CN112858495A - Construction method of HPLC (high Performance liquid chromatography) characteristic spectrum of bear gall Keming tablets - Google Patents
Construction method of HPLC (high Performance liquid chromatography) characteristic spectrum of bear gall Keming tablets Download PDFInfo
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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
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- 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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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G01N30/02—Column chromatography
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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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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Abstract
The invention provides a construction method of an HPLC (high performance liquid chromatography) characteristic spectrum of a bear gall Keming tablet, which comprises the following steps: A) grinding fel Ursi Keming tablet, and dissolving in methanol to obtain solution to be tested; B) measuring the solution to be measured by high performance liquid chromatography to obtain characteristic chromatogram of fel Ursi Keming tablet HPLC; the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is sodium dihydrogen phosphate solution, and gradient elution is carried out. The invention adopts the high performance liquid chromatography, selects the methanol-sodium dihydrogen phosphate solution as the mobile phase for gradient elution, establishes the HPLC characteristic spectrum of the bear gall Keming tablet, has good repeatability and precision, stable and reliable method, and can control the quality of the bear gall Keming tablet.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a construction method of an HPLC (high performance liquid chromatography) characteristic spectrum of a bear gall Keming tablet.
Background
The Chinese patent medicine bear gall Kaiming tablet is made up by using 8 Chinese medicinal materials of bear gall powder, abalone shell, chrysanthemum flower, abalone shell (calcined), lycium berry, alisma tuber (roasted), gentian and motherwort fruit, etc. The product has the main functions of clearing liver heat, nourishing yin and improving vision. Can be used for treating heat stagnation in liver and gallbladder, and pupil tightness due to yin essence deficiency, and cataract due to wind-evil with symptoms of conjunctival congestion, pain, photophobia, lacrimation, blurred vision, dysphoria, irritability, bitter taste, and dry throat; acute iridocyclitis and primary open-angle glaucoma with the above symptoms. The current executed standard is collected in the national drug standard, and the standard number is as follows: YBZ02172007, the quality standard of the product comprises the properties of tablet; identifying rhizoma Alismatis, fructus Lycii and radix Gentianae with thin layer; and (4) measuring the content of the bezoar ursodesoxycholic acid. The quality standard only controls a qualitative and quantitative detection method of one medicine, and the product quality is difficult to be integrally and comprehensively controlled.
The Chinese herbal compound is the main form of clinical medication of the traditional Chinese medicine, and the quality of the Chinese herbal compound is the key of the clinical curative effect. How to establish a scientific and reasonable quality control method which embodies the characteristics of a Chinese herbal compound is very important, and the quality control method of the Chinese herbal compound is one of the hot research directions in the field of Chinese herbal scientific research. How to scientifically and reasonably select characteristic effective index components for quality control to effectively control the quality of the Chinese herbal compound is the key point for establishing a quality control method of the Chinese herbal compound.
The bear gall Keming tablet is a traditional Chinese medicine compound preparation developed according to the theory and the medication experience of the traditional Chinese medicine and by combining the research results of the modern medicine of China, and is clinically applied to the treatment of acute iridocyclitis and primary open-angle glaucoma. The compound preparation is composed of eight medicines of bear gall powder, abalone shell, calcined abalone shell, chrysanthemum, gentian, medlar, rhizoma alismatis, motherwort fruit and the like, wherein the bear gall powder is taken as a monarch medicine, the abalone shell and the chrysanthemum are taken as ministerial medicines, and the medlar and the like are taken as assistant and guide medicines.
The current executed standard is collected in the national drug standard, and the standard number is as follows: YBZ02172007, the quality standard of the product comprises the properties of tablet; identifying rhizoma Alismatis, fructus Lycii and radix Gentianae with thin layer; and (4) measuring the content of the bezoar ursodesoxycholic acid. The quality standard only controls a qualitative and quantitative detection method of one medicine, and the product quality is difficult to be integrally and comprehensively controlled.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a method for constructing an HPLC feature map of an xiong dan kaiming tablet, which is stable and reliable, and can control the quality of the xiong dan kaiming tablet.
The invention provides a construction method of an HPLC (high performance liquid chromatography) characteristic spectrum of a bear gall Keming tablet, which comprises the following steps:
A) grinding fel Ursi Keming tablet, and dissolving in methanol to obtain solution to be tested;
B) measuring the solution to be measured by high performance liquid chromatography to obtain characteristic chromatogram of fel Ursi Keming tablet HPLC;
the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is sodium dihydrogen phosphate solution, and gradient elution is carried out.
Preferably, the method further comprises preparing a reference solution: dissolving one or more of gentiopicrin, chlorogenic acid and luteolin in methanol to obtain reference solutions;
measuring the reference substance solution by adopting a high performance liquid chromatography to respectively obtain chromatograms of the reference substances; and according to the chromatogram of the reference substance, the components of the HPLC characteristic spectrum of the fel Ursi open-mingming tablet are determined.
