CN109709222B - Component detection method of Ganmaoling and compound Ganmaoling - Google Patents
Component detection method of Ganmaoling and compound Ganmaoling Download PDFInfo
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Abstract
The invention provides a method for detecting components of Ganmaoling and compound Ganmaoling, which is characterized in that the content of chlorogenic acid, luteoloside, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine in Ganmaoling and compound Ganmaoling are measured by adopting a high performance liquid chromatography; the chromatographic conditions are as follows: filling agent: octadecylsilane chemically bonded silica, column temperature: 25-35 ℃, detector: diode array detector, detection wavelength: 272nm, 327nm, 334nm and 350nm, mobile phase: and taking acetonitrile as an A phase and 0.1-0.3% phosphoric acid aqueous solution as a B phase for gradient elution.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a detection method suitable for simultaneously detecting various components in a Chinese and western medicine compound preparation, namely Ganmaoling or compound Ganmaoling.
Background
Ganmaoling and compound Ganmaoling are classic compound preparations of Chinese and Western medicines, have wide clinical application and are commonly used for treating various symptoms caused by cold. The main dosage forms in the market comprise three types, namely granules, capsules and tablets, such as Ganmaoling granules, Ganmaoling capsules, compound Ganmaoling tablets and the like. As a model of the combination of Chinese and western medicines, the Ganmaoling and the compound Ganmaoling are widely accepted in continuous clinical practice. In the two compound preparations, the traditional Chinese medicine and the western medicine (small molecular chemical medicine) have synergistic effect, take effect quickly, and restrict the toxicity or adverse reaction of the western medicine components to a certain extent. However, the chemical components of the compound preparation of Chinese and western medicines become complex, and the analysis and research of multiple components and multiple indexes are crucial to the product quality and scientific medicine.
The cold-treating preparation and the compound cold-treating preparation have the same raw materials, and are acetaminophen, caffeine and chlorphenamine maleate; but the two are different in the aspect of traditional Chinese medicine raw materials: the Chinese medicinal materials of the preparation for treating common cold comprise thin evodia leaf, herba Sidae Rhombifoliae, flos Chrysanthemi Indici and flos Ilicis Asprellae, and the preparation for treating compound common cold comprises flos Lonicerae, mandarin orange, flos Chrysanthemi Indici, thin evodia leaf, rhizoma Et radix Baphicacanthis Cusiae and flos Ilicis Asprellae. In the prior art, a quality control and detection method of Ganmaoling and a compound Ganmaoling preparation takes one or more of western medicine components of acetaminophen, caffeine and chlorphenamine maleate as indexes, for example, the detection of the pharmaceutical components of acetaminophen, chlorphenamine maleate, caffeine and the like in Ganmaoling tablets is reported by Caitao et al (Caitao et al. quality standard research of Ganmaoling tablets [ J ] Chinese patent medicine, 2003, 25 (12): 964 + 967); or only detecting chemical components from traditional Chinese medicines, such as Pengxiapeng, in the quality control research of Ganmaoling granules, detecting index components such as neochlorogenic acid, cryptochlorogenic acid, luteolin, isochlorogenic acid C, linarin and the like (Pengxiapeng. Ganmaoling granule quality control research [ D ]. Guangdong pharmaceutical university, 2016); further, as the invention patent of "a method for detecting fingerprint of Ganmaoling granule" (grant publication No. CN 104181248B, published on 2015, 12 months and 2 days), the invention discloses a method for detecting fingerprint of chemical components of traditional Chinese medicine of Ganmaoling granule based on high performance liquid chromatography.
However, the Ganmaoling and the compound Ganmaoling both generate curative effect by the synergistic action of traditional Chinese medicine and chemical medicine, and the detection of a single component (traditional Chinese medicine or chemical medicine) can not fully reflect the inherent quality of the product. Therefore, for the quality control of the two preparations, the two raw materials of the traditional Chinese medicine and the chemical medicine can be simultaneously considered. In addition, in the prior art, the quality control and component detection methods for the two preparations are only specific to a single dosage form, such as the Ganmaoling tablet and Ganmaoling granule mentioned above, and an analysis and detection method which can be universally used for various dosage forms of Ganmaoling and compound Ganmaoling does not exist.
