CN109030641B - Method for constructing fingerprint of Yangweishu granules - Google Patents
Method for constructing fingerprint of Yangweishu granules Download PDFInfo
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- CN109030641B CN109030641B CN201810596195.9A CN201810596195A CN109030641B CN 109030641 B CN109030641 B CN 109030641B CN 201810596195 A CN201810596195 A CN 201810596195A CN 109030641 B CN109030641 B CN 109030641B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
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Abstract
The invention discloses a method for constructing a fingerprint of Yangweishu granules, which comprises the steps of taking Yangweishu granules as a test solution through extraction, measuring at least 10 batches of qualified Yangweishu granules according to optimized chromatographic conditions to obtain corresponding chromatograms, and introducing the corresponding chromatograms into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system specified by the State pharmacopoeia Committee to obtain the fingerprint of the Yangweishu granules. The method has the characteristics of simplicity, convenience, good stability, high precision and good reproducibility, can comprehensively reflect the material information of the traditional Chinese medicine preparation, and provides a new analysis method for the quality detection and quality control of the Yangweishu granules.
Description
Technical Field
The invention relates to a method for constructing a Yangweishu granule fingerprint, which can be used for quality evaluation of different batches of Yangweishu granule samples and belongs to the field of pharmaceutical analysis.
Background
The traditional Chinese medicine fingerprint refers to a chromatogram or a spectrogram which can mark the characteristics of a traditional Chinese medicine or a traditional Chinese medicine and is obtained by adopting a certain analysis method after the traditional Chinese medicine or the traditional Chinese medicine is properly processed in a certain producing area. Wherein the chromatographic method is mainly used.
The stomach nourishing Shu granule is a new product developed according to scientific achievements of professor of Jinzheng of Jieming Jun 105 Hospital and professor of Su Tong Lang of Anhui Chinese medicine institute awarded by national major scientific and technological achievements of traditional Chinese medicine, and has the effects of nourishing yin and nourishing stomach. Can be used for treating chronic gastritis, burning gastric cavity and latent operation pain, and is a core variety in the pharmaceutical industry of the combined fat Huarun Shenlu. Since the approval of the market in 1985, the clinical application of 30 years shows unique clinical application advantages, and thus much honor is obtained.
The YANGWEISHU granule is a compound Chinese medicinal preparation prepared from rhizoma Dioscoreae, radix Codonopsis, Atractylodis rhizoma (parched), fructus crataegi (parched), pericarpium Citri Tangerinae, Zingiberis rhizoma, radix scrophulariae, radix Glehniae, rhizoma Polygonati (steamed), semen Cuscutae and mume fructus. The formula preparation contains complex chemical components, and the currently executed quality standard is as follows: the eighteenth volume of the ministerial standard Chinese medicine prescription preparation and the 028 number of the pharmacopoeia industry (2000). Due to the limitation of the experimental conditions at that time, the standard formulation is simple, and the requirement of quality evaluation cannot be well met. The research on the fingerprint spectrum of the Yangweishu granules is not reported at home and abroad, and a method capable of comprehensively and quickly detecting the quality of the Yangweishu granules is established for ensuring the uniformity and stability of the medicine quality and the safety and effectiveness of the medicine application of patients, so that the method has important significance for comprehensive quality detection and overall quality control. Based on the method, the invention establishes a method for detecting the fingerprint of Yangweishu granules.
Disclosure of Invention
The invention aims to provide a construction method of a fingerprint of Yangweishu granules, which is simple and convenient to operate, good in reproducibility and high in precision, and can be used for quality detection and quality control of Yangweishu granules.
The stomach nourishing Shu granule comprises the raw medicines of Chinese yam, codonopsis pilosula, bighead atractylodes rhizome (fried), hawthorn fruit (fried), dried orange peel, dried ginger, figwort root, coastal glehnia root, sealwort (steamed), Chinese dodder seed and dark plum fruit.
