CN110596266B - Method for detecting gypenoside A in gelan Xinning soft capsule by adopting HPLC-UV method - Google Patents
Method for detecting gypenoside A in gelan Xinning soft capsule by adopting HPLC-UV method Download PDFInfo
- Publication number
- CN110596266B CN110596266B CN201910847188.6A CN201910847188A CN110596266B CN 110596266 B CN110596266 B CN 110596266B CN 201910847188 A CN201910847188 A CN 201910847188A CN 110596266 B CN110596266 B CN 110596266B
- Authority
- CN
- China
- Prior art keywords
- gypenoside
- solution
- methanol
- soft capsule
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/26—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
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The invention relates to a method for detecting gypenoside A in a gelan Xinning soft capsule by adopting an HPLC-UV method46H74O17) Calculated, the content of the active ingredient should not be less than 0.5 mg. The detection method has good specificity, instrument precision, linearity, precision, accuracy and durability, and the method has simple, convenient and accurate processing and detection processes, and is suitable for detection application in the production of the gelan Xinning soft capsule.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicines, and particularly relates to a method for detecting gypenoside A in a gelan Xinning soft capsule by adopting an HPLC-UV method.
Background
The gelan Xinning soft capsule is a traditional Chinese medicine variety produced by the company Limited in the Xian Qian standing grain pharmaceutical industry of the applicant, and clinical application of hospitals all over the country shows that the gelan Xinning soft capsule has obvious clinical effect on treating coronary heart disease and angina caused by blood stasis and obstruction. Preparing the gelan Xinning soft capsule: 200g of pueraria flavonid, 60g of hawthorn extract, 20g of gypenosides, 254g of salad oil, 6g of beeswax, 20g of hydrogenated palm oil, 10g of soybean phospholipid and 0.5g of methyl silicone oil, and the mixture is prepared into 1000 granules; the preparation method comprises the following steps: adding Cera flava and hydrogenated palm oil into salad oil, heating to dissolve, cooling, adding soybean phospholipid, stirring, adding radix Puerariae total flavone, fructus crataegi extract and herba Gynostemmatis total glycoside, stirring, colloid grinding, adding methyl silicone oil, removing bubbles under reduced pressure, and making into soft capsule.
The variety uses puerarin as a reference to perform TLC identification and content determination of the total flavonoid components of the kudzuvine root, and uses gypenoside A as a reference to perform identification of the gypenoside components. In the quality standard of the gypenoside, gypenoside A is used as a reference, and the content of the gypenoside A in a sample is measured by adopting a visible spectrophotometry.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a method for detecting gypenoside A in a gelan Xinning soft capsule by adopting an HPLC-UV method, and solves the problem that the content of an effective medicinal component, namely gypenoside component, is not determined in the original quality standard of the gelan Xinning soft capsule.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting gypenoside A in a Kulan Xinning soft capsule by adopting an HPLC-UV method comprises the following steps of directly pretreating the Kulan Xinning soft capsule, and measuring by adopting a high performance liquid chromatograph to obtain the content of gypenoside A in each Kulan Xinning soft capsule, wherein the content of gypenoside A is not less than 0.5mg, and the specific steps are as follows:
(1) chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the volume ratio of (C) to (B) is 60-80: 20-40 of 0.1% phosphoric acid-acetonitrile as a mobile phase; an ultraviolet detector: the detection wavelength is 203 nm; the flow rate is 1 ml/min; the column temperature is 25-35 ℃; calculated according to the peak of the gypenoside A, the content of the gypenoside A is not lower than 5000;
(2) preparation of test solution
Taking 8-12 Kulanxinning soft capsules, adding 80-120 ml of 10% methanol, weighing, refluxing in a water bath, cooling, weighing again, supplementing the lost weight with 10% methanol, shaking up, and centrifuging; centrifuging, taking supernatant, removing an oil layer, weighing 20-30 ml of a water layer, placing the water layer in a separating funnel, extracting for 1-2 times by shaking 20-30 ml of methyl tert-butyl ether, removing ether liquid, extracting water liquid for 3-5 times by shaking water saturated n-butyl alcohol, combining the n-butyl alcohol liquid, washing with 20-30 ml of an ammonia test solution, removing the ammonia liquid, washing with 2-3 times by using water saturated n-butyl alcohol, removing the water liquid by 20-30 ml each time, evaporating the n-butyl alcohol liquid to dryness, adding a proper amount of methanol into residues for dissolving, transferring to a 10ml measuring flask, adding methanol to a scale, shaking uniformly, and filtering to obtain a sample solution;
(3) preparation of standard solution
Precisely weighing a certain amount of gypenoside A reference substance, and preparing into solution containing 0.2mg per 1ml with methanol to obtain standard solution;
(4) and (3) sample analysis: respectively sucking 5-20 mul of reference solution and sample solution, and injecting into a liquid chromatograph for determination;
(5) and (3) measuring results: the content of gypengning total glycosides in each soft capsule is not less than 0.5mg (calculated as gypenoside A).
