CN111103362A - Detection method of compound folium isatidis composition - Google Patents
Detection method of compound folium isatidis composition Download PDFInfo
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- CN111103362A CN111103362A CN201811256536.4A CN201811256536A CN111103362A CN 111103362 A CN111103362 A CN 111103362A CN 201811256536 A CN201811256536 A CN 201811256536A CN 111103362 A CN111103362 A CN 111103362A
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Classifications
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Abstract
The invention discloses a detection method of a compound folium isatidis composition, which belongs to the technical field of quality detection of traditional Chinese medicine preparations.
Description
Technical Field
The invention relates to the technical field of extract quality detection, and particularly relates to a detection method of a compound folium isatidis composition.
Background
The compound folium isatidis composition has the functions of dispelling wind and clearing heat, detoxifying and reducing swelling, and cooling blood and benefiting gallbladder, and can be used for treating symptoms such as cold, fever and headache, throat red swelling, ear swelling and pain, hypochondriac pain and jaundice and the like, and symptoms such as influenza, parotitis and acute viral hepatitis.
The composition of this class of drugs is currently being studied in the literature. The applicant has already filed a Chinese patent with patent number 200610072997.7 and discloses a detection method of a new compound folium isatidis preparation, which comprises the steps of content identification and content determination of contained components. The content is a compound of Chinese and western medicines, the identification of the content is thin-layer identification of folium Isatidis, radix et rhizoma Rhei, vitamin C, and Notopterygii rhizoma, and the content determination of chlorogenic acid, acetaminophen, caffeine and amobarbital is performed by high performance liquid chromatography. The method takes 10ml of oral liquid, or corresponding crude drug granule, tablet, and capsule to detect multiple Chinese medicinal components one by one, and has the advantages of complicated detection process and long time consumption
The applicant also applies for the invention patent of patent No. 200910230003.3 and discloses a preparation and quality control method of compound folium isatidis preparation, wherein the quality control method comprises content identification and content determination of contained components, the content identification comprises identification of folium isatidis, flos lonicerae or flos lonicerae, radix et rhizoma rhei, notopterygium root by thin layer identification method and content determination of emodin and chrysophanol in radix et rhizoma rhei and content determination of chlorogenic acid by high performance liquid chromatography. This patent is through getting the product content, detects one by one to single product component many times, and the testing process is loaded down with trivial details, the time that consumes is long.
The compound dyers woad leaf composition is a semi-finished product prepared by processing processes of dyers woad leaf, honeysuckle, notopterygium root, purple ginseng and rhubarb through extraction, concentration, drying and the like, the prior art detects the extracted composition of multiple components for multiple times, each detection is only carried out on one component, the detection result is single, incomplete, complex and the like, lawless persons who steal labor, reduce materials, participate in adulteration and cause fakes and the like can take the opportunity, and a simple, efficient and comprehensive detection technology needs to be established to ensure that the effective monitoring of the multiple components can be carried out at one time in the same map. The research aims to establish a detection technology of an HPLC characteristic spectrum of the compound folium isatidis composition so as to achieve the aim of comprehensively controlling the product quality of the compound folium isatidis composition.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a detection method of a compound folium isatidis composition.
The technical scheme aims at the characteristic detection technology of the multiple components of the compound folium isatidis composition, can visually reflect the biological characteristics of the multiple components of the composition, is the characteristic which can not be replaced by single component combination, and has specificity on comprehensively reflecting the related substances of the specific traditional Chinese medicine composition.
In order to achieve the purpose, the invention adopts the following technical scheme:
a detection method of a compound folium isatidis composition is used, the compound folium isatidis composition is detected by adopting a high performance liquid chromatography, and the detection method comprises the following steps:
s1, chromatographic conditions: the chromatographic column is C18A column, wherein methanol is used as a mobile phase A, 0.2% phosphoric acid aqueous solution is used as a mobile phase B, the detection wavelength is 254nm, the flow rate is 0.1ml/min, and the column temperature is 35 ℃;
s2, preparation of reference solution: precisely weighing chlorogenic acid as a control solution, and adding a methanol solution into the chlorogenic acid;
s3, preparation of a test solution: precisely weighing compound folium Isatidis composition powder, adding methanol, ultrasonic extracting, and filtering;
s4, precisely sucking 20ul of reference substance solution and sample solution respectively, and injecting the reference substance solution and the sample solution into a liquid chromatograph for chromatographic analysis.
