mobile phase A and acetonitrile as a gradient eluent consisting of a mobile phase B, the flow rate is 1.0ml/min, the detection wavelength is 212nm, the sample injection amount is 10ul, and the theoretical plate number is not less than 7000.
Alternatively, the elution procedure is: 0-15 min, 10-20% of mobile phase A and 90-80% of mobile phase B; 15-20 min, 20-10% of mobile phase A and 80-90% of mobile phase B.
The beneficial effects of the application include, but are not limited to:
1. according to the method for measuring the content of allantoin and adenosine in the yam by adopting the one-measurement-multiple-evaluation method, the detection time is only 20min, the operation is simple, the time is short, the retention capacity of allantoin and adenosine components in a chromatographic column is relatively enhanced, the retention time of allantoin and adenosine is prolonged, the interference of the peak of other large-polarity components in the yam decoction pieces on the chromatographic peak of a target component is avoided, and the specificity is good.
2. According to the method for measuring the content of allantoin and adenosine in the yam by adopting the one-measurement-multiple-evaluation method, macromolecules such as starch in the yam are fully precipitated by limiting the centrifugal rotation speed and the centrifugal time, so that the anti-interference performance of a sample to be measured is improved.
3. According to the method for measuring the content of allantoin and adenosine in the yam by adopting the one-measurement-multiple-evaluation method, the quantitative limit of the allantoin is 31.25ug/ml, and the quantitative limit of the adenosine is 5.66ug/ml, so that the content detection requirement of the yam decoction pieces is met.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute a limitation on the application. In the drawings:
FIG. 1 is a graph of a specific spectrum in an embodiment of the application;
FIG. 2 is a linear pattern of allantoin in an embodiment of the present application;
FIG. 3 is a linear graph of adenosine in an embodiment of the present application.
Detailed Description
The present application is described in detail below with reference to examples, but the present application is not limited to these examples.
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. The reagents or materials used in the present application may be purchased in conventional manners, and unless otherwise indicated, they may be used in conventional manners in the art or according to the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present application. The preferred methods and materials described in this patent are illustrative only.
Example 1
1. Instrument and materials
Waters high performance liquid chromatograph (Waters Co., model: waters 2695), agilent high performance liquid chromatograph (model: agilent 1260 Infinicity II), chromatographic column (cosmosil hilc (250 mm. Times.4.6 mm,5 um)), adenosine control (lot number: 8179, shanghai Shiadad standard technical service Co., ltd., purity: 98.0%), allantoin control (lot number: 5702, shanghai Shiadad standard technical service Co., ltd., purity: 98.8%), allantoin control (lot number: 111501-200202, chinese food and drug assay institute, purity: 100.0%), chinese yam sheet (fresh cut at place of production, henan Coke, wen county Yuxiangtang Chinese herbal medicine Co., ltd.);
reagent: acetonitrile chromatographic purity, methanol as analytically pure water as purified water of the chen type.
2. Chromatographic conditions and system suitability test: column temperature: 25 ℃; detection wavelength: 212nm; mobile phase: and (3) performing gradient elution by using water as a mobile phase A and acetonitrile as a gradient eluent consisting of a mobile phase B, wherein the elution conditions are shown in table 1, the flow rate is 1.0ml/min, the detection wavelength is 212nm, the sample injection amount is 10ul, and the theoretical plate number is not less than 7000.
TABLE 1 elution conditions
3. Experimental methods and results
3.1 measurement method
(1) Preparation of test solution: pulverizing rhizoma Dioscoreae into powder, sieving with No. 5 sieve, precisely weighing 2g of the sieved powder, precisely weighing, placing in 100ml measuring flask, adding 50ml of 10% methanol water solution, sealing with sealing film, ultrasonic extracting for 60min, shaking, collecting part of the extractive solution to centrifuge tube, centrifuging at 5000r/min for 10min, and filtering the supernatant with 0.45um microporous membrane.
(2) Preparation of a control solution: taking appropriate amounts of adenosine and allantoin reference substances, precisely weighing, adding 10% methanol water solution, and dissolving to obtain mixed reference solution containing 80ug of adenosine and 200ug of allantoin per 1 ml.