It is preferable that: a method for detecting gypenoside A in a gelan Xinning soft capsule by adopting an HPLC-UV method comprises the following steps:
(1) chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the volume ratio is 65-75: 25-35 of 0.1% phosphoric acid-acetonitrile as a mobile phase; an ultraviolet detector: the detection wavelength is 203 nm; the flow rate is 1 ml/min; the column temperature is 25-35 ℃; calculated according to the peak of the gypenoside A, the content of the gypenoside A is not lower than 5000;
(2) preparation of test solution
Taking 10 Kulanxinning soft capsules, placing in a conical flask with a plug, adding 100ml of 10% methanol, weighing, refluxing in water bath, cooling, weighing again, supplementing the weight loss with 10% methanol, shaking up, and centrifuging; centrifuging, collecting supernatant, removing oil layer, weighing 25ml of water layer, placing in a separating funnel, extracting with 25ml of methyl tert-butyl ether under shaking for 1 time, removing ether solution, extracting water solution with water saturated n-butanol under shaking for 4 times, each time 25ml, mixing n-butanol solutions, washing with 25ml of ammonia test solution, removing ammonia solution, washing with n-butanol saturated water for 2 times, each time 25ml, removing water solution, evaporating n-butanol solution to dryness, dissolving residue with appropriate amount of methanol, transferring to 10ml measuring flask, adding methanol to scale, shaking, and filtering to obtain sample solution;
(3) preparation of standard solution
Precisely weighing a certain amount of gypenoside A reference substance, and preparing into solution containing 0.2mg per 1ml with methanol to obtain standard solution;
(4) sample analysis
Respectively sucking 20 μ l of reference solution and sample solution, and measuring in liquid chromatograph;
(5) measurement results
The content of gypengning total glycosides in each soft capsule is not less than 0.5mg (calculated as gypenoside A).
It is preferable that: and the water bath reflux time in the step 2 is 1-3 hours.
It is preferable that: the analysis conditions of the liquid chromatograph are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the ratio of 0.1 percent phosphoric acid to acetonitrile is 70:30 is a mobile phase; an ultraviolet detector: the detection wavelength is 203 nm; the flow rate is 1 ml/min; the column temperature is 30 ℃; calculated according to the peak of the gypenoside A, the content of the gypenoside A is not lower than 5000.
Compared with the prior art, the invention has the following beneficial technical effects:
1. according to the detection method for determining the gypenoside component in the gelan Xinning soft capsule by adopting the HPLC-UV method, the content in the gelan Xinning soft capsule does not need to be taken out in advance in the treatment process of a test sample, the complete soft capsule granules can be directly subjected to heating reflux treatment, the operation is more convenient, in addition, a common fat-soluble extraction solvent diethyl ether in the extraction and separation process is replaced by methyl tert-butyl ether with higher safety and more convenient management, and the defects of low diethyl ether boiling point and inconvenience in use due to the fact that the diethyl ether belongs to a control reagent are overcome.
2. The detection method disclosed by the invention is verified by the method, the specificity, the instrument precision, the linearity, the precision, the accuracy and the durability are good, the processing and detection processes of the method are simple, convenient and accurate, and the method is suitable for detection application in the production of the gelan Xinning soft capsule.
3. The detection method provided by the invention is based on the existing detection method of the gelan Xinning soft capsule, the detection of the active pharmaceutical ingredient, namely the gypenoside ingredient in the gelan Xinning soft capsule is added, the defects of the prior art are overcome, the quality of the gelan Xinning soft capsule is more comprehensively and effectively controlled, and more powerful guarantee is provided for the safety and effectiveness of the gelan Xinning soft capsule used by patients.
Drawings
FIG. 1 is a high performance liquid chromatogram of a gypenoside A reference solution;
FIG. 2 is a high performance liquid chromatogram of a test sample solution;
FIG. 3 is a high performance liquid chromatogram of a negative sample solution.
Detailed Description
In order that the above objects, features and advantages of the present invention can be more clearly understood, a detailed description of the present invention will be given below with reference to the accompanying drawings and specific embodiments. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
In the following description, numerous specific details are set forth to provide a thorough understanding of the present invention, and the described embodiments are merely a subset of the embodiments of the present invention, rather than a complete embodiment. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: a detection method for determining gypenoside components in a gelan Xinning soft capsule by adopting an HPLC-UV method comprises the following steps:
the method comprises the following steps: preparation of gelan Xinning soft capsule and negative control capsule
1. Preparation of gelan Xinning soft capsule
The prescription of the gelan Xinning soft capsule comprises: 200g of pueraria flavonid, 60g of hawthorn extract, 20g of gypenosides, 254g of salad oil, 6g of beeswax, 20g of hydrogenated palm oil, 10g of soybean phospholipid and 0.5g of methyl silicone oil, and the prepared 1000 granules are prepared.