The invention relates to a method for constructing a fingerprint of Yangweishu granules, which comprises the following steps:
step 1: preparation of test solution
Weighing 5.0-10.0g stomach nourishing granule, adding into 25-100mL methanol water solution, performing ultrasonic treatment at 10-60 deg.C for 10-60min, cooling, and filtering to obtain filtrate as sample solution;
step 2: preparation of control solutions
Weighing hesperidin, harpagoside and chlorogenic acid with methanol as solvent to respectively prepare reference solutions;
and step 3: chromatographic detection
Respectively sucking 5-20 μ L of test solution and reference solution, injecting into high performance liquid chromatograph, and detecting to obtain chromatogram of the test solution and the reference solution;
the chromatographic detection parameters were as follows:
octadecylsilane chemically bonded silica is used as a filling agent; performing gradient elution by using acetonitrile as a mobile phase A and using a phosphoric acid aqueous solution with the volume fraction of 0.1% as a mobile phase B; the detection wavelength is 275-285 nm; the column temperature is 25-40 ℃; the flow rate is 0.5-1.5 mL/min.
The gradient elution program was set up as follows:
0min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B;
0-10min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B;
for 10min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B;
the volume of the mobile phase A is gradually increased from 10% to 28% within 10-35min, and the balance is the mobile phase B;
35min, the volume of the mobile phase A is 28%, and the balance is the mobile phase B;
the volume of the mobile phase A is gradually increased from 28% to 40% within 35-45min, and the balance is the mobile phase B;
45min, the volume of the mobile phase A is 40%, and the balance is the mobile phase B;
gradually increasing the volume of the mobile phase A from 40% to 80% within 45-46min, and taking the rest as the mobile phase B;
46min, wherein the volume of the mobile phase A is 80%, and the balance is the mobile phase B;
46-55min, the volume of the mobile phase A is 80%, and the balance is the mobile phase B;
55min, the volume of the mobile phase A is 80%, and the balance is the mobile phase B;
the volume of the mobile phase A is gradually reduced from 80% to 10% after 55-60min, and the balance is the mobile phase B;
for 60min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B;
the volume of the mobile phase A is 10 percent, and the balance is the mobile phase B, 60-65 min;
65min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B.
And 4, step 4: and (3) respectively carrying out data import, multipoint correction and data matching on the chromatograms of the test solution and the reference solution by utilizing a Chinese medicine chromatogram fingerprint similarity evaluation system of the State pharmacopoeia Committee, and obtaining the fingerprint by adopting a median method and the time width of 0.3 min.
Preferably, in step 1, 5.0-8.0g of Yangweishu granules are weighed and added into 25-50mL of 40-80% methanol aqueous solution by volume concentration, ultrasonic treatment is carried out for 30-50min at 10-60 ℃, cooling and filtering are carried out, and the obtained filtrate is the test solution.
Further preferably, in step 1, 7.5g of yangweishu granules are weighed and added into 25mL of methanol aqueous solution with volume concentration of 50-70%, ultrasonic treatment is carried out for 45min at 10-60 ℃, cooling and filtering are carried out, and the obtained filtrate is the test solution.
Preferably, in step 2, the concentration of the hesperidin solution in the control solution is 0.06mg/mL, the concentration of harpagoside in the control solution is 0.05mg/mL, and the concentration of chlorogenic acid in the control solution is 0.05 mg/mL.
Preferably, in step 3, the sample amount of the test solution and the reference solution is 5 to 15 μ L, and more preferably 10 μ L; the detection wavelength is 280 nm; the column temperature is 30 ℃; the flow rate was 1.0 mL/min.
Preferably, in step 3, a 250mm × 4.6mm, 5 μm Agilent Eclipse XDB-C18 chromatography column is used for the chromatographic detection.
The fingerprint spectrum of the Yangweishu granules obtained by the invention has 13 common peaks, and the comparison of the comparison product spectrum determines that the peak No. 2 is chlorogenic acid, the peak No. 8 is hesperidin and the peak No. 11 is harpagoside; taking hesperidin peak 8 as a reference peak, and the relative retention time of each peak is as follows: peak No. 1 0.307, peak No. 2 0.346, peak No. 3 0.374, peak No. 4 0.397, peak No. 5 0.522, peak No. 6 0.610, peak No. 7 0.931, peak No. 8 1.000, peak No. 9 1.031, peak No. 10 1.246, peak No. 11 1.259, peak No. 12 1.309, and peak No. 13 1.380.