Preferably, the chromatographic conditions and system suitability test in S1: the chromatographic column is Kromasil 100-5C18The specification of the chromatographic column is as follows: 4.6mm × 250mm, 5 um; the phosphoric acid aqueous solution was subjected to gradient elution by adjusting the pH to 3.0 with a 10% sodium hydroxide solution.
Preferably, the elution conditions of the gradient elution are as follows: the volume fraction of the methanol is 3-35% in 0-30 min, 35-65% in 30-50 min, 65% in 50-60 min, 65-3% in 60-61 min and 3% in 61-65 min.
Preferably, the preparation of the reference solution in S2: precisely weighing appropriate amount of chlorogenic acid reference substance, and adding methanol to obtain 30 μ g/ml solution.
Preferably, the preparation of the test solution in S3: precisely weighing 0.2g of compound folium isatidis composition powder, adding 30% methanol, performing ultrasonic extraction for 30min with the ultrasonic extraction power of 250W, and filtering to obtain the compound folium isatidis composition powder.
The invention has the beneficial effects that:
1. according to the invention, the compound folium isatidis composition is detected by adopting a high performance liquid chromatography method, the process is simple and rapid, the result is accurate, the content of the compound folium isatidis composition can be determined and qualitatively analyzed, the effective control on the quality of the compound folium isatidis composition can be improved, and the use safety of the compound folium isatidis composition is ensured.
2. According to the invention, the accuracy of the detection result can be improved by carrying out comparison detection on the compound folium isatidis composition and the five medicinal materials.
Drawings
FIG. 1 is a standard characteristic spectrum of a detection method of a compound folium isatidis composition provided by the invention;
fig. 2 is a peak detection diagram of 5 batches of compound folium isatidis compositions taken from a production workshop in the detection method of the compound folium isatidis composition provided by the invention;
FIG. 3 is a detection peak diagram of 20ul each of the honeysuckle extract solution, the indigowoad leaf extract solution, the purple ginseng extract solution, the notopterygium root extract solution and the rhubarb extract solution according to the detection method of the compound indigowoad leaf composition provided by the invention;
fig. 4 is a detection peak diagram of a method for detecting a compound folium isatidis composition, which is provided by the invention, by taking a honeysuckle extract, a folium isatidis extract, a radix codonopsis pilosulae extract, a notopterygium root extract and a rhubarb extract respectively.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
The invention is determined according to high performance liquid chromatography (pharmacopoeia appendix VI D, 2010 edition).
The test sample characteristic map should have 15 peaks, the peak corresponding to the reference substance peak is the S peak, the relative retention time and the relative peak area of each characteristic peak and the S peak are calculated, the relative retention time is within + -10% of the specified value, the relative retention time is within 10% of the relative retention time value,
the relative retention time of each of the other characteristic peaks is specified as: 0.21 (peak 1), 0.40 (peak 2), 0.45 (peak 3), 0.60 (peak 4), 0.67 (peak 5), 0.73 (peak 6), 1.00[ peak 7 (S) ], 1.08 (peak 8), 1.30 (peak 9), 1.46 (peak 10), 1.64 (peak 11), 1.74 (peak 12), 1.78 (peak 13), 1.82 (peak 14), 2.19 (peak 15), and the characteristic map is shown in FIG. 1.
The first embodiment is as follows: taking 5 batches of the compound folium isatidis composition from a production workshop for detection, and detecting the compound folium isatidis composition by adopting high performance liquid chromatography (the high performance liquid chromatography adopts Kromasil 100-5C)18Detection on a chromatographic column, and C18The length of the chromatographic column is 250mm, C18Inner diameter of chromatographic column of 4.6mm, C18The diameter of the filler particles of the chromatographic column is 5 um), and the detection method comprises the following steps:
s1, chromatographic conditions: the chromatographic column is C18A column, methanol is used as a mobile phase A, 0.2% phosphoric acid aqueous solution (the pH is adjusted to be 3.0 by 10% sodium hydroxide solution) is used as a mobile phase B, the detection wavelength is 254nm, the flow rate is 0.1ml/min, and the column temperature is 35 ℃;
s2, preparation of reference solution: precisely weighing chlorogenic acid as a control solution, and adding methanol solution into the chlorogenic acid to make the concentration of the chlorogenic acid be 30 ug/ml;
s3, preparation of a test solution: respectively taking flos Lonicerae, folium Isatidis, radix Ginseng Indici, Notopterygii rhizoma and radix et rhizoma Rhei 0.2g each, precisely adding 30% methanol 25ml, ultrasonic extracting for 30min to obtain sample solution of each medicinal material, and injecting 20 μ l each into chromatograph for detection according to the above chromatographic conditions;
s4, precisely sucking 20ul of reference substance solution and sample solution respectively, and injecting the reference substance solution and the sample solution into a liquid chromatograph for chromatographic analysis.