The preparation method of the gelan Xinning soft capsule comprises the following steps: adding Cera flava and hydrogenated palm oil into salad oil, heating to dissolve, cooling, adding soybean phospholipid, stirring, adding radix Puerariae total flavone, fructus crataegi extract and herba Gynostemmatis total glycoside, stirring, colloid grinding, adding methyl silicone oil, removing bubbles under reduced pressure, and making into soft capsule.
2. Preparation of negative control capsules
Removing gypenoside according to the prescription: 200g of pueraria flavonid, 60g of hawthorn extract, 254g of salad oil, 6g of beeswax, 20g of hydrogenated palm oil, 10g of soybean lecithin and 0.5g of methyl silicone oil, and the prepared 1000 granules are prepared.
The preparation method comprises the following steps: adding Cera flava and hydrogenated palm oil into salad oil, heating to dissolve, cooling, adding soybean phospholipid, stirring, adding radix Puerariae total flavone and fructus crataegi extract, stirring, colloid grinding, adding methyl silicone oil, removing bubbles under reduced pressure, and making into soft capsule to obtain negative control capsule. The negative control sample solution can judge whether an interfering substance which influences the actual detection of the detected component exists in the detection process.
Step two: selection of chromatographic conditions
Table 1: selection of mobile phase
| Numbering | Mobile phase |
| 1 | Acetonitrile and water 35:65 |
| 2 | Acetonitrile-0.5% phosphoric acid solution 38: 72 |
| 3 | Methanol-0.4% aqueous phosphoric acid 50:50 |
| 4 | 0.1% phosphoric acid-acetonitrile 70:30 |
The results of comparing four different mobile phases show that: acetonitrile to water in a volume ratio of 35:65 has poor phase separation effect as a mobile phase; the tailing phenomenon occurs in the acetonitrile-0.5 percent phosphoric acid solution with the volume ratio of 38: 72; the peak is too early and the peak shape is very poor by using a methanol-0.4 percent phosphoric acid aqueous solution with the volume ratio of 50: 50; and the peak shape of the mobile phase of 0.1 percent phosphoric acid-acetonitrile with the volume ratio of 70:30 is better, the separation degree is higher, and the required peak-out time is proper, so that the mobile phase of 0.1 percent phosphoric acid-acetonitrile can be used as a determination method of the content of the gypenoside components, the detection is more convenient, and the retention time is also proper.
Step three: establishment of methodology for detecting gypenoside component in gelan Xinning soft capsule by HPLC-UV method
1. HPLC-UV chromatography conditions:
a chromatographic column: 4.6X 250mm, 5 μm; octadecylsilane chemically bonded silica is used as a filling agent;
the volume ratio of mobile phase composition is as follows: 0.1% phosphoric acid: acetonitrile 70:30, of a nitrogen-containing gas; detection wavelength: 203 nm;
flow rate: 1 ml/min; column temperature: 30 ℃; sample introduction amount: 20 mu l of the mixture;
the number of theoretical plates: calculated according to the peak of the gypenoside A, the content of the gypenoside A is not lower than 5000.
2. Solution preparation and determination:
a: preparation of control solutions: taking a proper amount of gypenoside A reference substance, precisely weighing, and adding methanol to obtain a solution containing 0.2mg per 1 ml.
B: preparation of a test solution: taking 10 Kuran Xinning soft capsules prepared in the step one, placing the capsules in conical flasks with stoppers, precisely adding 100ml of 10% methanol, weighing the weight, refluxing in water bath for 1 hour, cooling, weighing the weight again, supplementing the loss weight with 10% methanol, shaking up, centrifuging, taking supernatant, discarding oil layer, precisely measuring 25ml of water layer, placing the capsules in a separating funnel, shaking up and extracting for 1 time with 25ml of methyl tert-butyl ether, discarding ether solution, shaking up and extracting the water solution with water-saturated n-butyl alcohol for 4 times, 25ml each time, combining n-butyl alcohol solutions, washing with 25ml of ammonia test solution, discarding ammonia solution, washing with 2 times of n-butyl alcohol-saturated water, 25ml each time, discarding water solution, evaporating the n-butyl alcohol solution to dryness, adding a proper amount of methanol to dissolve residues, transferring to 10ml measuring flasks, adding methanol to the scale, shaking up and filtering to obtain a test solution.
Preparation of negative control sample solution: taking 10 negative control capsules prepared in the step one, placing the negative control capsules into a conical flask with a plug, precisely adding 100ml of 10% methanol, weighing the weight, refluxing in a water bath for 1 hour, cooling, weighing again, supplementing the weight loss with 10% methanol, shaking uniformly, centrifuging, taking supernatant, discarding an oil layer, precisely measuring 25ml of a water layer, placing the water layer into a separating funnel, shaking and extracting for 1 time by using 25ml of methyl tert-butyl ether, discarding ether liquid, shaking and extracting water liquid for 4 times by using water-saturated n-butyl alcohol, 25ml each time, combining n-butyl alcohol liquid, washing with 25ml of ammonia test solution, discarding ammonia liquid, washing n-butyl alcohol for 2 times by using saturated water, 25ml each time, discarding water liquid, evaporating the n-butyl alcohol liquid to dryness, adding methanol into residues for dissolving, transferring to a 10ml measuring flask, adding methanol to a scale, shaking uniformly, and filtering to obtain a negative control sample solution.