Disclosure of Invention
The invention aims to provide a component detection method which is universal for different preparations of Ganmaoling and compound Ganmaoling (including but not limited to Ganmaoling granules, Ganmaoling capsules, compound Ganmaoling tablets, compound Ganmaoling granules and the like) aiming at the defects of the prior art. The detection method is based on a high performance liquid chromatography, combines traditional Chinese medicine and chemical raw materials in the preparation, can simultaneously carry out quantitative detection on seven chemical components in the preparation, such as chlorogenic acid, galuteolin, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen, caffeine and the like, improves the detection efficiency, and has short time consumption, thereby reducing the detection cost.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
a method for detecting components of GANMAOLING and FUFANGGANMAOLING; the detection method comprises determining contents of chlorogenic acid, luteoloside, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine in GANMAILING and FUFANGGANLING by high performance liquid chromatography; the chromatographic conditions were as follows:
filling agent: octadecylsilane chemically bonded silica;
column temperature: 25-35 ℃;
a detector: a diode array detector;
detection wavelength: 272nm, 327nm, 334nm and 350 nm;
mobile phase: taking acetonitrile as an A phase and 0.1-0.3% phosphoric acid aqueous solution as a B phase, and performing gradient elution according to the following procedures:
| Time | phase A | Phase B |
| 0→10min | 13％ | Balance of |
| 10→15min | 13％→20％ | Balance of |
| 15→20min | 20％ | Balance of |
| 20→40min | 20％→30％ | Balance of |
| 40→ |
30％→60％ | Balance of |
Flow rate: 0.8-1.2 ml/min.
Preferably, the phase B of the mobile phase is 0.1-0.2% phosphoric acid aqueous solution, and more preferably 0.1% phosphoric acid aqueous solution.
Preferably, the component detection method of the present invention further comprises preparing a reference solution, specifically:
precisely weighing chlorogenic acid, galuteolin, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine, putting into a same volumetric flask, dissolving and diluting with methanol, and mixing into a reference solution with the concentration of 20-200 μ g/ml respectively.
Preferably, the component detection method of the present invention further comprises preparing a test solution, specifically:
pulverizing Ganmaoling or Compound Ganmaoling, ultrasonic extracting with methanol, filtering, and collecting filtrate to obtain test solution.
More preferably, the preparation of the test solution comprises the following specific steps: taking the Ganmaoling or the compound Ganmaoling, removing sugar coating of tablets, taking the content of capsules, putting the capsules into a mortar for grinding, weighing 1-5 g, putting the tablets into a conical bottle with a plug, adding 20-50 ml of methanol, sealing the plug, weighing, carrying out ultrasonic extraction for 30-60 min, cooling, weighing, supplementing the weight loss with the methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the Ganmaoling.
Further preferably, the preparation of the test solution comprises the following specific steps: taking GANMAOLING or FUFANGGANMAOLING, removing sugar coating of tablet, taking capsule, grinding in mortar, precisely weighing 3g, placing in conical flask with plug, precisely adding 25ml methanol, sealing, weighing, ultrasonically extracting for 30min, cooling, weighing, supplementing with methanol, shaking, filtering, and collecting filtrate
Preferably, the component detection method of the present invention specifically comprises: and respectively sucking 10-20 μ l of the test solution and the reference solution, injecting into a liquid chromatograph, measuring, and calculating the content of chlorogenic acid, luteoloside, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine in the test sample by an external standard method.
In the present invention, the "Ganmaoling" refers to a preparation prepared by taking thin evodia leaf, bidens biternata, wild chrysanthemum flower, roughhaired holly root, acetaminophen, caffeine and chlorphenamine maleate as raw materials (can also comprise peppermint oil), adding pharmaceutically acceptable auxiliary materials and preparing by a conventional method in the field, and comprises Ganmaoling tablets, Ganmaoling capsules and Ganmaoling granules. The three preparations are collected in the book of national Chinese patent drug standards compilation, department of internal medicine lung system (I) (State drug administration, 2002) or the book of the thirteenth Chinese patent medicine prescription preparation.
In the present text, the "compound Ganmaoling" refers to a preparation prepared by taking honeysuckle, citrus unshiu, wild chrysanthemum flower, thin evodia leaf, south isatis root, roughhaired holly root, acetaminophen, caffeine and chlorphenamine maleate as raw materials, adding pharmaceutically acceptable auxiliary materials and adopting a conventional method in the field, and the preparation comprises Ganmaoling tablets, Ganmaoling capsules and Ganmaoling granules. The three preparations are collected in the book of national Chinese patent drug standards compilation, department of internal medicine lung system (I) (State drug administration, 2002) or the book of twelfth book of Chinese medicinal preparations.