The application of the fingerprint of the Yangweishu granules constructed by the invention is to detect the quality of Yangweishu granules preparation by using the fingerprint constructed by the invention, and the application specifically comprises the following steps:
step 1: preparation of test substance solution
Precisely weighing 5.0-10.0g of Yangweishu granules to be measured, adding 25-100mL of methanol solution with volume concentration of 0-100%, performing ultrasonic treatment at 10-60 ℃ for 10-60min, cooling, and filtering to obtain filtrate, namely solution to be measured;
step 2: preparation of control solutions
Methanol is used as a solvent to prepare reference substance solutions with the hesperidin solution concentration of 0.06mg/mL, the harpagoside concentration of 0.05mg/mL and the chlorogenic acid concentration of 0.05mg/mL respectively.
And step 3: chromatographic detection
Respectively sucking 5-20 μ L of the solution to be detected and the reference solution, injecting into a high performance liquid chromatograph, and detecting to obtain chromatograms of the solution to be detected and the reference solution;
and 4, step 4: comparing the obtained fingerprint of the to-be-detected product with the constructed standard fingerprint by using a Chinese medicine chromatography fingerprint similarity evaluation system of the State pharmacopoeia Committee, calculating the similarity of the fingerprints, if the similarity is more than or equal to 0.9 and 13 characteristic peaks in the standard fingerprint appear, and the peak corresponding to the reference peak is an S peak, and setting the retention time as follows according to the principle that the deviation of the retention time is not more than +/-5 percent:
peak No. 1: 0.307 +/-0.015; peak No. 2: 0.346 +/-0.017; peak No. 3: 0.374 +/-0.019; peak No. 4: 0.397 +/-0.020; peak No. 5: 0.522 plus or minus 0.026; peak No. 6: 0.610 plus or minus 0.030; peak No. 7: 0.931. + -. 0.047; peak No. 8: 1.000; peak No. 9: 1.031 ± 0.052; peak No. 10: 1.246 plus or minus 0.062; peak No. 11: 1.259 plus or minus 0.063; peak No. 12: 1.309 +/-0.065; peak No. 13: 1.380 +/-0.069. Wherein the peak No. 2 is chlorogenic acid, the peak No. 8 is hesperidin, and the peak No. 11 is harpagoside.
And if the relative retention time of the common peaks is within the range, the quality of the Yangweishu granules to be detected is qualified, otherwise, the quality of the Yangweishu granules to be detected is unqualified.
The Yangweishu granules adopted by the invention are 10 batches of samples which are qualified by the current standard inspection.
The invention provides a construction method of a fingerprint of Yangweishu granules, which is simple and convenient to operate, good in reproducibility and high in precision, can be used for controlling the quality of a preparation through the construction of the fingerprint, is good in stability and repeatability, can be used for monitoring the production process of Yangweishu granules and detecting in quality evaluation, is beneficial to comprehensive quality detection and overall quality control of the Yangweishu granules, and ensures that the quality of the Yangweishu granules meets the standard.
The preparation method of the test sample is simple and convenient, and the chromatographic conditions are easy to realize.
The method for establishing the fingerprint of Yangweishu granules has the advantages of high stability, high precision, good repeatability and the like, and the established fingerprint can be used for monitoring the production process and controlling the quality of Yangweishu granules, so that the stability and the uniformity of the quality of Yangweishu granules can be ensured, and the effectiveness and the safety of clinical medication of the medicine can be improved.
Drawings
FIG. 1 is a liquid chromatogram of a hesperidin reference solution in Experimental example 3 of the present invention;
FIG. 2 is a liquid chromatogram of a harpagoside reference solution in Experimental example 3;
FIG. 3 is a liquid chromatogram of a chlorogenic acid control solution in Experimental example 3 of the present invention;
FIG. 4 is a liquid chromatogram of a test solution (lot number: 1705202) of Yangweishu granules according to Experimental example 3 of the present invention;
fig. 5 is a superposition chromatogram derived from 10 batches of yangweishu granule test solution introduced into fingerprint similarity calculation software in experimental example 3;
fig. 6 is a fingerprint of yangweishu granules established in experimental example 3 of the present invention.
Detailed Description
In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.
The instrument and reagent of the embodiment of the invention are as follows:
the instrument comprises the following steps: FA2104 ten-thousandth electronic balance (manufactured by shanghai balance); AU-220 one ten thousandth electronic balance (Shimadzu, Japan); ultrasonic instruments (Tianjin Orthon precision instruments Co.); LC-20AT high performance liquid chromatograph, SPD-20A detector (Shimadzu corporation, Japan); waters e2695 high performance liquid chromatograph, 2998PDA detector (Watts corporation, USA).