Preferably, the mobile phase A is methanol, and the mobile phase B is 0.01mol/L sodium dihydrogen phosphate solution.
Preferably, the gradient elution is specifically:
0-5 min, 5-15% of mobile phase A and 95-85% of mobile phase B;
5-40 min, 15-15% of mobile phase A and 85-85% of mobile phase B;
40-70 min, 15-65% of mobile phase A and 85-35% of mobile phase B;
70-100 min, 65-40% of mobile phase A and 35-60% of mobile phase B;
100-101 min, 40-5% of mobile phase A and 60-95% of mobile phase B;
101-110 min, 5-5% of mobile phase A and 95-95% of mobile phase B.
Preferably, the chromatographic column is an Agilent ZORBAX SB-C18 column with the specification of 5 μm and 4.6X 250 mm;
the flow rate of the mobile phase is 0.5-1.5 mL/min; the column temperature is 20-40 ℃, and the detection wavelength is 190-410 nm; the sample injection amount is 5-15 mu L; the theoretical plate number is not less than 3000 calculated according to gentiopicroside peak.
Preferably, the flow rate of the mobile phase is 0.7 mL/min; the column temperature was 25 ℃ and the detection wavelength was initially 287nm and was adjusted to 335nm by 70 minutes.
Preferably, a traditional Chinese medicine chromatogram fingerprint similarity evaluation system is adopted to evaluate the similarity of the HPLC characteristic spectrum of the ursolic acid keming tablets, so as to obtain the HPLC standard characteristic spectrum of the ursolic acid keming tablets consisting of 10 characteristic peaks, wherein the No. 3 peak is a gentiopicroside peak, the No. 4 peak is a chlorogenic acid peak, and the No. 8 peak is luteolin; the 10 characteristic peaks were assigned: 1. the attribution of No. 3 characteristic peak is gentiana scabra medicinal material, the attribution of No. 2 characteristic peak is bear gall powder medicinal material, the attribution of No. 4, No. 6, No. 7 and No. 8 characteristic peak is chrysanthemum medicinal material, the attribution of No. 5 characteristic peak is rhizoma alismatis medicinal material, the attribution of No. 9 characteristic peak is medlar medicinal material, and the attribution of No. 10 characteristic peak is motherwort fruit medicinal material.
Preferably, in the standard feature map, taking the gentiopicroside peak as a reference peak S peak, calculating the relative retention time of each feature peak and the S peak, wherein the relative retention time is within +/-5% of specified values: 0.57-Peak 1, 0.62-Peak 2, 1.00-Peak 3, 1.20-Peak 4, 1.42-Peak 5, 1.77-Peak 6, 1.88-Peak 7, 2.00-Peak 8, 2.25-Peak 9, 2.42-Peak 10.
Preferably, ultrasonic treatment is performed after the methanol is dissolved in the step A), and the ultrasonic treatment time is 20-40 min.
Preferably, the ratio of the mass g of the ursolic acid guanamine tablets in the step A) to the volume mL of the methanol is (0.5-1.5): (20-70).
Compared with the prior art, the invention provides a construction method of an HPLC (high performance liquid chromatography) characteristic spectrum of a bear gall Keming tablet, which comprises the following steps: A) grinding fel Ursi Keming tablet, and dissolving in methanol to obtain solution to be tested; B) measuring the solution to be measured by high performance liquid chromatography to obtain characteristic chromatogram of fel Ursi Keming tablet HPLC; the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is sodium dihydrogen phosphate solution, and gradient elution is carried out. The invention adopts the high performance liquid chromatography, selects the methanol-sodium dihydrogen phosphate solution as the mobile phase for gradient elution, establishes the HPLC characteristic spectrum of the bear gall Keming tablet, has good repeatability and precision, stable and reliable method, and can control the quality of the bear gall Keming tablet.
Drawings
FIG. 1 is an HPLC characteristic spectrum of a XIONGDANKANGMING tablet prepared in example 2 of the present invention;
FIG. 2 is the HPLC characteristic spectrum of the XIONGDANKAMING tablet prepared in example 3 of the present invention;
FIG. 3 is a feature map and common mode of 10 batches of XIONGDANKANGMING tablets in example 4 of the present invention;
fig. 4 is an HPLC standard control profile of the sikkaimine tablets, wherein the peak S: peak No. 3, gentiopicroside peak.
Detailed Description
The invention provides a construction method of HPLC characteristic map of a bear gall Keming tablet, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a construction method of an HPLC (high performance liquid chromatography) characteristic spectrum of a bear gall Keming tablet, which comprises the following steps:
A) grinding fel Ursi Keming tablet, and dissolving in methanol to obtain solution to be tested;
B) measuring the solution to be measured by high performance liquid chromatography to obtain characteristic chromatogram of fel Ursi Keming tablet HPLC;
the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is sodium dihydrogen phosphate solution, and gradient elution is carried out.