In the present specification, unless otherwise specified, the percentage concentration refers to the volume percentage concentration, and for example, the B phase of the mobile phase is 0.1 to 0.3% phosphoric acid aqueous solution, that is, the phosphoric acid aqueous solution with the volume percentage concentration of 0.1 to 0.3%.
The detection method provided by the invention is based on a high performance liquid and diode array detector, can quantitatively detect seven components from traditional Chinese medicine and chemical medicine raw materials in the Ganmaoling and the compound Ganmaoling at one time, and can comprehensively reflect the quality of a detected preparation. The methodological investigation proves that the method has high sensitivity, good repeatability and good recovery rate, and can be used as one of the methods for controlling the quality of the Ganmaoling or the compound Ganmaoling.
In addition, tests prove that the detection method provided by the invention is universal and is suitable for detecting the components of various common preparations of the Ganmaoling and the compound Ganmaoling.
Drawings
The invention will be further described with reference to the accompanying drawings, in which:
fig. 1A to 1G are HPLC charts of sample solutions under different mobile phase conditions in example 1 of the present invention, in which the mobile phase of 1A is methanol-water, the mobile phase of 1B is methanol-0.1% acetic acid aqueous solution, the mobile phase of 1C is methanol-0.1% phosphoric acid aqueous solution, the mobile phase of 1D is methanol-0.1% phosphoric acid aqueous solution, the mobile phase of 1E is acetonitrile-water, the mobile phase of 1F is acetonitrile-0.1% acetic acid aqueous solution, and the mobile phase of 1G is acetonitrile-0.2% acetic acid aqueous solution; in the figure: the absorption peak of chlorogenic acid is denoted by 1. The reference numeral 2 is the luteolin absorption peak, the reference numeral 3 is the isochlorogenic acid B absorption peak, the reference numeral 4 is the isochlorogenic acid C absorption peak, the reference numeral 5 is the linarin absorption peak, and the reference numeral 6 is the acetaminophen absorption peak.
Fig. 2A to 2D are HPLC chromatograms of the sample solution under different gradient elution procedures in example 1 of the present invention, wherein 2A is a chromatogram under the gradient elution procedure 1, 2B is a chromatogram under the gradient elution procedure 2, 2C is a chromatogram under the gradient elution procedure 3, and 2D is a chromatogram under the gradient elution procedure 4; the absorption peak of chlorogenic acid is marked with 1 in the figure. The reference numeral 2 is the luteolin absorption peak, the reference numeral 3 is the isochlorogenic acid B absorption peak, the reference numeral 4 is the isochlorogenic acid C absorption peak, the reference numeral 5 is the linarin absorption peak, and the reference numeral 6 is the acetaminophen absorption peak.
FIGS. 3A to 3D are HPLC profiles of the sample solution under different column temperature conditions in example 1 of the present invention, wherein 3A is a chromatogram at a column temperature of 25 ℃, 3B is a chromatogram at a column temperature of 30 ℃, 3C is a chromatogram at a column temperature of 35 ℃, and 3D is a chromatogram at a column temperature of 40 ℃; in the figure, the number 1 is the absorption peak of chlorogenic acid. The reference numeral 2 is the luteolin absorption peak, the reference numeral 3 is the isochlorogenic acid B absorption peak, the reference numeral 4 is the isochlorogenic acid C absorption peak, the reference numeral 5 is the linarin absorption peak, and the reference numeral 6 is the acetaminophen absorption peak.
FIGS. 4A to 4C are HPLC profiles of the sample solution under different flow rate conditions in example 1 of the present invention, wherein 4A is a chromatogram at a flow rate of 0.8ml/min, 4B is a chromatogram at a flow rate of 1.0ml/min, 4C is a chromatogram at a flow rate of 1.2ml/min, and the absorption peak of chlorogenic acid is denoted by 1 in the figures. The reference numeral 2 is the luteolin absorption peak, the reference numeral 3 is the isochlorogenic acid B absorption peak, the reference numeral 4 is the isochlorogenic acid C absorption peak, the reference numeral 5 is the linarin absorption peak, and the reference numeral 6 is the acetaminophen absorption peak.
