CN115508465A - Method for simultaneously determining content of 3 nucleoside components in radix glehniae - Google Patents
Method for simultaneously determining content of 3 nucleoside components in radix glehniae Download PDFInfo
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- 239000000243 solution Substances 0.000 claims abstract description 48
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- 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
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- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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Abstract
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for simultaneously determining the content of 3 nucleoside components in radix glehniae. The method specifically comprises the following steps: preparing a reference solution; preparing a test solution; precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring; the method adopts 0.1 percent phosphoric acid solution for extraction, ensures the stability and the accuracy of the nucleoside components in the radix glehniae, adopts an HPLC method for content determination, and solves the key problem of completing the content determination of the nucleoside components in the radix glehniae by using the same extraction method and the same determination system.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for simultaneously determining the content of 3 nucleoside components in radix glehniae.
Background
Radix glehniae is the dry root of glehnia littoralis Fr.Schmidt ex Miq. which is an umbelliferae plant, and listed as the top grade in Shen nong Ben Cao Jing, which is one of the traditional Chinese medicinal materials in large quantities commonly used in China. Sweet, slightly bitter and slightly cold, entering lung and stomach meridians, has the efficacies of nourishing yin, clearing lung, benefiting stomach and promoting fluid production, and is used for treating dry cough due to lung dryness, overstrain cough with phlegm and blood, deficiency of stomach yin, body fluid impairment due to fever, dry throat, thirst and other symptoms. Wild radix glehniae is a second-level important protection wild plant in China, and is a mainstream cultivated species in the market, mainly produced in Hebei Anguo, shandong Laiyang, inner Mongolia Chifeng and other places. The existing quality standard of radix glehniae is recorded in the first part of the 2020 edition of Chinese pharmacopoeia, and only a method for microscopically identifying characters and cross sections is adopted. The main active ingredients in the radix glehniae are polysaccharides, nucleosides, coumarins, polyacetylenes and the like, the literature mostly refers to the research on the polysaccharides, coumarins and polyacetylenes in the radix glehniae, and the research on the nucleosides in the radix glehniae is not seen. The radix glehniae is decocted with water, the preparation is also prepared by a water extraction process, and nucleoside ingredients in water-soluble ingredients have strong antiviral, antibacterial and endotoxin resisting effects, so that the radix glehniae can be used as one of quality control indexes of the radix glehniae. At present, no method which can rapidly and simply measure the content of adenosine, guanosine and uridine in radix glehniae while having good sensitivity, repeatability and accuracy exists.
Disclosure of Invention
Aiming at the problems, the invention provides a method for simultaneously determining the content of 3 nucleoside components in radix glehniae, which comprises the following steps:
A. preparing a reference solution;
B. preparing a test solution: weighing 1g of radix glehniae powder, weighing, placing in a conical flask with a plug, adding 25ml of 0.1% phosphoric acid solution, sealing the plug, weighing, ultrasonically treating for 50 minutes, cooling, weighing again, supplementing the loss weight with 0.1% phosphoric acid solution, shaking up, and filtering to obtain the radix glehniae powder.
C. Sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring;
chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; taking methanol as a mobile phase A and 0.1 percent phosphoric acid solution as a mobile phase B to carry out gradient elution; the detection wavelength is 253nm; the column temperature was 30 ℃. The number of theoretical plates is not less than 10000 calculated by uridine peak.
Preferably, the preparation method of the reference solution comprises the following steps: weighing uridine control, adenosine control, and guanosine control, and adding 0.1% phosphoric acid solution to obtain mixed solution containing uridine 20 μ g, adenosine 10 μ g, and guanosine 20 μ g per 1 ml.
Preferably, in the step B, the ultrasonic treatment power is 500W, and the frequency is 40kHz; the water temperature is below 30 ℃.
Preferably, the gradient elution procedure is as follows:
the invention has the beneficial effects that:
the method established by the invention is rapid, simple and convenient, has good sensitivity, repeatability and accuracy, solves the key problem of the extraction stability of the nucleoside in the radix glehniae, can fill the blank of the determination of the content of the nucleoside in the radix glehniae, and solves a series of problems for the quality control of the radix glehniae and the preparation thereof.