The mobile phase gradient elution table for the chromatographic analysis is shown below:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0-30 | 3-35 | 97-65 |
| 30-50 | 35-65 | 65-35 |
| 50-60 | 65 | 35 |
| 60-61 | 65-3 | 35-97 |
| 61-65 | 3 | 97 |
The detection results are shown in FIG. 2;
from the results of the tests in FIG. 2, it can be seen that the folium Isatidis compositions of lot 5 contained peaks from numbers 1 to 15.
Example two: respectively taking honeysuckle, indigowoad leaf, radix salviae miltiorrhizae, notopterygium root and rhubarb to carry out chromatographic detection, wherein the detection method comprises the following steps:
s1, chromatographic conditions: the chromatographic column is C18Column, methanol as mobile phase A, 0.2% phosphoric acid in water (10% sodium hydroxide solution)Adjusting pH to 3.0) to be a mobile phase B, detecting the wavelength to be 254nm, the flow rate to be 0.1ml/min and the column temperature to be 35 ℃;
s2, preparation of reference solution: precisely weighing chlorogenic acid as a control solution, and adding methanol solution into the chlorogenic acid to make the concentration of the chlorogenic acid be 30 ug/ml;
s3, preparation of a test solution: respectively taking flos Lonicerae, folium Isatidis, radix Ginseng Indici, Notopterygii rhizoma and radix et rhizoma Rhei 0.2g each, precisely adding 30% methanol 25ml, ultrasonic extracting for 30min to obtain sample solution of each medicinal material, and injecting 20 μ l each into chromatograph for detection according to the above chromatographic conditions;
s4, precisely sucking 20ul of reference solution and sample solution respectively, injecting the reference solution and sample solution into a liquid chromatograph for chromatographic analysis,
the mobile phase gradient elution table for the chromatographic analysis is shown below:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0-30 | 3-35 | 97-65 |
| 30-50 | 35-65 | 65-35 |
| 50-60 | 65 | 35 |
| 60-61 | 65-3 | 35-97 |
| 61-65 | 3 | 97 |
The detection results are shown in FIG. 3;
from the detection results in fig. 3, it can be found that among 15 known chromatographic peaks, 8 are from honeysuckle (1, 2, 5, 7-chlorogenic acid, 8, 9, 12, 13), 7 are from dyers woad leaf (1, 2, 4, 6, 10, 11, 13), 3 are from salvia chinensis (1, 3, 4), 4 are from notopterygium root (1, 2, 13, 14), and 3 are from rhubarb (11, 13, 15), and the specific distribution is shown in table 1:
| peak number | Corresponding |
| 1 | Flos Lonicerae, folium Isatidis, radix Salviae Miltiorrhizae, and rhizoma Et radix Notopterygii |
| 2 | Honeysuckle flower, isatis leaf and |
| 3 | Radix Salviae Miltiorrhizae |
| 4 | Folium Isatidis and radix Salviae Miltiorrhizae |
| 5 | Honeysuckle |
| 6 | Folium isatidis |
| 7-chlorogenic acid | Honeysuckle |
| 8 | Honeysuckle |
| 9 | Honeysuckle |
| 10 | Folium isatidis |
| 11 | Folium Isatidis and radix et rhizoma Rhei |
| 12 | Honeysuckle |
| 13 | Honeysuckle flower, isatis leaf, notopterygium root and rhubarb |
| 14 | Notopterygium |
| 15 | Radix et rhizoma Rhei |
Table 1: the distribution of chromatographic peaks corresponding to the five Chinese medicinal materials of honeysuckle, dyers woad leaf, radix adenophorae, notopterygium root and rhubarb
Example three: respectively taking honeysuckle, indigowoad leaf, radix salviae miltiorrhizae, notopterygium root and rhubarb for detection, wherein the detection method comprises the following steps:
s1, chromatographic conditions: the chromatographic column is C18Column, methanol as mobile phase A, 0.2% phosphoric acid water solution (pH adjusted to 3.0 with 10% sodium hydroxide solution) as mobile phase B, detection wavelength of 254nm, flow rate of 0.1ml/min, column temperature of 35 deg.C
S2, preparation of reference solution: precisely weighing chlorogenic acid as a control solution, and adding methanol solution into the chlorogenic acid to make the concentration of the chlorogenic acid be 30 ug/ml;