FIGS. 5A-5D are HPLC integral contrast charts of a mixed control solution and a test solution under the preferred detection method established in example 1 of the present invention, wherein 5A and 5C are chromatograms of the mixed control solution, and 5B and 5D are chromatograms of the test solution; the absorption peak of chlorogenic acid is marked with 1 in the figure. The reference numeral 2 is an absorption peak of luteoloside, the reference numeral 3 is an absorption peak of isochlorogenic acid B, the reference numeral 4 is an absorption peak of isochlorogenic acid C, the reference numeral 5 is an absorption peak of linarin, the reference numeral 6 is an absorption peak of acetaminophen, and the reference numeral 7 is an absorption peak of caffeine.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified. Wherein, some reagents and instruments are purchased as follows:
high performance chromatograph: agilent 1260 high performance liquid chromatograph, Agilent technologies (china);
a chromatographic column: eclipse Plus C18(4.6 mm. times.250 mm, 5 μm) chromatography column, Agilent technologies (China) Inc.;
an ultrasonic processor: KQ-600VDB model double-frequency numerical control ultrasonic cleaner, Kunshan ultrasonic Instrument Co., Ltd
Acetaminophen (batch No.: 100018-201409), caffeine (batch No.: 171215-201509), chlorogenic acid (batch No. 110753-201415), luteolin (batch No. 111720-200905): purchased from China pharmaceutical biologicals institute;
isochlorogenic acid B (batch number MUST-18031602), isochlorogenic acid C (batch number MUST-18031603), linarin (batch number MUST-17071710): purchased from Dowman Stokes Biometrics Ltd.
The information of the Ganmaoling and Compound Ganmaoling samples is shown in Table 1.
TABLE 1 Ganmaoling and Compound Ganmaoling sample information
The invention provides a component detection method of Ganmaoling and compound Ganmaoling, which is based on high performance liquid chromatography and a diode array detector and is used for determining the content of chlorogenic acid, luteoloside, isochlorogenic acid B, isochlorogenic acid C, linalooside, acetaminophen and caffeine in Ganmaoling and compound Ganmaoling; the chromatographic conditions were as follows:
filling agent: octadecylsilane chemically bonded silica;
column temperature: 25-35 ℃;
a detector: a diode array detector;
detection wavelength: 272nm, 327nm, 334nm and 350 nm;
mobile phase: taking acetonitrile as an A phase and 0.1-0.3%, preferably 0.1-0.2%, more preferably 0.1% phosphoric acid aqueous solution as a B phase, and performing gradient elution according to the following procedures:
| Time | phase A | Phase B |
| 0→10min | 13％ | Balance of |
| 10→15min | 13％→20％ | Balance of |
| 15→20min | 20％ | Balance of |
| 20→40min | 20％→30％ | Balance of |
| 40→50min | 30％→60％ | Balance of |
Flow rate: 0.8-1.2 ml/min;
theoretical plate number: calculated according to chlorogenic acid, galuteolin, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine peak, respectively, not less than 3000;
the method comprises the following steps:
preparation of control solutions: precisely weighing chlorogenic acid, galuteolin, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine reference substances respectively, placing into a same volumetric flask, adding methanol to dissolve and dilute to obtain mixed reference substance solutions with concentrations of 43 μ g/ml (chlorogenic acid), 35 μ g/ml (galuteolin), 42 μ g/ml (isochlorogenic acid B), 42 μ g/ml (isochlorogenic acid C), 64 μ g/ml (linarin), 200 μ g/ml (acetaminophen) and 25 μ g/ml (caffeine), storing in a refrigerator at 4 deg.C for later use, and filtering with 0.22 μm microporous membrane before use to obtain the final product;
preparation of a test solution: taking Ganmaoling or Compound Ganmaoling, removing sugar coating of tablet, taking capsule, grinding in mortar, precisely weighing 3g, placing in conical flask with plug, precisely adding 25ml of methanol, sealing, weighing, ultrasonically extracting for 30min, cooling, weighing, adding methanol to compensate lost weight, shaking, filtering, and taking subsequent filtrate;
and (3) determination: respectively sucking 10 μ l of test solution and reference solution, injecting into liquid chromatograph, recording chromatogram, and calculating content of each index component in the test solution by external standard method.
The establishment of the detection method of the present invention and a methodological examination are described in detail below by way of example 1.