Reagent testing: methanol is analytically pure (Shanghai Suyi chemical reagent Co., Ltd.); acetonitrile is chromatographically pure (osmic Barker environmental chemical, Sweden); phosphoric acid is analytically pure (Shanghai Suyi chemical reagent Co., Ltd.); the ultrapure water is self-made by a water purifier; the Yangweishu granules are respectively 1705202, 1704213, 1704208, 1703220, 1703216, 1703207, 1701219, 1701210, 1701206 and 1701201.
Example 1: preparation of test solution
1. Examination of extraction solvent and sample amount
Taking about 7.5g and 4 parts of Yangweishu granule sample (1705202), respectively placing into 150mL conical flasks with plugs, respectively precisely adding methanol, 75% methanol, 50% methanol and 25mL water, respectively, ultrasonically extracting for 30min, standing to room temperature, filtering with 0.45 μm filter membrane, and collecting the filtrate. The samples were injected into 20. mu.L and 10. mu.L, respectively. The peak area differences per unit mass of different solvents were compared.
The result shows that the peak shape of the sample injection amount of 20 muL is poor, the peak shape of the sample injection amount of 10 muL is good, the theoretical pedal number is high, and therefore the sample injection amount of 10 muL is selected; the peak area of the main chromatographic peak unit mass of the sample extracted by the 50% methanol solution is larger, and the extraction is more complete; extraction with methanol resulted in sample encapsulation and lower extraction yields, so 50% methanol was chosen as the extraction solvent.
2. Investigation of extraction time
Taking about 7.5g and 3 parts of Yangweishu granule sample (1705202), respectively placing into 150mL conical flasks, precisely adding 25mL of 50% methanol, and respectively ultrasonically extracting for 30min, 45min and 60 min. Cooling to room temperature, filtering with 0.45 μm filter membrane, and collecting the filtrate. Comparing peak area differences of unit mass of different extraction time.
The results show that the ultrasonic extraction is more complete in 45min, the peak area per unit mass does not increase any more in 60min, and therefore the extraction time is selected to be 45 min.
Example 2: chromatographic condition optimization
1. Investigation of detection wavelength
According to the reference of the literature, the content of chlorogenic acid, vitexin rhamnoside and hesperidin in the Yangweishu granules is measured simultaneously by an HPLC method, the maximum absorption wavelengths of the chlorogenic acid, the vitexin rhamnoside and the hesperidin are 327nm, 335nm and 283nm respectively, a Yangweishu granule sample (1705202) is measured in the wavelength range of 220nm-400nm by a PDA detector, chromatograms at 220nm, 254nm, 280nm, 300nm and 330nm are extracted respectively, comparison of the different wavelengths shows that chromatographic peak information at 220nm and 280nm is most abundant, a 280nm base line is better, and a plurality of main chromatographic peaks (RT (39.745), 31.891 and 39.954) have maximum absorption near 280nm, so that 280nm is selected as the detection wavelength of the fingerprint of the Yangweishu granules.
2. Elution gradient investigation
Because the Yangweishu granules contain various polar compounds, octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.1% phosphoric acid water solution as mobile phase; column temperature: and (3) optimizing a gradient elution program under the basic conditions that the detection wavelength is 220nm and the sample injection amount is 10 mu L at the temperature of 30 ℃, and preferably selecting the optimal elution gradient as follows:
example 3: fingerprint construction of Yangweishu granules
Taking 10 batches of Yangweishu particle samples which are respectively 1705202(S1), 1704213(S2), 1704208(S3), 1703220(S4), 1703216(S5), 1703207(S6), 1701219(S7), 1701210(S8), 1701206(S9) and 1701201(S10) qualified by current standard detection, and carrying out Yangweishu particle fingerprint detection according to optimized detection conditions, wherein the method specifically comprises the following steps:
(1) preparation of a test solution: precisely weighing 7.5g of Yangweishu granule powder, adding 25mL of 50% methanol solution, performing ultrasonic treatment for 45min, cooling, filtering, and collecting the filtrate to obtain a test solution;
(2) preparation of control solutions: accurately weighing hesperidin control, and adding methanol to obtain 0.06mg/mL control solution as reference solution. Methanol is respectively added into a proper amount of harpagoside and chlorogenic acid to prepare a reference substance solution of 0.05 mg/mL;
(3) chromatographic conditions are as follows: using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A, and a phosphoric acid aqueous solution with volume fraction of 0.1% as a mobile phase B, and performing gradient elution according to the following procedures:
0min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B;
0-10min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B;
for 10min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B;
the volume of the mobile phase A is gradually increased from 10% to 28% within 10-35min, and the balance is the mobile phase B;
35min, the volume of the mobile phase A is 28%, and the balance is the mobile phase B;
the volume of the mobile phase A is gradually increased from 28% to 40% within 35-45min, and the balance is the mobile phase B;
45min, the volume of the mobile phase A is 40%, and the balance is the mobile phase B;
gradually increasing the volume of the mobile phase A from 40% to 80% within 45-46min, and taking the rest as the mobile phase B;
46min, wherein the volume of the mobile phase A is 80%, and the balance is the mobile phase B;
46-55min, the volume of the mobile phase A is 80%, and the balance is the mobile phase B;
55min, the volume of the mobile phase A is 80%, and the balance is the mobile phase B;
the volume of the mobile phase A is gradually reduced from 80% to 10% after 55-60min, and the balance is the mobile phase B;
for 60min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B;
the volume of the mobile phase A is 10 percent, and the balance is the mobile phase B, 60-65 min;
65min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B.