The invention provides a construction method of an HPLC (high performance liquid chromatography) characteristic spectrum of a pandemic cholestyril tablet. Preferably, the preparation method comprises grinding fel Ursi Kaiming tablet, dissolving in methanol, ultrasonic treating, cooling, adding methanol to reduce weight, shaking, and filtering.
In the present invention, the grinding is not limited, and the powder may be obtained by grinding. And carrying out ultrasonic treatment after the methanol is dissolved, wherein the ultrasonic treatment time is 20-40 min.
Wherein the ratio of the mass g of the ursolic acid guanamine tablet to the volume mL of methanol is preferably (0.5-1.5): (20-70); more preferably (0.8 to 1.2): (25-45).
The invention also includes preparing a reference solution: dissolving one or more of gentiopicrin, chlorogenic acid and luteolin in methanol to obtain reference solutions respectively.
Wherein the concentration of the gentiopicrin is preferably 50 mug/mL; the concentration of the chlorogenic acid is preferably 50 mug/mL; the concentration of the luteolin is preferably 50 mug/mL.
And (4) measuring the liquid to be measured by adopting a high performance liquid chromatography to obtain the HPLC characteristic spectrum of the bear gall Keming tablet.
Measuring the reference substance solution by adopting a high performance liquid chromatography to respectively obtain chromatograms of the reference substances; and according to the chromatogram of the reference substance, the components of the HPLC characteristic spectrum of the fel Ursi open-mingming tablet are determined.
The mobile phase A is methanol, and the mobile phase B is a sodium dihydrogen phosphate solution; preferably 0.01mol/L sodium dihydrogen phosphate solution; adjusting the pH value to 2.8-3.2 by using phosphoric acid; gradient elution.
The gradient elution of the invention is preferably specifically:
0-5 min, 5-15% of mobile phase A and 95-85% of mobile phase B;
5-40 min, 15-15% of mobile phase A and 85-85% of mobile phase B;
40-70 min, 15-65% of mobile phase A and 85-35% of mobile phase B;
70-100 min, 65-40% of mobile phase A and 35-60% of mobile phase B;
100-101 min, 40-5% of mobile phase A and 60-95% of mobile phase B;
101-110 min, 5-5% of mobile phase A and 95-95% of mobile phase B.
The invention has good baseline separation, good separation degree of each peak and stable baseline under the elution gradient.
The chromatographic column is an Agilent ZORBAX SB-C18 column with the specification of 5 mu m and 4.6 multiplied by 250 mm;
the flow rate of the mobile phase is preferably 0.5-1.5 mL/min; more preferably 0.7-1.2 mL/min; most preferably 0.7 mL/min.
The chromatographic peak condition under the flow rate of 0.5-1.5 ml/min is inspected, the chromatographic peaks are found to be disordered and difficult to separate, and tests show that the chromatographic peaks under the flow rate of 0.7ml/min are better separated and the peak shapes are more symmetrical, so that the method is the most preferable scheme.
The column temperature of the present invention is preferably from 20 ℃ to 40 ℃, more preferably from 25 ℃ to 35 ℃, and most preferably 25 ℃.
The invention investigates the separation effect of chromatographic peaks at different column temperatures. The chromatographic peak separation is better, the peak shape is more symmetrical, and the most preferable scheme is that the column temperature is 25 ℃ because the chromatographic peak separation is difficult to control at 20 ℃ and the column life is not suitable for the chromatographic columns at 30 ℃ and 35 ℃.
The detection wavelength of the invention is preferably 190 nm-410 nm; more preferably, the detection wavelength is initially 287nm and is adjusted to 335nm by 70 minutes.
The inventor finds that the large-polarity unknown component has stronger response at 220nm and the gentiopicroside peak response value is low; at 370nm, the peak area of most components is small, and the response value is low; the chromatographic information is rich at 287nm and 335nm, each component has better absorption, the response value is moderate, each peak separation degree is better, the base line is stable, and according to the corresponding peak appearance condition, the single wavelength is difficult to present, so in the most preferable scheme of the invention, the detection wavelength is 287nm at the beginning and is adjusted to 335nm after 70 minutes.
The sample injection amount is 5-15 mu L; preferably 10. mu.L; the theoretical plate number is not less than 3000 calculated according to gentiopicroside peak.
The invention has the advantages that under the condition of liquid chromatography, the fingerprint spectrum is used for controlling the substance group of the fel Ursi Keming tablet, and the fingerprint spectrum is positioned by the gentiopicrin, chlorogenic acid and luteolin reference; and calculating the content of the effective components by adopting a dual-wavelength external standard method. Not only the content result is accurate and the identification effect is better, but also the detection cost can be greatly reduced, and qualitative detection is realized.