Example 1Establishment of the detection method of the invention
In this example, a control solution was prepared as follows:
precisely weighing 1.071mg of chlorogenic acid, 0.879mg of luteoloside, 1.052mg of isochlorogenic acid B, 1.054mg of isochlorogenic acid C, 1.607mg of linalooside, 5.002mg of acetaminophen and 0.623mg of caffeine as reference substances, respectively, placing in a 25ml volumetric flask, adding methanol to dissolve and dilute to obtain a mixed reference substance solution with concentrations of 42.8 μ g/ml (chlorogenic acid), 35.2 μ g/ml (luteoloside), 42.1 μ g/ml (isochlorogenic acid B), 42.2 μ g/ml (isochlorogenic acid C), 64.3 μ g/ml (linalooside), 200.1 μ g/ml (acetaminophen) and 24.9 μ g/ml (caffeine), storing in a refrigerator at 4 deg.C, and filtering with 0.22 μm microporous membrane before use.
The test solution was prepared as follows:
taking a sample with the batch number of 170101 (Ganmaoling granules, Limited liability company of Chinese medicine factory in Sichuan province), grinding in a mortar, precisely weighing about 3g, placing in a conical flask with a plug, precisely adding 25ml of methanol, sealing the plug, weighing, ultrasonically extracting for 30min, cooling, weighing, supplementing the weight loss with methanol, shaking, filtering, and taking the subsequent filtrate.
1. Chromatographic conditions and System suitability test
1.1 selection of the Mobile phase
Under the detection wavelength of 300nm (target peak information can be seen more comprehensively), the influence of gradient elution of mobile phases of different systems such as methanol-water, methanol-0.1% acetic acid aqueous solution, methanol-0.1% phosphoric acid aqueous solution, acetonitrile-water, acetonitrile-0.1% acetic acid aqueous solution, acetonitrile-0.1% phosphoric acid aqueous solution and the like on the separation of each component in the test solution is considered, the elution procedure is shown in table 5, and the chromatogram is respectively shown in 1A-1G of fig. 1. According to the comparison chromatogram, when the acetonitrile-phosphoric acid aqueous solution is used as a mobile phase, the separation effect is best; while the elution systems of the acetonitrile-0.1 percent phosphoric acid aqueous solution and the acetonitrile-0.2 percent phosphoric acid aqueous solution have no obvious difference, and in order to reduce the damage to the chromatographic column, the elution system with low phosphoric acid concentration, namely the acetonitrile-0.1 percent phosphoric acid aqueous solution, is selected.
1.2 examination of elution procedure
Under the detection wavelength of 300nm, acetonitrile (A) -0.1% phosphoric acid water solution (B) is used as a mobile phase, and the separation effects of different elution procedures on the relevant components of the sample solution are compared. The gradient elution procedures are shown in tables 2-5, and the chromatograms are shown in FIGS. 2A-2D, respectively.
Table 2 gradient elution procedure 1
| Time | Phase A | Phase B |
| 0→ |
6％→13％ | Balance of |
| 5→15min | 13％→20％ | Balance of |
| 15→25min | 20％→30％ | Balance of |
| 25→30min | 30％→60％ | Balance of |
| 30→45min | 60％→95％ | Balance of |
| 45→50min | 95％ | Balance of |
Table 3 gradient elution procedure 2
Table 4 gradient elution procedure 3
| Time | Phase A | Phase B |
| 0→10min | 13％ | Balance of |
| 10→20min | 13％→20％ | Balance of |
| 20→25min | 20％ | Balance of |
| 25→40min | 20％→30％ | Balance of |
| 40→50min | 30％→60％ | Balance of |
Table 5 gradient elution procedure 4
| Time | Phase A | Phase B |
| 0→10min | 13％ | Balance of |
| 10→15min | 13％→20％ | Balance of |
| 15→20min | 20％ | Balance of |
| 20→40min | 20％→30％ | Balance of |
| 40→50min | 30％→60％ | Balance of |
Comparing fig. 2A-2D, the results show that the chromatogram obtained under gradient elution procedure 4 has a better degree of separation and a suitable retention time, so that the gradient elution procedure 4 is selected as the elution condition of the detection method of the present invention.
1.2 selection of detection wavelength
The detection method of the invention adopts a diode array detector (PAD detector), and can simultaneously detect the absorption of the sample at all wavelengths. By scanning the reference solution at full wavelength and analyzing spectrograms at various wavelengths based on spectral absorption, literature comparison, compound polarity and the like, the maximum absorption wavelength of the linarin is 334nm, the maximum absorption wavelength of the luteoloside is 350nm, and the maximum absorption wavelengths of the chlorogenic acid, the isochlorogenic acid B and the isochlorogenic acid C are 327 nm. The baseline of acetaminophen and caffeine at 272nm is stable, the chromatogram peak shape is good, and the separation degree is good, so 327nm, 334nm, 350nm and 272nm are selected as detection wavelengths, and the component information of traditional Chinese medicine components and chemical medicine components is fully displayed.