(3) Respectively precisely sucking 10ul of the reference substance solution and 10ul of the test substance solution, injecting into a liquid chromatograph, and calculating the contents of allantoin and adenosine in the test substance according to the external standard method and peak area.
3.2 investigation of sample concentration
Sample solutions of 2.17mg/ml, 5.05mg/ml, 10.01mg/ml, 20.15mg/ml and 40.10mg/ml were prepared respectively, and injected into a liquid chromatograph, and the contents of allantoin and adenosine and the signal-to-noise ratio were calculated under the respective concentration conditions, and the best results were found when the concentration of the sample was determined to be 40mg/ml, and the results are shown in Table 2 in detail.
TABLE 2 examination of sample concentrations
3.3 investigation of sample extraction methods
Accurately weighing 1000mg of Chinese yam slice powder into a conical flask, accurately transferring into 50ml of 10% methanol diluent, preparing 3 parts in parallel, carrying out ultrasonic treatment on sample 1 and sample 2 for 60 minutes, carrying out ultrasonic treatment on sample 3 for 30 minutes, respectively centrifuging to obtain supernatant after ultrasonic extraction, centrifuging sample 1 for 5000 revolutions for 10 minutes, centrifuging sample 2 and sample 3 for 8000 revolutions for 10 minutes, filtering supernatant to obtain subsequent filtrate, sampling, comparing the ratio of peak area to mass, and determining that the ultrasonic time of a sample to be tested is 60 minutes and the centrifugal rotating speed is 5000rpm. The results are detailed in Table 3.
TABLE 3 investigation of sample extraction methods
3.4 methodological verification
1) Specialization of
To examine whether the diluent and other impurities in the sample interfere with the content measurement results, blank solvent, sample solution and reference solution were measured separately, and the results are shown in fig. 1.
The results show that the blank solvent has no chromatographic peak at the retention time corresponding to the allantoin and the adenosine in the test sample, and the blank solvent has no interference to the content measurement of the allantoin and the adenosine in the yam decoction pieces.
2) Repeatability of
6 parts of test solutions were prepared in parallel according to the method of 3.1, and the contents of allantoin and adenosine in the test were calculated to examine the reproducibility of the method. The results are detailed in Table 4.
TABLE 4 repeatability test results
| Sample of | Allantoin peak area | Allantoin content% | Adenosine peak area | Adenosine content% |
| Test sample solution 1-1 | 3104.522 | 1.08 | 578.826 | 0.038 |
| Test sample solutions 1 to 2 | 3087.099 | 1.08 | 596.372 | 0.039 |
| Test sample solution 2-1 | 3187.464 | 1.11 | 594.487 | 0.039 |
| Test sample solution 2-2 | 3183.199 | 1.11 | 590.590 | 0.039 |
| Sample solution 3-1 | 3188.606 | 1.11 | 591.203 | 0.039 |
| Sample solution 3-2 | 3192.803 | 1.11 | 586.233 | 0.039 |
| Sample solution 4-1 | 3193.354 | 1.11 | 584.773 | 0.038 |
| Sample solution 4-2 | 3187.080 | 1.11 | 592.128 | 0.039 |
| Sample solution 5-1 | 3204.351 | 1.11 | 587.676 | 0.038 |
| Sample solution 5-2 | 3206.022 | 1.11 | 567.699 | 0.037 |
| Sample solution 6-1 | 3204.216 | 1.11 | 589.981 | 0.039 |
| Sample solution 6-2 | 3209.481 | 1.11 | 569.794 | 0.037 |
| Mean value of | - | 1.10 | - | 0.039 |
| RSD％ | - | 1.02 | - | 1.76 |
The results show that the RSD of the two component contents is less than 2%, and the repeatability meets the requirement.
3) Linearity of
Respectively precisely sucking appropriate amount of allantoin reference substance mother solution and adenosine reference substance mother solution, diluting with 10% methanol to obtain 6 series of test substance solutions, and linearly regressing with reference substance peak area and its corresponding concentration. The results are shown in Table 5, and the linear patterns are shown in FIG. 2 and FIG. 3.