s3, preparation of a test solution: respectively taking flos Lonicerae, folium Isatidis, radix Ginseng Indici, Notopterygii rhizoma and radix et rhizoma Rhei 0.2g each, precisely adding 30% methanol 25ml, ultrasonic extracting for 30min to obtain sample solution of each medicinal material, and injecting 20 μ l each into chromatograph for detection according to the above chromatographic conditions;
s4, precisely sucking 20ul of reference solution and sample solution respectively, injecting the reference solution and sample solution into a liquid chromatograph for chromatographic analysis,
the mobile phase gradient elution table for the chromatographic analysis is shown below:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0-30 | 3-35 | 97-65 |
| 30-50 | 35-65 | 65-35 |
| 50-60 | 65 | 35 |
| 60-61 | 65-3 | 35-97 |
| 61-65 | 3 | 97 |
The detection results are shown in FIG. 4;
from the detection results in fig. 4, it can be found that the specific distribution of the corresponding herbs in the known 15 chromatographic peaks is shown in table 2 (the numbers of the chromatographic peaks in table 2 and fig. 4 are not consistent with those of the chromatographic peaks in table 1 and fig. 3, and there is no corresponding relationship):
table 2: the distribution of chromatographic peaks corresponding to the medicinal material extracts of the five medicines of honeysuckle, indigowoad leaf, purple ginseng, notopterygium root and rhubarb
As can be seen from fig. 4 and table 2, 7 of the 17 peaks were derived from honeysuckle (2, 6, 7-chlorogenic acid, 8, 10, 11, 13'), 3 were derived from isatis leaf (1, 2, 4), 5 were derived from salvia chinensis (3-gallic acid, 5, 6, 7-chlorogenic acid, 8), 4 were derived from notopterygium root (1, 2, 8, 17), and 9 were derived from rhubarb (3-gallic acid, 9, 10, 11, 12, 13, 14, 15, 16).
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (5)
1. A detection method of a compound folium isatidis composition is characterized in that the compound folium isatidis composition is detected by adopting a high performance liquid chromatography, and the detection method comprises the following steps:
s1, chromatographic conditions: the chromatographic column is C18A column, wherein methanol is used as a mobile phase A, 0.2% phosphoric acid aqueous solution is used as a mobile phase B, the detection wavelength is 254nm, the flow rate is 0.1ml/min, and the column temperature is 35 ℃;
s2, preparation of reference solution: precisely weighing chlorogenic acid as a control solution, and adding a methanol solution into the chlorogenic acid;
s3, preparation of a test solution: precisely weighing compound folium Isatidis composition powder, adding methanol, ultrasonic extracting, and filtering;
s4, precisely sucking 20ul of reference substance solution and sample solution respectively, and injecting the reference substance solution and the sample solution into a liquid chromatograph for chromatographic analysis.
2. The method of claim 1, wherein the chromatographic conditions and system applicability test in S1 is as follows: the chromatographic column is Kromasil 100-5C18The specification of the chromatographic column is as follows: 4.6mm × 250mm, 5 um; the phosphoric acid aqueous solution was subjected to gradient elution by adjusting the pH to 3.0 with a 10% sodium hydroxide solution.
3. The method for detecting a compound folium isatidis composition according to claim 2, wherein the gradient elution conditions are as follows: the volume fraction of the methanol is 3-35% in 0-30 min, 35-65% in 30-50 min, 65% in 50-60 min, 65-3% in 60-61 min and 3% in 61-65 min.
4. The method for detecting a compound folium isatidis composition according to claim 1, wherein the preparation of the reference solution in S2: precisely weighing appropriate amount of chlorogenic acid reference substance, and adding methanol to obtain 30 μ g/ml solution.
5. The method for detecting a compound folium isatidis composition according to claim 1, wherein the preparation of the test solution in S3: precisely weighing 0.2g of compound folium isatidis composition powder, adding 30% methanol, performing ultrasonic extraction for 30min with the ultrasonic extraction power of 250W, and filtering to obtain the compound folium isatidis composition powder.
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