1.3 chromatographic columns
Agilent Eclipse Plus C18(4.6 mm. times.250 mm, 5 μm) chromatography column.
1.4 selection of column temperature
Under the optimized mobile phase and elution program, 327nm, 334nm, 350nm and 272nm are used as detection wavelengths, the influence of four column temperatures of 25 ℃, 30 ℃, 35 ℃ and 40 ℃ on the separation of each component in the test solution is examined, and chromatograms are respectively shown in fig. 3A-3D. The chromatogram shows that the separation degree of each component is better under different column temperatures to be inspected, so that the detection method can be carried out at room temperature (25-35 ℃) without taking special measures to ensure the column temperature.
1.5 investigation of flow Rate
Under the optimized mobile phase and elution program, 327nm, 334nm, 350nm and 272nm are taken as detection wavelengths, the influence of three flow rates of 0.8ml/min, 1.0ml/min and 1.2ml/min on the separation of each component in the test solution is examined, and chromatograms are respectively shown in fig. 4A-4C. The chromatogram shows that the separation degree of each component is better under different flow rates, so that the flow rate of the detection method can be 0.8-1.2 ml/min.
2. Examination of extraction method
Methanol, water and acetonitrile are used as extraction solvents, Ganmaoling granules (Ganmaoling granules, Limited liability company of Chinese medicine factory in Sichuan province, batch number 170101) are extracted by ultrasonic, and test sample solutions (chromatogram is omitted) obtained by measuring different extraction solvents under the optimized spectrogram condition. As a result, methanol is used as an extraction solvent, and compared with water or ethanol, the methanol extraction solvent has the advantages of less extracted impurities and better chromatographic peak pattern. Methanol is therefore preferred as the extraction solvent. And comparing the influence of different ultrasonic treatment time (10min, 20min, 30min, 40min and 60min) on chromatographic peak in the test solution. As a result, it was found that the peak area of the chromatographic peak of each index component increased with the increase of the ultrasonic time, and the peak area increased only insignificantly after 30min of extraction (the chromatogram was slight). Therefore, the optimal ultrasonic extraction time of the methanol is determined to be 30 min.
3. Optimized post-established detection method
Through the investigation and the test, the chromatographic conditions of the component detection methods of the Ganmaoling and the compound Ganmaoling and the preparation method of the test solution are preferably selected, and the specific steps are as follows:
the detection method is based on high performance liquid chromatography and diode array detector, and is used for determining the content of chlorogenic acid, luteoloside, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine in Ganmaoling and Compound Ganmaoling; the chromatographic conditions were as follows:
filling agent: octadecylsilane chemically bonded silica;
column temperature: 25-35 ℃;
a detector: a diode array detector;
detection wavelength: 272nm, 327nm, 334nm and 350 nm;
mobile phase: using acetonitrile as phase A and 0.1% phosphoric acid water solution as phase B, and performing gradient elution according to the following procedures:
| Time | phase A | Phase B |
| 0→10min | 13％ | Balance of |
| 10→15min | 13％→20％ | Balance of |
| 15→20min | 20％ | Balance of |
| 20→40min | 20％→30％ | Balance of |
| 40→50min | 30％→60％ | Balance of |
Flow rate: 0.8-1.2 ml/min;
theoretical plate number: calculated according to chlorogenic acid, galuteolin, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine peak, respectively, not less than 3000;
preparation of a test solution: taking Ganmaoling or Compound Ganmaoling, removing sugar coating of tablet, taking capsule, grinding in mortar, precisely weighing 3g, placing in conical flask with plug, precisely adding 25ml of methanol, sealing, weighing, ultrasonically extracting for 30min, cooling, weighing, adding methanol to compensate lost weight, shaking, filtering, and taking subsequent filtrate.
The chromatograms of the above test sample and mixed reference are shown in FIGS. 5A-5D.
4. Methodology validation
4.1 Linear investigation
Precisely sucking 2, 4, 6, 8, 10 and 12 μ l of the mixed control solution, respectively, injecting into a liquid chromatograph, and measuring according to the chromatographic conditions under item "3". The sample amount was taken as the abscissa and the peak area as the ordinate, and regression analysis was performed, the regression equation being shown in table 6.