The detection wavelength is 280nm, the column temperature is 30 ℃, and the flow rate is 1.0 mL/min;
(4) accurately sucking 10 μ L of each of the sample solution and the reference solution, injecting into a high performance liquid chromatograph, and measuring to obtain chromatograms of the sample solution and the reference solution;
(5) a comparison characteristic spectrum is generated by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system in fingerprint similarity evaluation software compiled by a pharmacopoeia committee. The liquid chromatogram of the reference solution, the liquid chromatogram of the Yangweishu granule test solution with lot number 1705202, the superimposed chromatogram derived after 10 batches of the Yangweishu granule test solution are introduced into the fingerprint similarity calculation software, and the established reference fingerprint of the Yangweishu granule are respectively shown in fig. 1-5.
(6) The common peaks in the fingerprint spectrum of the Yangweishu granules are detected as follows: peak 1, peak 2 chlorogenic acid, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8 hesperidin, peak 9, peak 10, peak 11 harpagoside, peak 12 and peak 13; relative retention times for the other peaks were calculated using peak No. 8 as the reference peak and relative retention time 1, and the results are shown in the table below.
TABLE 110 batch Yangweishu granules sharing peak relative retention time
From table 1, it follows: taking hesperidin peak 8 as a reference peak, wherein the average relative retention time of each peak is as follows: peak No. 1 0.307, peak No. 2 0.346, peak No. 3 0.374, peak No. 4 0.397, peak No. 5 0.522, peak No. 6 0.610, peak No. 7 0.931, peak No. 8 1.000, peak No. 9 1.031, peak No. 10 1.246, peak No. 11 1.259, peak No. 12 1.309, and peak No. 13 1.380. The relative standard deviations are all small. According to the principle that the deviation of the retention time is not more than +/-5%, the retention time is defined as:
peak No. 1: 0.307 +/-0.015; peak No. 2: 0.346 +/-0.017;
peak No. 3: 0.374 +/-0.019; peak No. 4: 0.397 +/-0.020;
peak No. 5: 0.522 plus or minus 0.026; peak No. 6: 0.610 plus or minus 0.030;
peak No. 7: 0.931. + -. 0.047; peak No. 8: 1.000;
peak No. 9: 1.031 ± 0.052; peak No. 10: 1.246 plus or minus 0.062;
peak No. 11: 1.259 plus or minus 0.063; peak No. 12: 1.309 +/-0.065;
peak No. 13: 1.380 plus or minus 0.069;
wherein the peak No. 2 is chlorogenic acid, the peak No. 8 is hesperidin, and the peak No. 11 is harpagoside.
Example 4: stomach nourishing granule fingerprint spectrum methodology verification
1. Precision test
Taking the same batch of Yangweishu granule samples (1705202), preparing a test sample solution according to the test sample solution preparation method, carrying out continuous sample injection for 6 times, and inspecting the relative retention time and the relative peak area of each common peak and an internal standard peak, wherein the results are shown in tables 1 and 2.