Step three: determination of results
1. Specificity test: precisely sucking 20 μ l of each of the reference solution, the test solution and the negative reference sample solution, and respectively injecting into a liquid chromatograph for determination. Chromatograms were recorded (see FIGS. 1, 2, 3).
The interference is at the corresponding position of the chromatographic peak of the gypenoside A reference substance, the same chromatographic peak is shown in the chromatogram of the test solution, the negative reference sample solution does not have an obvious corresponding chromatographic peak, and the negative is not interfered.
2. Precision test of instrument
The gypenoside A reference solution (0.19835mg/ml) was sampled 6 times for measurement, the results are shown in Table 2, the calculated peak area RSD value was 0.1%, indicating that the instrument precision was good.
Table 2: results of instrumental precision test
3. Investigation of linear relationships
The formulation of each linear solution is shown in Table 4. Taking linear solutions with different concentration levels, respectively sucking 20 mul of the linear solutions, injecting the linear solutions into a liquid chromatograph, measuring, performing linear regression analysis on the concentration (x) by using a peak area (y), and drawing a linear relation graph, wherein a linear regression equation is as follows: y 6370915.6x +3635.0 and R0.9999. The result shows that the gypenoside A has a good linear relation with the peak area within the concentration range of 0.00992-0.38769 mg/ml. The results of the linearity test are shown in Table 3.
TABLE 3 Linear test results of gypenoside A
TABLE 4 Linear solution formulation method
4. Precision test
A. Repeatability test
6 parts of the test solution were prepared in parallel according to the above test solution preparation method, and 20. mu.l of each solution was precisely pipetted and injected into a liquid chromatograph, and the results of the measurements are shown in Table 5. The result shows that the average content of the results measured by 6 test sample solutions is 0.6106 mg/capsule, the specification of the product is 0.58 g/capsule, and the content level of gypenoside A is 0.11%. According to the guiding principle of the ChP 'analysis method for the quality standards of drugs' 2015 edition (general rule 9101), the content of the component to be detected in the sample is 0.1%, and the acceptable RSD value of repeatability is 3%; the RSD value of the method is 2.1 percent, which accords with the regulation and shows that the method has better repeatability.
TABLE 5 results of the repeatability tests
5. Intermediate precision test
In the same laboratory, different experimenters used different chromatographs and different chromatographic columns to perform repeated experiments on different dates, and the results are shown in table 6. The RSD of the gypenoside A content measured by 6 test solutions is 2.0%.
TABLE 6 intermediate precision test results
The measurement results of 12 parts of solution with repeatability and intermediate precision are counted, and the result shows that the RSD value of the detection result of 12 parts of test solution is 2.6 percent, which indicates that the precision of the method is good.
6. Accuracy survey
Stock solutions of the samples of gypenoside A were prepared according to the methods shown in Table 7.
TABLE 7 accuracy loading control stock solution configuration information
Precisely measuring 5ml of a gypenoside A sample-adding reference substance stock solution, placing the sample-adding reference substance stock solution into a conical flask with a plug, volatilizing the solvent, and cooling, wherein 3 parts of sample-adding level are parallelly operated, and 9 parts of sample-adding level are calculated in total. Taking 5 capsules of gelan Xinning soft capsule prepared in example 1 with known content, adding into each conical flask with a plug, adding 100ml of 10% methanol precisely, preparing the sample solution according to the above sample solution preparation method, sucking 20 μ l precisely, injecting into high performance liquid chromatograph, measuring, calculating content by external standard method, and calculating recovery rate.
Recovery (%). percent (total amount measured-content in sample)/control addition X100%
The results are shown in Table 8. The recovery rate of the 9 samples of the gypenoside A is between 96.5 and 104.8 percent, and the RSD value is 2.7 percent. According to the guidelines of the ChP drug quality standard analysis methods of 2015 edition (general rule 9101), the content of the component to be detected in the sample is 0.1%, the acceptable range of the recovery rate is 90% -108%, and the test result shows that the method has better accuracy.
TABLE 8 recovery test results
7. Durability examination
A. Stability of solution
The test solution 2 was left at room temperature for 0, 21, 22, 23, and 24 hours under the repeatability test items, and 20. mu.l of the solution was precisely aspirated and injected into a HPLC, and the peak area was measured, the results are shown in Table 9.
TABLE 9 stability test results of solutions
The result shows that the RSD value of the peak area of the test solution within 24 hours is less than 2.0 percent, which indicates that the test solution is stable within 24 hours under the room temperature condition.
B. Durability of phosphoric acid concentration in mobile phase
The concentrations of phosphoric acid in the mobile phase were adjusted to 0.09%, 0.10%, and 0.11%, and 20. mu.l of the same reference solution and sample solution were injected, and the peak area measurement results are shown in Table 10.