Drawings
FIG. 1 is a spectrum of a uridine control solution;
FIG. 2 is a spectrum of an adenosine control solution;
FIG. 3 is a spectrum of a uridine control solution;
FIG. 4 is a chromatogram of a control solution;
FIG. 5 is a chromatogram of a test solution;
FIGS. 6 to 8 are chromatograms of the sample solution in the case of using a column I, a column II, and a column III, respectively, in which: peak 1, uridine; peak 2, adenosine; peak 3, guanosine;
FIGS. 9 to 11 are chromatograms of the test solution at 25 ℃, 30 ℃ and 35 ℃, respectively, wherein: peak 1, uridine; peak 2, adenosine; peak 3, guanosine;
FIGS. 12-14 are chromatograms of the test solution at flow rates of 0.9ml/min, 1.0ml/min, 1.1ml/min, respectively, wherein: peak 1, uridine; peak 2, adenosine; peak 3, guanosine;
FIG. 15 is a chromatogram of example 1;
FIG. 16 is a chromatogram of example 2;
FIG. 17 is a chromatogram of example 3.
Detailed Description
In order to make the aforementioned features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below, but the present invention is not limited thereto.
In the description of the present invention, reference to "one embodiment" means that a particular feature, structure, or parameter, step, or the like described in the embodiment is included in at least one embodiment according to the present invention. Thus, appearances of the phrases such as "in one embodiment," "in one embodiment," and the like in this specification are not necessarily all referring to the same embodiment, nor are other phrases such as "in another embodiment," "in a different embodiment," and the like. Those of skill in the art will understand that the particular features, structures or parameters, steps, etc., disclosed in one or more embodiments of the present description may be combined in any suitable manner.
Example 1
Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 253nm; the column temperature was 30 ℃. The number of theoretical plates should not be less than 10000 calculated as uridine peaks.
Preparation of control solutions: precisely weighing appropriate amount of uridine control, adenosine control, and guanosine control, and adding 0.1% phosphoric acid solution to obtain mixed solution containing uridine 20 μ g, adenosine 10 μ g, and guanosine 20 μ g per 1 ml.
Preparation of a test solution: weighing about 1g of the powder (passing through a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 0.1% phosphoric acid solution, sealing the plug, weighing, performing ultrasonic treatment (power 500W, frequency 40kHz, water temperature below 30 ℃) for 50 minutes, cooling, weighing again, supplementing the lost weight with 0.1% phosphoric acid solution, shaking uniformly, and filtering to obtain the product.
The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring.
Example 2
Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 253nm; the column temperature was 30 ℃. The number of theoretical plates should not be less than 10000 calculated as uridine peaks.
Preparation of control solutions: precisely weighing appropriate amount of uridine control, adenosine control, and guanosine control, and adding 0.05% phosphoric acid solution to obtain mixed solution containing uridine 20 μ g, adenosine 10 μ g, and guanosine 20 μ g per 1 ml.
Preparation of a test solution: weighing about 1g of the powder (passing through a third sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 0.05% phosphoric acid solution, sealing the plug, weighing, performing ultrasonic treatment (power 500W, frequency 40kHz, water temperature below 30 ℃) for 50 minutes, cooling, weighing again, supplementing the weight loss with 0.05% phosphoric acid solution, shaking uniformly, and filtering to obtain the product.
The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring.
Example 3
Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 253nm; the column temperature was 30 ℃. The number of theoretical plates should not be less than 10000 calculated as uridine peaks.
Preparation of control solutions: precisely weighing appropriate amount of uridine control, adenosine control, and guanosine control, and adding 0.15% phosphoric acid solution to obtain mixed solution containing uridine 20 μ g, adenosine 10 μ g, and guanosine 20 μ g per 1 ml.
Preparation of a test solution: taking about 1g of the powder (passing through a No. three sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 0.15% phosphoric acid solution, sealing the plug, weighing, performing ultrasonic treatment (power 500W, frequency 40kHz, water temperature below 30 ℃) for 50 minutes, cooling, weighing again, complementing the weight loss with 0.15% phosphoric acid solution, shaking uniformly, and filtering to obtain the product.
The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Results of content determination of three examples of the same batch of samples
The above embodiments are all verified by methodology according to pharmacopeia requirements, the result display method is accurate, reliable, sensitive and exclusive, each index meets the quality control requirements, and the content determination method can be suitable for the quality control of radix glehniae.
1 Instrument and reagent
1.1 instruments
High performance liquid chromatograph: LC-40AT, shimadzu, japan;
a chromatographic column: agilent ZORBAXSB-Aq C 18 (5μm，4.6mm×250mm)；
An electronic balance: mettler AE163; mettler AE240;
ultra-pure water instrument: integral 10 (MILLIPORE).