The invention adopts a Chinese medicine chromatogram fingerprint similarity evaluation system of the State pharmacopoeia Committee to evaluate the similarity of the HPLC characteristic spectrum of the bear gall Keming tablet, and obtains the HPLC standard characteristic spectrum of the bear gall Keming tablet consisting of 10 characteristic peaks. Wherein the No. 3 peak is gentiopicroside peak, the No. 4 peak is chlorogenic acid peak, and the No. 8 peak is luteolin. The 10 characteristic peaks were assigned: 1. the attribution of No. 3 characteristic peak is gentiana scabra medicinal material, the attribution of No. 2 characteristic peak is bear gall powder medicinal material, the attribution of No. 4, No. 6, No. 7 and No. 8 characteristic peak is chrysanthemum medicinal material, the attribution of No. 5 characteristic peak is rhizoma alismatis medicinal material, the attribution of No. 9 characteristic peak is medlar medicinal material, and the attribution of No. 10 characteristic peak is motherwort fruit medicinal material.
In the standard characteristic map, taking a gentiopicroside peak as a reference peak S peak, calculating the relative retention time of each characteristic peak and the S peak, wherein the relative retention time is within +/-5% of a specified value, and the specified values are respectively: 0.57-Peak 1, 0.62-Peak 2, 1.00-Peak 3, 1.20-Peak 4, 1.42-Peak 5, 1.77-Peak 6, 1.88-Peak 7, 2.00-Peak 8, 2.25-Peak 9, 2.42-Peak 10.
Quality judgment standard: and (3) taking a sample of the bear gall Keming tablet, operating according to the same method to obtain a characteristic spectrum of the bear gall Keming tablet, and analyzing the standard characteristic spectrum and the sample characteristic spectrum of the bear gall Keming tablet by adopting the 2012 version of the similarity evaluation system of the traditional Chinese medicine chromatographic fingerprint spectrum of the State Committee of pharmacopoeia, wherein the similarity is more than 0.90.
The invention establishes an HPLC characteristic spectrum common mode of the Chinese patent medicine 'bear gall Keming tablets', calibrates 10 characteristic peaks, and the established characteristic spectrum comprehensively reflects the types and the quantity of chemical components, thereby avoiding the singleness and the sidedness of the quality control of the bear gall Keming tablets and being beneficial to the comprehensive control of the product quality. The initial detection wavelength of the invention is 287nm, and the adjustment is 335nm in 70 minutes, the detection has many peaks, good peak shape, easy identification, high similarity, good stability, accuracy and reliability. The invention has the characteristics of convenience, rapidness, stability, high precision, good reproducibility and the like, and can effectively control the quality of the bear gall yiming tablet.
The invention provides a construction method of an HPLC (high performance liquid chromatography) characteristic spectrum of a bear gall Keming tablet, which comprises the following steps: A) grinding fel Ursi Keming tablet, and dissolving in methanol to obtain solution to be tested; B) measuring the solution to be measured by high performance liquid chromatography to obtain characteristic chromatogram of fel Ursi Keming tablet HPLC; the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is sodium dihydrogen phosphate solution, and gradient elution is carried out. The invention adopts the high performance liquid chromatography, selects the methanol-sodium dihydrogen phosphate solution as the mobile phase for gradient elution, establishes the HPLC characteristic spectrum of the bear gall Keming tablet, has good repeatability and precision, stable and reliable method, and can control the quality of the bear gall Keming tablet.
In order to further illustrate the present invention, the method for constructing HPLC profile of luchongkeming tablet provided by the present invention is described in detail below with reference to the following examples.
Example 1: HPLC (high Performance liquid chromatography) characteristic spectrum construction method of bear gall Keming tablets
The instrument comprises the following steps: agilent 1200 model HPLC, MS205DU model analytical balance
Reagent testing: the gentiopicroside reference substance is prepared from Gentiana Longissima pharmaceutical Limited liability company by taking methanol as chromatographic purity for liquid chromatography, taking other reagents as analytical purity, taking water as ultrapure water, and taking fel Ursi Keming tablets.
Preparation of a test solution: precisely weighing 1.0g of fel Ursi KANGMING tablet fine powder, placing in a conical flask with a plug, precisely adding 25ml of methanol, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the weight loss with methanol, shaking, filtering, and collecting the filtrate.
Preparation of reference solutions: respectively taking appropriate amount of gentiopicroside reference substance, precisely weighing, and adding methanol to obtain solutions containing 50 μ g of gentiopicroside per 1 ml.
1 bear gall Kaiming tablet characteristic spectrum experiment condition selection
1.1 Mobile phase selection
By selecting methanol as the main mobile phase through experiments and referring to relevant documents, methanol-0.2% phosphoric acid and different gradient elution effects are experimentally examined (table 1), and finally the gradient elution program is determined, as shown in table 2.
Table 1 mobile phase selection
TABLE 2 conditions of mobile phase elution
1.2 wavelength selection
In the experiment, a DAD detector is adopted to examine the detection condition in the range of 200-400 nm, and 4 wavelengths of 220nm, 287nm, 335nm and 370nm are mainly examined. The large-polarity unknown component has stronger response at 220nm, and the gentiopicroside peak response value is low; at 370nm, the peak area of most components is small, and the response value is low; the chromatographic information is rich at 287nm and 335nm, each component has better absorption, the response value is moderate, the separation degree of each peak is better, the base line is stable, and the initial detection wavelength is 287nm and is adjusted to 335nm after 70 minutes according to the corresponding peak output condition.