TABLE 10 phosphoric acid concentration durability test results
The results show that the peak areas and the contents of the gynostemma pentaphylla soap a in the reference solution and the test solution are stable, which indicates that the phosphoric acid concentration in the mobile phase is in the range of 0.09-0.11%, and the durability is good.
8. Durability of column temperature
The column temperature of the chromatographic column was adjusted to 25 deg.C, 30 deg.C and 35 deg.C, and 20. mu.l of the same reference solution and sample solution were injected, respectively, and the peak area measurement results are shown in Table 11.
TABLE 11 column temperature durability test results
The results show that the peak areas and the contents of the gypenoside A of the reference solution and the test solution are stable, which indicates that the column temperature is in the range of 25-35 ℃ and the durability is good.
9. Determination of content
Preparing 3 batches of gelan Xinning soft capsule samples (batch numbers: 20160831, 20160832 and 20160833) according to the prescription and the preparation method in the step one, measuring according to the optimal measuring method, and calculating the content results to be shown in table 12, wherein the results show that the content of gypenoside A in the 3 batches of gelan Xinning soft capsules is about 0.6 mg.
TABLE 123 determination results of gypenoside A in Kulan Xinning Soft Capsule
10. Content limitation formulation
Taking 0.2g of gypenosides, accurately weighing, placing in a 50ml measuring flask, adding appropriate amount of methanol, ultrasonic dissolving, diluting to scale, shaking, filtering with 0.45 μm filter membrane, measuring the filtrate by sampling with the above method, and calculating by external standard method to obtain the result shown in Table 13.
TABLE 133 determination of gypenoside A content
The result shows that the gypenoside A content in 3 batches of gypenoside is 3.7Between% and 5.7%. The prescription of the gelan Xinning soft capsules shows that each soft capsule contains 20mg of gypenoside A, and theoretically, each soft capsule contains 0.74-1.14 mg of gypenoside A according to the content of gypenoside A in 3 batches of gypenoside A. Considering the difference of raw materials, combining the detection results of 3 batches of samples, formulating gypengnin-containing gypenoside A (C) for each granule of the gelan Xinning soft capsule46H74O17) Calculated, the content of the active ingredient should not be less than 0.5 mg.
In conclusion, the detection method for determining the gypenoside component in the gelan Xinning soft capsule by adopting the HPLC-UV method has the advantages of strong specificity, good precision, high accuracy and good durability, can accurately and effectively determine the content of the gypenoside component, namely gypenoside A, in the gelan Xinning soft capsule, makes up the defects of the prior art, improves the quality control of the gelan Xinning soft capsule, and provides a powerful guarantee for the safety and the effectiveness of the gelan Xinning soft capsule.
Example 2: a method for detecting gypenoside A in a gelan Xinning soft capsule by adopting an HPLC-UV method is characterized by comprising the following steps: pretreating the Kulan Xinning soft capsule, and directly determining with high performance liquid chromatography (HPLC-UV) to obtain each Kulan Xinning soft capsule containing gypenoside A (C)46H74O17) In terms of the content of the active ingredients, the content of the active ingredients is not less than 0.5mg, and the specific steps are as follows:
(1) preparation of a test solution: taking 8 Kulanxinning soft capsules, adding 90ml of 10% methanol, weighing, refluxing in water bath for 1 hour, cooling, weighing again, supplementing the lost weight with 10% methanol, shaking up, and centrifuging;
centrifuging, collecting supernatant, removing oil layer, weighing 23ml of water layer, placing in a separating funnel, extracting with 23ml of methyl tert-butyl ether under shaking for 1 time, removing ether solution, extracting water solution with water saturated n-butanol under shaking for 3 times, each time 23ml, mixing n-butanol solutions, washing with 23ml of ammonia test solution, removing ammonia solution, washing with n-butanol saturated water for 2 times, each time 23ml, removing water solution, evaporating n-butanol solution to dryness, dissolving residue with appropriate amount of methanol, transferring to 10ml measuring flask, adding methanol to scale, shaking, and filtering to obtain sample solution;
(2) preparing a standard solution;
(3) and (3) sample analysis: respectively sucking 5 μ l of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for determination;
the analysis conditions of the liquid chromatograph are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the ratio of 0.1 percent phosphoric acid to acetonitrile is 60: 20 is a mobile phase; an ultraviolet detector: the detection wavelength is 180 nm; the flow rate is 1 ml/min; the column temperature was 25 ℃; calculated according to the peak of the gypenoside A, the content of the gypenoside A is not lower than 5000;
(4) and (3) measuring results: each soft capsule contains gypenoside A (C) and gypenoside A46H74O17) Calculated, the content of the active ingredient should not be less than 0.5 mg.