TABLE 5
| Composition of the components | Regression equation | Linear range/(ug/ml) | r |
| Allantoin | y＝7.0809x+9.4448 | 31.25～1000 | 1.0000 |
| Adenosine | y＝38,364.7496x+45,973.1208 | 2.83～362.404 | 1.0000 |
The result shows that the allantoin has good linear relationship in the range of 31.25ug/ml to 1000ug/ml, the quantitative limit is 31.25ug/ml, the adenosine has good linear relationship in the range of 11.33 ug/ml to 362.404ug/ml, the quantitative limit is 5.66ug/ml, and the requirement of content detection is met.
4) Recovery rate
6 parts of the same batch of test samples with known yam component content are taken, 200mg of each part is precisely added with a certain amount of adenosine reference substance and allantoin reference substance respectively to prepare test sample solutions, the test sample solutions are measured according to the chromatographic conditions, and the average sample adding recovery rate and RSD% value are calculated, and the results are shown in Table 6.
TABLE 6 recovery test results
The result shows that the recovery rate of allantoin is 94.40% -98.61% and the average recovery rate is 96.24%. The recovery rate of the adenosine is within the range of 89.66-103.84 percent, the average recovery rate is 96.93 percent, and the recovery rate meets the standard specified in pharmacopoeia.
5) Solution stability
The result shows that the peak area of the reference substance is not obviously changed after the sample is placed at room temperature for 24 hours, the content of the sample is basically unchanged, and the daily detection requirement is met. The results are shown in Table 7.
TABLE 7 results of solution stability experiments
6) Durability of
The results show that when the column temperature is within + -2 ℃, the wavelength is within + -2 nm, the flow rate is within + -0.1 ml/min, the data result is not changed significantly, and the durability of the method is good. The results are shown in Table 8.
Table 8 durability test results
The above description is only an example of the present application, and the scope of the present application is not limited to the specific examples, but is defined by the claims of the present application. Various modifications and variations of the present application will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present application should be included in the protection scope of the present application.
Claims (3)
1. A method for measuring the content of allantoin and adenosine in yam by a multi-evaluation method, which is characterized by comprising the following steps:
(1) Preparation of test solution: pulverizing rhizoma Dioscoreae into powder, sieving, precisely weighing, adding methanol water solution, sealing, ultrasonic extracting, shaking, centrifuging part of the extractive solution, centrifuging, collecting supernatant, and filtering;
(2) Preparation of a control solution: taking appropriate amounts of adenosine and allantoin reference substances, precisely weighing, adding appropriate amount of methanol water solution for dissolving, and preparing into mixed reference solution;
(3) Precisely sucking the reference substance solution and the test substance solution respectively, injecting into a liquid chromatograph, and calculating the contents of allantoin and adenosine in the test substance according to an external standard method and peak area;
the method comprises the steps of (1) grinding yam slices into powder, sieving with a No. 5 sieve, precisely weighing 2g of the sieved powder, placing into a measuring flask, adding 10% methanol water solution with volume fraction, sealing with a sealing film, ultrasonically extracting for 50-70min, shaking uniformly, taking part of extract into a centrifuge tube, centrifuging, and sucking supernatant, and filtering with a microporous filter membrane to obtain the Chinese yam powder;
the centrifugal speed in the step (1) is 4000-6000r/min, the centrifugal time is 8-12min, and the aperture of the microporous filter membrane is 0.45 mu m;
gradient elution is carried out by taking water as a mobile phase A and acetonitrile as a gradient eluent consisting of a mobile phase B; the elution procedure was: 0-15 min, 10-20% of mobile phase A and 90-80% of mobile phase B; 15-20 min, 20-10% of mobile phase A and 80-90% of mobile phase B;
the chromatographic column conditions were: the chromatographic column is Cosmosil HILIC,250mm×4.6mm,5 μm, and the column temperature is 25deg.C; the detection wavelength is 212nm.
2. The method according to claim 1, wherein the step (2) is to take a proper amount of the adenosine and allantoin reference substances, precisely measure them, and add 10% aqueous methanol solution to dissolve them, so as to prepare a mixed reference solution containing 80 μg of adenosine and 200 μg of allantoin per 1 ml.
3. The method according to claim 2, wherein the flow rate is 1.0ml/min, the sample introduction amount is 10. Mu.l, and the theoretical plate number is not less than 7000.
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