1.3 chromatographic column temperature selection
The results of chromatographic peak separation at column temperature of 20 deg.C, 25 deg.C, 30 deg.C, and 35 deg.C were examined. The separation of various chromatographic peaks is better, the peak shapes are more symmetrical, and the column temperature is selected to be 25 ℃ because the chromatographic column is difficult to control at 20 ℃ and the service life of the chromatographic column is not suitable at 30 ℃ and 35 ℃.
1.4 flow Rate selection
According to the experimental reference literature, the chromatographic peak conditions under the traditional flow rate of 0.9-1.1 ml/min are examined, and the chromatographic peaks are found to be disordered and not easy to separate, so that the flow rate is reduced in the experiment, and the chromatographic peak separation under the flow rate of 0.7ml/min is better than other flow rates and the peak shapes are more symmetrical, so that the flow rate of 0.7ml/min is selected.
1.5 extraction solvent selection
Experiments compare the difference of high performance liquid chromatograms of various extraction solvents of methanol, 50% methanol and ethanol by ultrasonic extraction. The ethanol does not extract various components, the components extracted by 50 percent of methanol and methanol have little difference, but the gentiopicroside content extracted by the methanol is the highest, so the methanol is selected as the extraction solvent.
1.6 extraction time study
Experiments compare the extraction efficiency of methanol ultrasonic extraction for 10min, 30min and 60min, the ultrasonic extraction for 10min is insufficient, the pattern repeatability is poor, and the ultrasonic extraction for 30min can completely extract all components, so the ultrasonic extraction for 30min is selected to extract the sample.
And finally determining:
chromatographic conditions are as follows: the chromatographic column is an Agilent ZORBAX SB-C18(5 μm, 4.6X 250mm) column, the mobile phase A is methanol, the mobile phase B is 0.01mol/L sodium dihydrogen phosphate (pH is adjusted to 2.8-3.2 by phosphoric acid), gradient elution is carried out, the flow rate is 0.7ml/min, the column temperature is 25 ℃, the detection wavelength is 287nm initially, and the detection wavelength is adjusted to 335nm by 70 minutes. The theoretical plate number is not less than 3000 calculated according to gentiopicroside peak. The volume specific concentration profile of the gradient elution procedure is shown in table 3:
TABLE 3
Preparation of a test solution: precisely weighing 1.0g of fel Ursi KANGMING tablet fine powder, placing in a conical flask with a plug, precisely adding 25ml of methanol, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the weight loss with methanol, shaking, filtering, and collecting the filtrate.
Preparation of reference solutions: accurately weighing appropriate amount of gentiopicrin reference substance, chlorogenic acid reference substance, and luteolin reference substance, and adding methanol to obtain solutions containing 50 μ g of gentiopicrin, chlorogenic acid, and luteolin per 1ml respectively.
The determination method comprises the following steps: precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into high performance liquid chromatograph, and recording chromatogram for 110 min.
1.7 methodological considerations
And (3) precision test: and continuously sampling the same sample solution for 6 times, recording the chromatogram, and comparing the relative retention time of each characteristic peak. As a result, the RSD value of the relative retention time of all characteristic peaks is less than 3.0%, indicating that the precision of the instrument is good. Detailed results are shown in table 4:
TABLE 4
And (3) repeatability test: taking 6 parts of the same batch (batch number: 160101) of the bear gall yiming tablets, preparing a test sample solution according to the test sample solution preparation method, injecting samples, and recording chromatograms. Results RSD values for relative retention times of all characteristic peaks were less than 3.0%, indicating good reproducibility of the method. Detailed results are shown in table 5:
TABLE 5
And (3) stability test: sampling the same sample solution of XIONGDANKANGMING tablet at 0, 4, 8, 12, 18, and 24 hr respectively, and recording chromatogram. Results RSD values for relative retention times of all characteristic peaks were less than 3.0%, indicating good 24 hour stability of the solution. Detailed results are shown in table 6:
TABLE 6
Example 2: HPLC (high Performance liquid chromatography) characteristic spectrum construction method of bear gall Keming tablets
The instrument comprises the following steps: agilent 1200 model HPLC, MS205DU model analytical balance
Reagent testing: the gentiopicroside reference substance is prepared from Gentiana Longissima pharmaceutical Limited liability company by taking methanol as chromatographic purity for liquid chromatography, taking other reagents as analytical purity, taking water as ultrapure water, and taking fel Ursi Keming tablets.
Preparation of a test solution: precisely weighing 1.5g of fel Ursi KANGMING tablet fine powder, placing in a conical flask with a plug, precisely adding 25ml of methanol, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the weight loss with methanol, shaking, filtering, and collecting the filtrate.