TABLE 6 Linear relationship of seven index components
4.2 precision test
Precisely sucking 10 mu l of mixed reference substance solution, continuously injecting samples for 6 times, calculating RSD values of peak areas of 7 components, and inspecting the precision of the instrument, wherein the results are shown in Table 7.
TABLE 7 results of precision test
The results in Table 6 show good precision of the instrument.
4.3 detection Limit
4.4 stability test
Preparing a test solution according to the method under the item '3', precisely absorbing 10 mu l of the test solution for 0, 2, 4, 6, 8, 10, 12 and 24 hours respectively, injecting the solution into a chromatograph for sample injection analysis, recording peak areas of 7 components, calculating RSD values of the peak areas, and inspecting the stability of the test solution, wherein the results are shown in a table 8.
TABLE 8 stability test results
The data in Table 7 show that the test solutions are stable over 24 hours.
4.5 repeatability test
6 parts of test solution were prepared in parallel by the method under the section "3", 10. mu.l of each sample was taken, the contents of 7 components were measured, the RSD value of the peak area was calculated, the reproducibility of the method was examined, and the results are shown in Table 9.
TABLE 9 results of the repeatability tests
The data in table 8 show that the method is very reproducible.
3.6 sample application recovery test
A suitable amount of a reference substance was precisely added to 6 parts of Ganmaoling (granule, from Limited liability company of Chinese medicine works, Sichuan province, lot No. 170101) powder of known content, each part was weighed to 1.5g, a test solution was prepared according to the method of item "3", the sample recovery rates and RSD values of the respective components were calculated according to the chromatographic conditions of item "3", and the results are shown in Table 10.
TABLE 10 sample recovery test results
The data in table 10 show that the average sample recovery rates of chlorogenic acid, galuteolin, isochlorogenic acid B, isochlorogenic acid C, linarin, p-acetylaminophenol and caffeine are 99.72%, 99.08%, 109.32%, 112.21%, 98.66%, 97.481% and 99.454% respectively, and the RSD are 3.42%, 4.64%, 4.95%, 3.77%, 3.02%, 2.50% and 2.06% respectively, indicating that the accuracy of the present invention is better.
In a word, the result of the methodology investigation shows that the detection method provided by the invention is sensitive, good in accuracy, high in stability and strong in operability.
Example 2Component detection of Ganmaoling granules
The Ganmaoling granules of different manufacturers and different batches shown in Table 1 were measured according to the detection method established in example 1, and the content of seven ingredients, i.e., chlorogenic acid, galuteolin, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine, were calculated by an external standard method, and the results are shown in Table 11.
TABLE 11 determination of Ganmaoling granules (mg/bag)
The results in table 11 show: 1) the detection method provided by the invention can detect the content of seven components, namely chlorogenic acid, luteoloside, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine, in different batches of Ganmaoling granules of different manufacturers. 2) The content difference of corresponding components of the Ganmaoling granules from different manufacturers and different batches is large, particularly five components of the traditional Chinese medicine are originated, and the analysis reason is probably caused by the difference of the quality of the traditional Chinese medicine raw materials and the preparation process conditions.
Example 3Component detection of Ganmaoling capsule
The Ganmaoling capsules of different manufacturers shown in Table 1 were taken, the content of the capsule was measured according to the detection method established in example 1, and the content of seven ingredients, i.e., chlorogenic acid, galuteolin, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine, was calculated by an external standard method, and the results are shown in Table 12.
TABLE 12 determination of Ganmaoling capsule (mg/granule)
The results in table 11 show: 1) the detection method provided by the invention can detect the content of seven components, namely chlorogenic acid, luteoloside, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine, in Ganmaoling capsules of different manufacturers. 2) The content difference of corresponding components of the Ganmaoling capsule from different manufacturers is large, particularly five components derived from traditional Chinese medicines, and the analysis reason is probably caused by the difference of the quality of the traditional Chinese medicine raw materials and the preparation process conditions.
Example 4Component detection of compound Ganmaoling granules
Taking the compound Ganmaoling granules of different manufacturers shown in Table 1, measuring the granules according to the detection method established in example 1, and calculating the contents of seven components including chlorogenic acid, galuteolin, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine by an external standard method, wherein the results are shown in Table 13.