TABLE 1 precision test-relative Retention time
TABLE 2 precision test-relative Peak area
The result shows that the relative retention time RSD is less than 1%, the relative peak area RSD is less than 3%, and the method has better precision.
2. Repeatability test
6 portions of the same lot of samples (1705202) were taken, and the sample solutions were prepared according to the sample solution preparation method, and the chromatograms were measured, and the results are shown in tables 3 and 4.
TABLE 3 repeatability tests-relative retention time
TABLE 4 repeatability tests-relative peak area
The result shows that the relative retention time RSD of each common chromatographic peak is less than 2%, the relative peak area RSD is less than 3%, and the method has good repeatability.
3. Stability test
The same test solution was taken and tested at 0, 2, 4, 8, 12h, 24h, respectively, and the retention time and peak area of each common chromatographic peak were recorded, the results are shown in tables 5 and 6.
TABLE 5 stability test-relative Retention time
TABLE 6 stability test-relative Peak area
The result shows that the relative retention time RSD of each common chromatographic peak within 24h is less than 1%, the relative peak area RSD is less than 3%, and the test solution is stable within at least 24 h.
Example 5: similarity evaluation of fingerprint spectrum of Yangweishu granules
Taking 10 batches of Yangweishu granule samples with the batch numbers of 1705202(S1), 1704213(S2), 1704208(S3), 1703220(S4), 1703216(S5), 1703207(S6), 1701219(S7), 1701210(S8), 1701206(S9) and 1701201(S10), detecting the fingerprints according to the method provided in example 3, introducing the 10 batches of samples into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system specified by the Committee pharmacopoeia, matching and calculating the similarity by taking 1705202 spectrogram as a reference spectrum and adopting a median method with the time width of 0.3 min. The similarity results are shown in table 7.
TABLE 7 fingerprint similarity results
As seen from the similarity results in table 7, the similarity between the 10 batches of yangweishu granules and the reference spectrum and the comparison spectrum generated according to the median method are both greater than 0.9, indicating that the similarity between different batches of yangweishu granules is better. The fingerprint of Yangweishu granule can be included in the quality evaluation, and the similarity is not less than 0.9.
Example 6:
the method for constructing the fingerprint of yangweishu granules in the embodiment is different from the method in the embodiment 3 only in that: in the preparation steps of the test solution, 8.0g of yangweishu granules are weighed, and the rest of experimental conditions and experimental operations are the same as those in example 3.
Example 7:
the method for constructing the fingerprint of yangweishu granules in the embodiment is different from the method in the embodiment 3 only in that: in the preparation step of the test solution, a methanol solution with a volume fraction of 70% was used as an extraction solvent, and the rest of the experimental conditions and experimental operations were the same as those of example 3.
Example 8:
the method for constructing the fingerprint of yangweishu granules in the embodiment is different from the method in the embodiment 3 only in that: in the preparation step of the test solution, the extraction ultrasound time was 40min, and the rest of the experimental conditions and experimental operations were the same as those in example 3.
Example 9: quality detection of Yangweishu granules
Sample batch number: 1706201, 1706202
Preparing a test solution, preparing a reference solution and carrying out chromatographic conditions: the same as in example 3.
Respectively and precisely sucking the equal amount of the test solution and the reference solution, injecting into a high performance liquid chromatograph, and recording for 65min according to the chromatographic conditions to obtain the liquid chromatogram of each test solution and reference solution. And evaluating the chromatogram by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system specified by a pharmacopoeia committee, and calculating the similarity with the reference fingerprint. The relative retention time of other characteristic peaks was calculated with the retention time of hesperidin as a control as 1, and the calculation results are shown in table 8.
TABLE 8 relative retention time of characteristic peaks and fingerprint similarity for lot Nos. 1706201 and 1706202 samples
Table 8 shows that the relative retention times of the characteristic peaks of the samples of lot numbers 1706201 and 1706202 are within the specified retention time range, and the similarity is > 0.9, which indicates that the quality of the two batches of yangweishu granules meets the requirement.