Preparation of reference solutions: taking appropriate amount of gentiopicroside reference substance, precisely weighing, and adding methanol to obtain a solution containing 50 μ g of gentiopicroside per 1 ml.
And (3) determination: chromatographic conditions are as follows: the chromatographic column is an Agilent ZORBAX SB-C18(5 μm, 4.6X 250mm) column, the mobile phase A is methanol, the mobile phase B is 0.01mol/L sodium dihydrogen phosphate (pH is adjusted to 2.8-3.2 by phosphoric acid), gradient elution is carried out, the flow rate is 0.7ml/min, the column temperature is 25 ℃, the detection wavelength is 287nm initially, and the detection wavelength is adjusted to 335nm by 70 minutes. The theoretical plate number is not less than 3000 calculated according to gentiopicroside peak. The volume specific concentration profile of the gradient elution procedure is shown in table 7:
TABLE 7
Precisely sucking 15 μ l of reference solution and sample solution respectively, injecting into high performance liquid chromatograph, and measuring by high performance liquid chromatography to obtain HPLC characteristic chromatogram of XIONGDANKANGMING tablet. As shown in fig. 1. FIG. 1 is an HPLC characteristic spectrum of a XIONGDANKANGMING tablet prepared in example 2 of the present invention;
example 3: HPLC (high Performance liquid chromatography) characteristic spectrum construction method of bear gall Keming tablets
The instrument comprises the following steps: daian U-3000 high performance liquid chromatograph, MS205DU analytical balance
Reagent testing: gentiopicrin reference substance, chlorogenic acid reference substance, and luteoloside reference substance, wherein the methanol for liquid chromatography is used as chromatographic purity, the other reagents are used as analytical purity, the water is ultrapure water, and the bear gall Kelaing tablet is provided by Jilin Changbai mountain pharmacy Limited liability company.
Preparation of a test solution: precisely weighing 1.0g of fel Ursi KANGMING tablet fine powder, placing in a conical flask with a plug, precisely adding 25ml of methanol, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the weight loss with methanol, shaking, filtering, and collecting the filtrate.
Preparation of reference solutions: accurately weighing appropriate amount of gentiopicrin reference substance, chlorogenic acid reference substance, and luteolin reference substance, and adding methanol to obtain solutions containing 50 μ g of gentiopicrin, chlorogenic acid, and luteolin per 1ml respectively.
And (3) determination: chromatographic conditions are as follows: the chromatographic column is an Agilent ZORBAX SB-C18(5 μm, 4.6X 250mm) column, the mobile phase A is methanol, the mobile phase B is 0.01mol/L sodium dihydrogen phosphate (pH is adjusted to 2.8-3.2 by phosphoric acid), gradient elution is carried out, the flow rate is 0.7ml/min, the column temperature is 25 ℃, the detection wavelength is 287nm initially, and the detection wavelength is adjusted to 335nm by 70 minutes. The theoretical plate number is not less than 3000 calculated according to gentiopicroside peak. The volume specific concentration profile of the gradient elution procedure is shown in table 8:
TABLE 8
Precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into high performance liquid chromatograph, and measuring by high performance liquid chromatography to obtain HPLC characteristic chromatogram of XIONGDANKANGMING tablet. As shown in fig. 2.
Example 4: determination of HPLC characteristic spectrum of bear gall Keming tablet
The instrument comprises the following steps: shimadzu 2010c high performance liquid chromatograph, MS205DU analytical balance
Reagent testing: gentiopicrin reference substance, chlorogenic acid reference substance, and luteoloside reference substance, wherein methanol for liquid chromatography is used as chromatographic pure, other reagents are used as analytical pure, water is ultrapure, and fel Ursi Keming tablet (batch numbers are 190101, 190102, 190103, 191201, 191202, 191203, 200101, 200102, 200103 and 201001 respectively) is provided by Jilin Changbai mountain pharmacy, Ltd.
Preparation of a test solution: precisely weighing 1.0g of fel Ursi KANGMING tablet fine powder, placing in a conical flask with a plug, precisely adding 50ml of methanol, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the weight loss with methanol, shaking, filtering, and collecting the filtrate.
Preparation of reference solutions: respectively taking appropriate amount of gentiopicroside reference substance, precisely weighing, and adding methanol to obtain solutions containing 50 μ g of gentiopicroside per 1 ml.
Chromatographic conditions are as follows: the chromatographic column is an Agilent ZORBAX SB-C18(5 μm, 4.6X 250mm) column, the mobile phase A is methanol, the mobile phase B is 0.01mol/L sodium dihydrogen phosphate (pH is adjusted to 2.8-3.2 by phosphoric acid), gradient elution is carried out, the flow rate is 0.7ml/min, the column temperature is 25 ℃, the detection wavelength is 287nm initially, and the detection wavelength is adjusted to 335nm by 70 minutes. The theoretical plate number is not less than 3000 calculated according to gentiopicroside peak. The volume specific concentration profile of the gradient elution procedure is shown in table 9:
TABLE 9
And (3) determination: precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into high performance liquid chromatograph, and measuring by high performance liquid chromatography.