1.2 reagents and reagents
Comparison products:
uridine (supplied by the midhouse, lot numbers 110887-201803);
adenosine (supplied by the midhouse, lot numbers 110879-201703);
guanosine (supplied by the midhouse, batch No. 111977-201501).
Reagent:
the methanol and the phosphoric acid are in chromatographic purity; the water is ultrapure water; other reagents were analytically pure.
2 method determination
2.1 determination of chromatographic conditions
2.1.1 selection of measurement wavelength uridine, adenosine and guanosine controls were weighed, respectively, and 0.1% phosphoric acid solution was used to prepare a control solution of appropriate concentration, and spectral scanning was performed at 200nm to 400 nm. The results show that uridine has a maximum absorption near 261nm, adenosine has a maximum absorption at 258nm, guanosine has a maximum absorption at 253nm, and the response value of the three components is compared, and for convenience of experiments, the maximum absorption wavelength 253nm of guanosine with a lower response value is selected as the measurement wavelength.
2.1.2 optimization of the Mobile phase
According to the investigation, the separation effect of using methanol as an organic phase is better than that of acetonitrile when a radix glehniae sample is determined, so that the methanol is selected as a mobile phase organic phase. The polarity of nucleoside components is larger, and a gradient elution mode with the initial proportion of pure water phase is selected. The aqueous phase was examined using pure water and a 0.1% phosphoric acid solution, and a 0.1% phosphoric acid solution that separated better and matched the extraction solvent was selected as the aqueous phase. Finally, the three components of uridine, adenosine and uridine are measured by using a text method, have symmetrical peak shapes and are well separated.
2.2 preparation of test solutions
2.2.1 examination of extraction solvent
In the process of examining the extraction solvent, the extraction is carried out by using water, 5% methanol solution, 10% methanol solution and the like, the obtained test sample solution has poor stability in the measurement process, and the extraction is carried out by using two solvents of 0.1% phosphoric acid solution and 0.05% trisodium phosphate respectively, and the result shows that the extraction rate of three components of uridine, adenosine and uridine in radix glehniae is high and the stability is good in 0.1% phosphoric acid solution (pH 2-3), and the extraction rate in 0.05% trisodium phosphate (pH 10-11) is slightly low, so that the phosphoric acid solution is selected as the extraction solvent. Taking appropriate amount of the same batch of samples, and respectively using 0.05% phosphoric acid solution, 0.1% phosphoric acid solution, and 0.15% phosphoric acid solution as extraction solvent, and testing according to the text method. The results show that the slightly changed phosphoric acid solution has no influence on the content determination of the sample, so that 0.1 percent phosphoric acid solution is selected as the extraction solvent, and the results are shown in Table 1.
TABLE 1 measurement results of five ingredients in different extraction solvents
2.2.2 examination of the extraction method
Taking a proper amount of samples of the same batch, performing ultrasonic treatment for 50 minutes and performing water bath reflux for 50 minutes respectively, and determining according to a text method. The results show that the ultrasonic treatment has higher extraction rate than the water bath reflux, because the water bath reflux adenosine is unstable, and in order to ensure good reproducibility, the extraction mode of the ultrasonic treatment is selected, and the temperature should be kept below 30 ℃ in the extraction process, and the results are shown in Table 2.
TABLE 2 measurement results of the contents of five components in different extraction modes
2.2.3 examination of extraction time
Taking appropriate amount of the same batch of samples, performing ultrasonic treatment for 40 min, 50 min and 60min respectively, and measuring according to text method. The results show that the ultrasonic treatment has no difference in the content of 40 min, 50 min and 60min, and the extraction time is selected to be 50 min in order to ensure complete extraction and save resources, and the results are shown in Table 3.
TABLE 3 measurement results of contents of five ingredients at different extraction times
3 methodological inspection
3.1 specificity test
Each solution was prepared according to the preparation method of the control solution and the preparation method of the test solution, and tested according to the text method. The results show that the method specificity is good.