Claims (4)
1. A method for constructing a fingerprint of Yangweishu granules is characterized by comprising the following steps:
step 1: preparation of test solution
Weighing 5.0-10.0g stomach nourishing granule, adding into 25-100mL methanol water solution, performing ultrasonic treatment at 10-60 deg.C for 10-60min, cooling, and filtering to obtain filtrate as sample solution;
step 2: preparation of control solutions
Weighing hesperidin, harpagoside and chlorogenic acid with methanol as solvent to respectively prepare reference solutions;
and step 3: chromatographic detection
Respectively sucking 5-20 μ L of test solution and reference solution, injecting into high performance liquid chromatograph, and detecting to obtain chromatogram of the test solution and the reference solution;
and 4, step 4: respectively carrying out data import, multipoint correction and data matching on chromatograms of a test solution and a reference solution by using a similarity evaluation system of a traditional Chinese medicine chromatogram fingerprint of the State pharmacopoeia Committee, and obtaining the fingerprint by adopting a median method and the time width of 0.3 min;
in the step 3, an Agilent Eclipse XDB-C18 chromatographic column of 250mm multiplied by 4.6mm and 5 mu m is used during chromatographic detection;
in step 3, the chromatographic detection parameters are as follows:
octadecylsilane chemically bonded silica is used as a filling agent; performing gradient elution by using acetonitrile as a mobile phase A and using a phosphoric acid aqueous solution with the volume fraction of 0.1% as a mobile phase B; the detection wavelength is 275-285 nm; the column temperature is 25-40 ℃; the flow rate is 0.5-1.5 mL/min;
the gradient elution program was set up as follows:
0min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B;
0-10min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B;
for 10min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B;
the volume of the mobile phase A is gradually increased from 10% to 28% within 10-35min, and the balance is the mobile phase B;
35min, the volume of the mobile phase A is 28%, and the balance is the mobile phase B;
the volume of the mobile phase A is gradually increased from 28% to 40% within 35-45min, and the balance is the mobile phase B;
45min, the volume of the mobile phase A is 40%, and the balance is the mobile phase B;
gradually increasing the volume of the mobile phase A from 40% to 80% within 45-46min, and taking the rest as the mobile phase B;
46min, wherein the volume of the mobile phase A is 80%, and the balance is the mobile phase B;
46-55min, the volume of the mobile phase A is 80%, and the balance is the mobile phase B;
55min, the volume of the mobile phase A is 80%, and the balance is the mobile phase B;
the volume of the mobile phase A is gradually reduced from 80% to 10% after 55-60min, and the balance is the mobile phase B;
for 60min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B;
the volume of the mobile phase A is 10 percent, and the balance is the mobile phase B, 60-65 min;
65min, the volume of the mobile phase A is 10%, and the balance is the mobile phase B.
2. The construction method according to claim 1, characterized in that:
in step 1, 5.0-8.0g of Yangweishu granules are weighed and added into 25-50mL of 40-80% methanol aqueous solution by volume concentration, ultrasonic treatment is carried out for 30-50min at 10-60 ℃, cooling and filtering are carried out, and the obtained filtrate is the test solution.
3. The construction method according to claim 1, characterized in that:
in the step 2, the concentration of the hesperidin solution is 0.06mg/mL, the concentration of harpagoside is 0.05mg/mL, and the concentration of chlorogenic acid is 0.05mg/mL in the reference solution.
4. Use of the fingerprint of yangweishu granules constructed by the method according to claim 1, wherein: detecting the quality of the Yangweishu granule preparation by using the constructed fingerprint; the method comprises the following steps:
step 1: preparation of test substance solution