Feature mapping
The method is adopted to measure 10 batches of the bear gall yiming tablets, the characteristic spectrum is recorded, the relative retention time is compared, and as a result, the RSD value of the relative retention time of the 10 batches of the bear gall yiming tablets is less than 3.0 percent, and the detailed results are as follows:
watch 10
After 10 batches of preparation chromatograms recorded under the proposed chromatographic condition are converted into numerical data and exported, traditional Chinese medicine chromatogram fingerprint similarity evaluation software 2012 edition (national pharmacopoeia committee) is adopted to conduct data import, matching and correction (figure 3), and a contrast characteristic spectrum which can comprehensively reflect all sample fingerprint characteristic information, namely an HPLC standard contrast characteristic spectrum of the ursolic acid tablets (figure 4) is generated. The similarity of the preparations of each batch was calculated by comparison with the control profile. Detailed results are shown in table 11:
TABLE 11
The experiment carries out characteristic spectrum research on the bear gall yiming tablets, and compares the relative retention time of 10 batches of samples, so that the RSD value of the relative retention time of 10 batches of the bear gall yiming tablets is less than 3.0 percent. And similarity software is adopted to evaluate chromatographic peak data, an HPLC standard control characteristic spectrum of the fel Ursi open-bright tablet is established, results show that the overall picture appearance of each batch of preparation peak groups is basically consistent, 10 common peaks are determined, and the similarity is over 0.9.
Example 5: HPLC (high Performance liquid chromatography) characteristic spectrum construction method of bear gall Keming tablets
The instrument comprises the following steps: agilent 1200 model HPLC, MS205DU model analytical balance
Reagent testing: the method comprises the steps of preparing a gentiopicrin reference substance, a chlorogenic acid reference substance and a luteoloside reference substance, using methanol as chromatographic purity for liquid chromatography analysis, using other reagents as analytical purity, using ultrapure water as water, and providing a fel Ursi Keming tablet sample, a fel Ursi powder medicinal material, a chrysanthemum medicinal material, a wolfberry medicinal material, an rhizoma alismatis medicinal material, a gentian medicinal material and a motherwort fruit medicinal material by Jilin Changbai mountain pharmacy Limited liability company.
Preparation of a test solution: precisely weighing 1.5g of fel Ursi KANGMING tablet fine powder, placing in a conical flask with a plug, precisely adding 40ml of methanol, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing the weight loss with methanol, shaking, filtering, and collecting the filtrate.
Preparing a bear gall powder medicinal material solution: and (3) taking bear gall powder with the amount of the medicinal materials converted from the prescription, and preparing a sample according to a test article treatment method to obtain a bear gall powder medicinal material solution.
Preparing chrysanthemum medicinal materials: taking chrysanthemum of which the prescription is converted into the amount of medicinal materials, and preparing a sample according to a test article treatment method to obtain a chrysanthemum medicinal material solution.
Preparing a wolfberry fruit medicinal material: taking the wolfberry fruit of which the prescription is converted into the amount of the medicinal material, and preparing a sample according to a test article treatment method to obtain a wolfberry fruit medicinal material solution.
Preparation of rhizoma alismatis: taking rhizoma alismatis of which the prescription is converted into the amount of the medicinal materials, and preparing a sample according to a test sample treatment method to obtain rhizoma alismatis medicinal material solution.
Preparing gentian: taking gentian with the amount of medicinal materials converted by the prescription, and preparing a sample according to a test article treatment method to obtain gentian medicinal material solution.
Preparation of motherwort fruit medicinal materials: collecting fructus Leonuri with reduced medicinal material amount, and preparing sample according to the sample treatment method to obtain fructus Leonuri medicinal material solution.
Preparation of reference solutions: accurately weighing appropriate amount of gentiopicrin reference substance, chlorogenic acid reference substance, and luteolin reference substance, and adding methanol to obtain solutions containing 50 μ g of gentiopicrin, chlorogenic acid, and luteolin per 1ml respectively.