Example 3: a method for detecting gypenoside A in a gelan Xinning soft capsule by adopting an HPLC-UV method is characterized by comprising the following steps: pretreating the Kulan Xinning soft capsule, and directly determining with high performance liquid chromatography (HPLC-UV) to obtain each Kulan Xinning soft capsule containing gypenoside A (C)46H74O17) In terms of the content of the active ingredients, the content of the active ingredients is not less than 0.5mg, and the specific steps are as follows:
(1) preparation of a test solution: taking 11 Kulanxinning soft capsules, adding 110ml of 10% methanol, weighing, refluxing in water bath, cooling, weighing again, supplementing lost weight with 10% methanol, shaking up, and centrifuging;
centrifuging, taking supernatant, removing an oil layer, weighing 27ml of a water layer, placing the water layer in a separating funnel, extracting for 2 times by shaking 27ml of methyl tert-butyl ether, removing ether liquid, extracting water liquid for 5 times by shaking water-saturated n-butyl alcohol, 27ml each time, combining the n-butyl alcohol liquid, washing with 23-27 ml of an ammonia test solution, removing the ammonia solution, washing for 3 times by using n-butyl alcohol-saturated water, 27ml each time, removing the water liquid, evaporating the n-butyl alcohol solution to dryness, adding a proper amount of methanol into residues for dissolving, transferring to a 10ml measuring flask, adding methanol to a scale, shaking uniformly, and filtering to obtain a test solution;
(2) preparing a standard solution;
(3) and (3) sample analysis: respectively sucking 20 μ l of reference solution and sample solution, and measuring in liquid chromatograph;
the analysis conditions of the liquid chromatograph are as follows: octadecylsilane chemically bonded silica is used as a filling agent; the ratio of 0.1 percent phosphoric acid to acetonitrile is 80: 40 is a mobile phase; an ultraviolet detector: the detection wavelength is 220 nm; the flow rate is 1 ml/min; the column temperature was 35 ℃; calculated according to the peak of the gypenoside A, the content of the gypenoside A is not lower than 5000;
(4) and (3) measuring results: each soft capsule contains gypenoside A (C) and gypenoside A46H74O17) Calculated, the content of the active ingredient should not be less than 0.5 mg.
Example 4: experiments on the selection of the fat-soluble component from the impurity-removed solvent
Taking 10 Kulanxinning soft capsules prepared in the step one of the example 1, placing the capsules in conical flasks with stoppers, precisely adding 100ml of 10% methanol, weighing the weight, refluxing in a water bath for 1 hour, cooling, weighing the weight again, supplementing the loss weight with 10% methanol, shaking up, centrifuging, taking supernatant, discarding oil layer, precisely measuring 25ml of water layer, placing the mixture in a separating funnel, operating the two parts in parallel, shaking and extracting one part with 25ml of diethyl ether for 1 time, shaking and extracting the other part with 25ml of methyl tert-butyl ether for 1 time, discarding ether liquid, shaking and extracting water liquid with water-saturated n-butanol for 4 times, 25ml each time, combining n-butanol liquid, washing with 25ml of ammonia test solution, discarding ammonia liquid, washing with n-butanol-saturated water for 2 times, 25ml each time, discarding water liquid, evaporating to dryness, dissolving residues with a proper amount of methanol, transferring to 10ml measuring bottles, adding methanol to the scales, shaking up, filtration, HPLC injection, chromatogram recording, and results are shown in Table 14. The results show that the use of diethyl ether and methyl tert-butyl ether as fat-soluble impurity removal solvents hardly affects the content measurement results, so that methyl tert-butyl ether with higher safety and more convenient management is selected as the fat-soluble impurity removal solvent.
TABLE 14 selection results of fat-soluble component impurity-removing solvent
Example 5: experiments on the selection of the number of n-butanol extractions
Taking 15 gelan Xinning soft capsules prepared in the step one of the example 1, placing the gelan Xinning soft capsules into a conical flask with a plug, precisely adding 100ml of 10% methanol, weighing the weight, refluxing in a water bath for 1 hour, cooling, weighing the weight again, supplementing the loss weight with 10% methanol, shaking uniformly, centrifuging, taking supernatant, discarding an oil layer, precisely weighing 25ml of a water layer, placing the water layer into a separating funnel, performing three parallel operations, performing shaking extraction with 25ml of methyl tert-butyl ether respectively, discarding ether liquid, performing shaking extraction with water-saturated n-butyl alcohol respectively for 3, 4 and 5 times, performing shaking extraction with 25ml of water-saturated n-butyl alcohol respectively, combining n-butyl alcohol liquid, washing with 25ml of ammonia test solution, discarding ammonia liquid, washing with 2 times of n-butyl alcohol-saturated water, performing 25ml each time, discarding water liquid, performing evaporation to dryness on the n-butyl alcohol liquid, adding methanol to dissolve residues appropriately, transferring to a 10ml measuring flask, adding methanol to scale, shaking uniformly, performing sample injection, recording a chromatogram, the extraction rate was calculated for each time with the peak area extracted 5 times as 100%, and the results are shown in Table 15. The result shows that the extraction amount of the gypenoside A is not greatly different when the n-butanol is extracted for 4 times and 5 times, so the extraction times are selected for 4 times.