3.2 Linear relationship inspection
A mixed control solution of uridine, adenosine and guanosine (uridine concentration 0.018134mg/ml, adenosine concentration 0.005112mg/ml, guanosine concentration 0.014199 mg/ml) was measured precisely at 1. Mu.l, 3. Mu.l, and 10. Mu.l, and a mixed control solution of uridine, adenosine, and guanosine (uridine concentration 0.18134mg/ml, adenosine concentration 0.05112mg/ml, guanosine concentration 0.14199 mg/ml) at 5. Mu.l, and 10. Mu.l were each injected into a liquid chromatograph, and measured under the proposed chromatographic conditions, and a standard curve was plotted with the control sample amount (μ g) as the abscissa and the peak area as the ordinate. The results show that uridine is in good linear relationship between 18.1338 and 1813.38 μ g, the regression equation is y =2154.5x +834.2 (r = 1), adenosine is in good linear relationship between 10.7487 and 1074.87 μ g, the regression equation is in good linear relationship between y =3174.8x-540.5 (r = 1), guanosine is in good linear relationship between 14.1996 and 1419.96 μ g, the regression equation is in y =2894.4x +179.2 (r = 1), and the experimental results are shown in table 4.
TABLE 4 investigation results of three-component linear relationship of radix Glehniae
3.3 repeatability test
Taking the same batch of samples, grinding, taking 0.5g, 1.0g and 1.5g of each three parts, precisely weighing, preparing into a test solution according to a test solution preparation method, and measuring according to a text method. The results show that the method has good repeatability, and the results are shown in Table 5.
TABLE 5 results of three-component repeatability test of radix Glehniae
3.4 recovery test
Taking 9 parts of sample powder with known content (the content is shown as a repeatability result), each part is about 0.5g, precisely weighing, and each three parts are taken as one group, respectively adding 25ml of mixed reference substance solution with low, medium and high concentrations, which is prepared by 0.1 percent phosphoric acid, and preparing the test solution according to the method under the preparation item of the test solution. Recovery was calculated as measured by the text method. The results are shown in Table 6. The method has better recovery rate.
TABLE 6-1 uridine recoveries test results
TABLE 6-2 adenosine recovery test results
TABLE 6-3 guanosine recovery test results
3.5 stability test
The same sample solution was taken, measurement was started at 0 hour, and the measurement was performed at intervals thereafter, and the peak area was recorded. The results show that the test solutions are stable for at least 24 hours, and the results are shown in Table 7.
TABLE 7 test results of stability of test solutions
3.6 durability test
According to the four parts of Chinese pharmacopoeia 2020 edition, the method is used for investigating chromatographic columns of different brands, the column temperature, the flow rate and the durability of a mobile phase, and calculating the content. The results show that the chromatographic peaks are well separated under the slight change of various conditions, the measurement results are basically consistent, and the durability is good, which is shown in Table 8.
TABLE 8 durability examination
Note:
a chromatographic column I: agilentZorbax SB-Aq, 4.6X 250mm,5 μm,
and (3) a chromatographic column II: waters SymmetryC 18 ，4.6×250mm,5μm,
A chromatographic column III: shiseidoMG II, 4.6X 250mm,5 μm.
Claims (4)
1. A method for simultaneously determining the content of 3 nucleoside components in radix glehniae is characterized by comprising the following steps:
A. preparing a reference solution;
B. preparing a test solution: weighing 1g of radix glehniae powder, weighing, placing into a conical flask with a plug, adding 25ml of 0.1% phosphoric acid solution, sealing the plug, weighing, ultrasonically treating for 50 minutes, cooling, weighing again, supplementing the loss weight with 0.1% phosphoric acid solution, shaking up, and filtering to obtain the radix glehniae powder;
C. sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring;
chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; taking methanol as a mobile phase A and taking 0.1% phosphoric acid solution as a mobile phase B to carry out gradient elution; the detection wavelength is 253nm; the column temperature was 30 ℃. The number of theoretical plates is not less than 10000 calculated by uridine peaks.
2. The method for simultaneously determining the contents of 3 nucleoside components in radix glehniae as claimed in claim 1, wherein: the preparation method of the reference substance solution comprises the following steps: weighing uridine control, adenosine control, and guanosine control, and adding 0.1% phosphoric acid solution to obtain mixed solution containing uridine 20 μ g, adenosine 10 μ g, and guanosine 20 μ g per 1 ml.
3. The method for simultaneously determining the contents of 3 nucleoside components in radix glehniae as claimed in claim 1, wherein: in the step B, the ultrasonic processing power is 500W, and the frequency is 40kHz; the water temperature is below 30 ℃.
4. The method for simultaneously determining the contents of 3 nucleoside components in radix glehniae as claimed in claim 1, wherein: the gradient elution procedure was as follows:
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