And (3) determination: chromatographic conditions are as follows: the chromatographic column is an Agilent ZORBAX SB-C18(5 μm, 4.6X 250mm) column, the mobile phase A is methanol, the mobile phase B is 0.01mol/L sodium dihydrogen phosphate (pH is adjusted to 2.8-3.2 by phosphoric acid), gradient elution is carried out, the flow rate is 0.7ml/min, the column temperature is 25 ℃, the detection wavelength is 287nm initially, and the detection wavelength is adjusted to 335nm by 70 minutes. The volume to volume concentration profile of the gradient elution procedure is shown in table 12:
TABLE 12
Respectively and precisely absorbing 10 mu l of reference substance solution and test sample solution, injecting into a high performance liquid chromatograph, recording a chromatogram, and determining the attribution of part of characteristic peaks and the relationship between the test sample and medicinal materials:
the reference peak is the No. 3 peak and is the gentiopicroside peak; peak No. 4 is the chlorogenic acid peak; peak 8 is luteolin. The 10 characteristic peaks were assigned: 1. the attribution of No. 3 characteristic peak is gentiana scabra medicinal material, the attribution of No. 2 characteristic peak is bear gall powder medicinal material, the attribution of No. 4, No. 6, No. 7 and No. 8 characteristic peak is chrysanthemum medicinal material, the attribution of No. 5 characteristic peak is rhizoma alismatis medicinal material, the attribution of No. 9 characteristic peak is medlar medicinal material, and the attribution of No. 10 characteristic peak is motherwort fruit medicinal material.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A construction method of an HPLC (high performance liquid chromatography) characteristic spectrum of a bear gall Keming tablet comprises the following steps:
A) grinding fel Ursi Keming tablet, and dissolving in methanol to obtain solution to be tested;
B) measuring the solution to be measured by high performance liquid chromatography to obtain characteristic chromatogram of fel Ursi Keming tablet HPLC;
the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 column; the mobile phase A is methanol, the mobile phase B is sodium dihydrogen phosphate solution, and gradient elution is carried out.
2. The method of claim 1, further comprising preparing a reference solution: dissolving one or more of gentiopicrin, chlorogenic acid and luteolin in methanol to obtain reference solutions;
measuring the reference substance solution by adopting a high performance liquid chromatography to respectively obtain chromatograms of the reference substances; and according to the chromatogram of the reference substance, the components of the HPLC characteristic spectrum of the fel Ursi open-mingming tablet are determined.
3. The method according to claim 1, wherein the mobile phase A is methanol and the mobile phase B is 0.01mol/L sodium dihydrogen phosphate solution.
4. The method according to claim 3, characterized in that the gradient elution is in particular:
0-5 min, 5-15% of mobile phase A and 95-85% of mobile phase B;
5-40 min, 15-15% of mobile phase A and 85-85% of mobile phase B;
40-70 min, 15-65% of mobile phase A and 85-35% of mobile phase B;
70-100 min, 65-40% of mobile phase A and 35-60% of mobile phase B;
100-101 min, 40-5% of mobile phase A and 60-95% of mobile phase B;
101-110 min, 5-5% of mobile phase A and 95-95% of mobile phase B.
5. The method of claim 4, wherein the chromatography column is an Agilent ZORBAX SB-C18 column, 5 μm, 4.6 x 250mm in size;
the flow rate of the mobile phase is 0.5-1.5 mL/min; the column temperature is 20-40 ℃, and the detection wavelength is 190-410 nm; the sample injection amount is 5-15 mu L; the theoretical plate number is not less than 3000 calculated according to gentiopicroside peak.
6. The method of claim 5, wherein the mobile phase flow rate is 0.7 mL/min; the column temperature was 25 ℃ and the detection wavelength was initially 287nm and was adjusted to 335nm by 70 minutes.
7. The method according to claim 1, wherein the similarity of the HPLC characteristic spectrum of the sikkimen tablet is evaluated by a traditional Chinese medicine chromatogram fingerprint similarity evaluation system to obtain the HPLC standard characteristic spectrum of the sikkimen tablet consisting of 10 characteristic peaks, wherein the No. 3 peak is a gentiopicroside peak, the No. 4 peak is a chlorogenic acid peak, and the No. 8 peak is luteolin; the 10 characteristic peaks were assigned: 1. the attribution of No. 3 characteristic peak is gentiana scabra medicinal material, the attribution of No. 2 characteristic peak is bear gall powder medicinal material, the attribution of No. 4, No. 6, No. 7 and No. 8 characteristic peak is chrysanthemum medicinal material, the attribution of No. 5 characteristic peak is rhizoma alismatis medicinal material, the attribution of No. 9 characteristic peak is medlar medicinal material, and the attribution of No. 10 characteristic peak is motherwort fruit medicinal material.
8. The method according to claim 7, wherein in the standard feature map, relative retention times of each feature peak to the S peak are calculated using the gentiopicroside peak as a reference peak, the S peak, the relative retention times being within ± 5% of defined values: 0.57-Peak 1, 0.62-Peak 2, 1.00-Peak 3, 1.20-Peak 4, 1.42-Peak 5, 1.77-Peak 6, 1.88-Peak 7, 2.00-Peak 8, 2.25-Peak 9, 2.42-Peak 10.
9. The method according to claim 1, wherein the methanol dissolution in the step A) is followed by ultrasonic treatment, and the ultrasonic treatment time is 20-40 min.
10. The method according to claim 1, wherein the ratio of the mass g of the ursocholoethamine tablets in step A) to the volume mL of methanol is (0.5-1.5): (20-70).
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Application publication date: 20210528 |