TABLE 15 selection of n-butanol extraction times
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The embodiments given above are preferable examples for implementing the present invention, and the present invention is not limited to the above-described embodiments. Any non-essential addition and replacement made by the technical characteristics of the technical scheme of the invention by a person skilled in the art belong to the protection scope of the invention.
Claims (2)
1. A method for detecting gypenoside A in a gelan Xinning soft capsule by adopting an HPLC-UV method is characterized by comprising the following steps: directly pretreating the Kulan Xinning soft capsule, and determining by high performance liquid chromatography to obtain the Kulan Xinning soft capsule containing gypenoside A not less than 0.5mg, wherein the Kulan Xinning soft capsule contains gypenoside A, and the method comprises the following steps:
(1) chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; in a ratio of 0.1% phosphoric acid to acetonitrile of 70:30 is a mobile phase; an ultraviolet detector: the detection wavelength is 203 nm; the flow rate is 1 ml/min; the column temperature is 30 ℃; calculated according to the peak of the gypenoside A, the content of the gypenoside A is not lower than 5000;
(2) preparation of test solution
Taking 8-12 Kulanxinning soft capsules, adding 80-120 ml of 10% methanol, weighing, refluxing in a water bath for 1-3 hours, cooling, weighing again, supplementing the weight loss with 10% methanol, shaking up, and centrifuging; centrifuging, taking supernatant, removing an oil layer, weighing 20-30 ml of a water layer, placing the water layer in a separating funnel, extracting for 1-2 times by shaking 20-30 ml of methyl tert-butyl ether, removing ether liquid, extracting water liquid for 3-5 times by shaking water saturated n-butyl alcohol, combining the n-butyl alcohol liquid, washing with 20-30 ml of an ammonia test solution, removing the ammonia liquid, washing with 2-3 times by using water saturated n-butyl alcohol, removing the water liquid by 20-30 ml each time, evaporating the n-butyl alcohol liquid to dryness, adding a proper amount of methanol into residues for dissolving, transferring to a 10ml measuring flask, adding methanol to a scale, shaking uniformly, and filtering to obtain a sample solution;
(3) preparation of standard solution
Precisely weighing a certain amount of gypenoside A reference substance, and preparing into solution containing 0.2mg per 1ml with methanol to obtain standard solution;
(4) and (3) sample analysis: respectively sucking 5-20 mul of reference solution and sample solution, and injecting into a liquid chromatograph for determination;
(5) and (3) measuring results: the content of gypengning total glycosides in each soft capsule is not less than 0.5mg (calculated as gypenoside A).
2. The method for detecting gypenoside A in the gelan Xinning soft capsule by adopting the HPLC-UV method as claimed in claim 1, which is characterized in that: the method comprises the following steps:
(1) chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; in a ratio of 0.1% phosphoric acid to acetonitrile of 70:30 is a mobile phase; an ultraviolet detector: the detection wavelength is 203 nm; the flow rate is 1 ml/min; the column temperature is 30 ℃; calculated according to the peak of the gypenoside A, the content of the gypenoside A is not lower than 5000;
(2) preparation of test solution
Taking 10 Kulanxinning soft capsules, placing in a conical flask with a plug, adding 100ml of 10% methanol, weighing, refluxing in water bath for 1 hour, cooling, weighing again, supplementing the weight loss with 10% methanol, shaking up, and centrifuging; centrifuging, collecting supernatant, removing oil layer, weighing 25ml of water layer, placing in a separating funnel, extracting with 25ml of methyl tert-butyl ether under shaking for 1 time, removing ether solution, extracting water solution with water saturated n-butanol under shaking for 4 times, each time 25ml, mixing n-butanol solutions, washing with 25ml of ammonia test solution, removing ammonia solution, washing with n-butanol saturated water for 2 times, each time 25ml, removing water solution, evaporating n-butanol solution to dryness, dissolving residue with appropriate amount of methanol, transferring to 10ml measuring flask, adding methanol to scale, shaking, and filtering to obtain sample solution;
(3) preparation of standard solution
Precisely weighing a certain amount of gypenoside A reference substance, and preparing into solution containing 0.2mg per 1ml with methanol to obtain standard solution;
(4) sample analysis
Respectively sucking 20 μ l of reference solution and sample solution, and measuring in liquid chromatograph;
(5) measurement results
The content of gypengning total glycosides in each soft capsule is not less than 0.5mg (calculated as gypenoside A).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910847188.6A CN110596266B (en) | 2019-09-09 | 2019-09-09 | Method for detecting gypenoside A in gelan Xinning soft capsule by adopting HPLC-UV method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910847188.6A CN110596266B (en) | 2019-09-09 | 2019-09-09 | Method for detecting gypenoside A in gelan Xinning soft capsule by adopting HPLC-UV method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110596266A CN110596266A (en) | 2019-12-20 |