Precisely weighing 5.0-10.0g of Yangweishu granules to be measured, adding 25-100mL of methanol solution with volume concentration of 0-100%, performing ultrasonic treatment at 10-60 ℃ for 10-60min, cooling, and filtering to obtain filtrate, namely solution to be measured;
step 2: preparation of control solutions
Using methanol as a solvent to respectively prepare reference substance solutions with the hesperidin solution concentration of 0.06mg/mL, the harpagoside concentration of 0.05mg/mL and the chlorogenic acid concentration of 0.05 mg/mL;
and step 3: chromatographic detection
Respectively sucking 5-20 μ L of the solution to be detected and the reference solution, injecting into a high performance liquid chromatograph, and detecting to obtain chromatograms of the solution to be detected and the reference solution;
and 4, step 4: comparing the obtained fingerprint of the to-be-detected product with the constructed standard fingerprint by using a Chinese medicine chromatography fingerprint similarity evaluation system of the State pharmacopoeia Committee, calculating the similarity of the fingerprints, if the similarity is more than or equal to 0.9 and 13 characteristic peaks in the standard fingerprint appear, and the peak corresponding to the reference peak is an S peak, and setting the retention time as follows according to the principle that the deviation of the retention time is not more than +/-5 percent:
peak No. 1: 0.307 +/-0.015; peak No. 2: 0.346 +/-0.017; peak No. 3: 0.374 +/-0.019; peak No. 4: 0.397 +/-0.020; peak No. 5: 0.522 plus or minus 0.026; peak No. 6: 0.610 plus or minus 0.030; peak No. 7: 0.931. + -. 0.047; peak No. 8: 1.000; peak No. 9: 1.031 ± 0.052; peak No. 10: 1.246 plus or minus 0.062; peak No. 11: 1.259 plus or minus 0.063; peak No. 12: 1.309 +/-0.065; peak No. 13: 1.380 plus or minus 0.069;
and if the relative retention time of the common peaks is within the range, the Yangweishu granules to be detected are qualified in quality.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101703748A (en) * | 2009-11-06 | 2010-05-12 | 合肥神鹿双鹤药业有限责任公司 | Method for detecting quality of Yangweishu granules |
| WO2016201299A1 (en) * | 2015-06-12 | 2016-12-15 | Indiana University Research & Technology Corporation | Predicting suicidality using a combined genomic and clinical risk assessment |
| CN106770785A (en) * | 2017-01-11 | 2017-05-31 | 合肥华润神鹿药业有限公司 | A kind of method of the finger-print for setting up madder rattan medicinal material |
-
2018
- 2018-06-11 CN CN201810596195.9A patent/CN109030641B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101703748A (en) * | 2009-11-06 | 2010-05-12 | 合肥神鹿双鹤药业有限责任公司 | Method for detecting quality of Yangweishu granules |
| WO2016201299A1 (en) * | 2015-06-12 | 2016-12-15 | Indiana University Research & Technology Corporation | Predicting suicidality using a combined genomic and clinical risk assessment |
| CN106770785A (en) * | 2017-01-11 | 2017-05-31 | 合肥华润神鹿药业有限公司 | A kind of method of the finger-print for setting up madder rattan medicinal material |
Non-Patent Citations (11)
| Title |
|---|
| A multicenter study of Chinese patent medicine wenweishu/yangweishu in the treatment of Helicobacter pylori positive patients with chronic gastritis and peptic ulcer;Hu, Fu-lian;《Zhonghua yi xue za zhi》;20101231;第90卷(第2期);第75-78页 * |
| HPLC-DAD法同时测定清肺宁嗽丸中4种成分的含量;李宪刚 等;《中国医药科学》;20160531;第6卷(第9期);第36-39页 * |
| HPLC法同时测定养胃舒颗粒中绿原酸、牡荆素鼠李糖苷及橙皮苷的含量;江萌 等;《中国药师》;20141231;第17卷(第3期);第412-414页 * |
| HPLC法同时测定银翘散煎液中七种有效成分含量;薛鑫 等;《2013年"好医生杯"中药制剂创新与发展论坛论文集》;20140725;第754-759页 * |
| Modeling Natural Anti-Inflammatory Compounds by Molecular Topology;María Galvez-Llompart 等;《International Journal of Molecular Sciences》;20111231;第12卷(第12期);第9481-9503页 * |
| 五参二连颗粒高效液相色谱指纹图谱研究及多成分定量分析;陈永刚 等;《中国医药工业杂志》;20131231;第44卷(第6期);第604-608页 * |
| 咳立止滴丸的指纹图谱研究;刘小花 等;《第三届中国中西部地区色谱学术交流会论文集》;20120327;第43-47页 * |
| 怀牛膝"性善下行"对通塞脉微丸中绿原酸、异甘草苷、哈巴俄苷和甘草素体内药代动力学影响研究;程健 等;《中国中药杂志》;20140430;第39卷(第8期);第1502-1508页 * |
| 急支糖浆指纹图谱鉴别方法研究;屈巧玲 等;《中国中药杂志》;20070430;第32卷(第8期);第681-683页 * |
| 高效液相色谱法测定养胃舒颗粒中橙皮苷的含量;彭文莉 等;《安徽医药》;20080430;第12卷(第4期);第311-312页 * |
| 麻黄宣肺止咳糖浆指纹图谱研究;胡怀明 等;《中国药师》;20171231;第20卷(第12期);第2127-2130页 * |
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