TABLE 13 Compound GANMAOLING granule determination results (mg/bag)
The results in table 12 show: 1) the detection method provided by the invention can detect the content of seven components, namely chlorogenic acid, luteoloside, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine, in the compound Ganmaoling granules of different manufacturers. 2) The content difference of corresponding components of the compound Ganmaoling granules from different manufacturers is large, particularly five components of the traditional Chinese medicine are originated, and the analysis reason is probably caused by the difference of the quality of the traditional Chinese medicine raw materials and the preparation process conditions.
Example 5Component detection of compound Ganmaoling tablet
Taking the compound Ganmaoling tablets of different manufacturers shown in Table 1, removing the sugar coating, measuring according to the detection method established in example 1, and calculating the contents of seven components including chlorogenic acid, galuteolin, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine by an external standard method, wherein the results are shown in Table 14.
TABLE 14 determination of Compound GANMAOLING tablet (mg/tablet)
The results in table 14 show: 1) the detection method provided by the invention can detect the content of seven components, namely chlorogenic acid, luteoloside, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine, in the compound Ganmaoling granules of different manufacturers. 2) The content difference of corresponding components of the compound Ganmaoling tablet from different manufacturers is large, particularly five components of the traditional Chinese medicine are originated, some index components can not be detected even in some tested products, and the analysis reason can be caused by the difference of the quality of the traditional Chinese medicine raw materials and the preparation process conditions.
The results of the embodiments 2-5 show that the detection method provided by the invention can be applied to the component detection of various Ganmaoling and compound Ganmaoling. The content difference of the corresponding components of the medicines of different manufacturers is obvious. Therefore, if only single components (both the components from the traditional Chinese medicine and the chemical components) are used, the real quality of the medicine cannot be reflected, and only different chemical (active) components of the compound preparation are considered, the quality of the medicine can be effectively reflected, so that the safety and the effectiveness of clinical medication are ensured.
Claims (4)
1. A method for detecting components of GANMAOLING and FUFANGGANMAOLING comprises determining contents of chlorogenic acid, luteoloside, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine by high performance liquid chromatography; the chromatographic conditions were as follows:
filling agent: octadecylsilane chemically bonded silica;
column temperature: 25-35 ℃;
a detector: a diode array detector;
detection wavelength: 272nm, 327nm, 334nm and 350 nm;
mobile phase: taking acetonitrile as an A phase and 0.1-0.3% phosphoric acid aqueous solution as a B phase, and performing gradient elution according to the following procedures:
Flow rate: 0.8-1.2 ml/min;
the preparation of the reference solution comprises the following specific steps:
precisely weighing chlorogenic acid, galuteolin, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine, putting the chlorogenic acid, galobatin, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine into a same volumetric flask, dissolving and diluting the chlorogenic acid, isochlorogenic acid C, isochlorogenic acid B, isochlorogenic acid C and caffeine into mixed reference solutions with respective concentrations of 20-200 μ g/ml independently;
the preparation of the test solution comprises the following specific operations:
taking Ganmaoling or compound Ganmaoling, wherein sugar coating of tablets needs to be removed, taking contents of capsules, putting the capsules into a mortar for grinding, weighing 1-5 g, putting the tablets into a conical bottle with a plug, adding 20-50 ml of methanol, sealing the plug, weighing, carrying out ultrasonic extraction for 30-60 min, weighing after cooling, complementing the weight loss by using the methanol, shaking up and filtering, and taking the subsequent filtrate to obtain the Ganmaoling;
the specific operation of the determination is as follows:
and respectively sucking 10-20 μ l of the test solution and the reference solution, injecting into a liquid chromatograph, measuring, and calculating the content of chlorogenic acid, luteoloside, isochlorogenic acid B, isochlorogenic acid C, linarin, acetaminophen and caffeine in the test sample by an external standard method.
2. The method for detecting a component according to claim 1, wherein the phase B of the mobile phase is a 0.1 to 0.2% phosphoric acid aqueous solution.
3. The method according to claim 2, wherein the phase B of the mobile phase is a 0.1% phosphoric acid aqueous solution.
4. The method for detecting a component according to claim 1, wherein the step of preparing the sample solution comprises the steps of:
taking Ganmaoling or Compound Ganmaoling, removing sugar coating of tablet, taking capsule, grinding in mortar, precisely weighing 3g, placing in conical flask with plug, precisely adding 25ml of methanol, sealing, weighing, ultrasonically extracting for 30min, cooling, weighing, adding methanol to compensate lost weight, shaking, filtering, and taking subsequent filtrate.
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