| CN110596266B true CN110596266B (en) | 2022-04-19 |
Family
ID=68858240
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910847188.6A Active CN110596266B (en) | 2019-09-09 | 2019-09-09 | Method for detecting gypenoside A in gelan Xinning soft capsule by adopting HPLC-UV method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110596266B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103969351A (en) * | 2013-01-31 | 2014-08-06 | 广州白云山和记黄埔中药有限公司 | Detection method and application of gynostemma pentaphyllum (thunb.) makino saponin A |
| CN104569165A (en) * | 2013-10-16 | 2015-04-29 | 西安千禾药业有限责任公司 | Detection method of pharmaceutical composition Gelan Xinning for treating coronary heart disease and angina |
| CN107998171A (en) * | 2017-12-11 | 2018-05-08 | 云南梁河民族制药有限公司 | A kind of Herb Gynostemmae Pentaphylli extract and its preparation process and detection method and purposes |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8486464B2 (en) * | 2000-12-22 | 2013-07-16 | Tasly Pharmaceutical Group Co. Ltd. | Herbal composition for angina pectoris, method to prepare same and uses thereof |
-
2019
- 2019-09-09 CN CN201910847188.6A patent/CN110596266B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103969351A (en) * | 2013-01-31 | 2014-08-06 | 广州白云山和记黄埔中药有限公司 | Detection method and application of gynostemma pentaphyllum (thunb.) makino saponin A |
| CN104569165A (en) * | 2013-10-16 | 2015-04-29 | 西安千禾药业有限责任公司 | Detection method of pharmaceutical composition Gelan Xinning for treating coronary heart disease and angina |
| CN107998171A (en) * | 2017-12-11 | 2018-05-08 | 云南梁河民族制药有限公司 | A kind of Herb Gynostemmae Pentaphylli extract and its preparation process and detection method and purposes |
Non-Patent Citations (2)
| Title |
|---|
| HPLC法测定绞股蓝中绞股蓝皂苷A的含量;张转平等;《西北药学杂志》;20080430;第23卷(第2期);第87页第2节 * |
| HPLC法测定绞股蓝总苷胶囊中绞股蓝皂苷B的含量;刘春祥等;《药物分析杂志》;20121231;第32卷(第9期);第1611-1613,1616页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110596266A (en) | 2019-12-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107153098B (en) | Method for measuring fingerprint spectrum of traditional Chinese medicine composition | |
| CN101028388B (en) | Quality inspection of Chinese-medicinal preparation for treating shortsighness and asthenopia | |
| CN106404942B (en) | A kind of construction method and its standard finger-print of kidney-healing particle finger-print | |
| CN101966223A (en) | Fingerprint detection method for compound wintercreeper preparation | |
| CN103808835B (en) | The method of 10 kinds of chemical composition contents in HPLC-DAD method Simultaneously test Siwu Tang decoction | |
| CN112684036A (en) | Fingerprint spectrum determination method of kidney-tonifying capsules containing leeches and application of fingerprint spectrum determination method | |
| CN109709222B (en) | Component detection method of Ganmaoling and compound Ganmaoling | |
| CN110596266B (en) | Method for detecting gypenoside A in gelan Xinning soft capsule by adopting HPLC-UV method | |
| CN113466382B (en) | Content determination method of traditional Chinese medicine preparation for treating cervicodynia | |
| CN113759057B (en) | Characteristic spectrum of allium macrostemon white water extract and preparation thereof and construction method thereof | |
| CN107764924B (en) | Detection method of effective components in asthma granules | |
| CN114636760B (en) | Fingerprint spectrum establishment method of traditional Chinese medicine composition for treating kidney diseases and application of fingerprint spectrum establishment method | |
| CN109142563A (en) | A kind of construction method of guilingji capsules UPLC finger-print and its application | |
| CN105067725B (en) | It is a kind of at the same determine shenmei stomach nourishing granule in the root of herbaceous peony, radix glycyrrhizae and the red sage root content method | |
| CN111398505B (en) | Method for simultaneously detecting contents of five components of traditional Chinese medicine for treating infantile enuresis | |
| CN101028474B (en) | Method for inspecting the quality of Chinese preparation with Yang-and kidney tonifying functions | |
| CN110470751B (en) | Method for simultaneously detecting contents of 7 components in stomach benefiting capsule | |
| CN114646690A (en) | Detection method for chemical components in immature bitter orange, longstamen onion bulb and cassia twig decoction and establishment method of fingerprint spectrum | |
| CN109085259B (en) | Fingerprint detection method of traditional Chinese medicine composition | |
| CN113341007A (en) | Method for measuring contents of multiple components in whole Chinese date seed nerve-soothing capsule based on HPLC (high performance liquid chromatography) characteristic spectrum | |
| CN102008541A (en) | Method for simultaneously detecting three main active ingredients in sugar-free type compound wintercreeper preparation | |
| CN112034085A (en) | HPLC fingerprint detection method for liver-soothing, stomach-harmonizing and pain-relieving traditional Chinese medicine | |
| CN113588808B (en) | Content determination method for simultaneously detecting multiple active ingredients in Xueping capsules | |
| CN109917041A (en) | The measuring method of a variety of active ingredients content in xiaoshuan tongluo tablet | |
| CN110274963B (en) | Fingerprint detection method for diabetes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |