CN108663454B - Rapid detection method capable of simultaneously quantifying gallic acid, andrographolide and dehydroandrographolide in compound andrographis tablet - Google Patents
Rapid detection method capable of simultaneously quantifying gallic acid, andrographolide and dehydroandrographolide in compound andrographis tablet Download PDFInfo
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- CN108663454B CN108663454B CN201810593637.4A CN201810593637A CN108663454B CN 108663454 B CN108663454 B CN 108663454B CN 201810593637 A CN201810593637 A CN 201810593637A CN 108663454 B CN108663454 B CN 108663454B
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- dehydroandrographolide
- andrographolide
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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
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The invention relates to a method for detecting active ingredients in compound andrographis tablet, in particular to a method for rapidly detecting gallic acid, andrographolide and dehydroandrographolide in compound andrographis tablet, which comprises the following steps: (1) preparing a standard solution; (2) preparing a test solution; (3) detecting the standard solution and the sample solution with supercritical chromatograph, and simultaneously obtaining the contents of gallic acid, andrographolide and dehydroandrographolide in the sample solution. The invention adopts supercritical chromatography for detection, the separation capability of the supercritical chromatography is strong, only extraction is needed when the test solution is prepared, the step of column chromatography purification is omitted, the experimental time is shortened from 4-5 hours to 40-50 minutes, the working efficiency is greatly improved, the time, the manpower and the material resources are saved, and simultaneously, the contents of gallic acid, andrographolide and dehydroandrographolide can be accurately quantified.
Description
Technical Field
The invention relates to a detection method of compound andrographis tablet, in particular to a rapid detection method capable of simultaneously quantifying gallic acid, andrographolide and dehydroandrographolide in the compound andrographis tablet.
Background
The compound herba Andrographitis tablet is prepared from 2 medicinal materials including herba Andrographitis and radix Ardisiae Japonicae, and has effects of clearing away heat and toxic materials, cooling blood, and promoting diuresis. The existing drug standard only has 2 identifications, lacks content determination items and cannot carry out comprehensive quality control on the product. In the 2015 edition of Chinese pharmacopoeia, a method for measuring the content of andrographolide and dehydroandrographolide controlled by standard components of medicinal material andrographis paniculata needs to extract and purify by neutral alumina column chromatography, then HPLC detection takes 4 hours, and due to the fact that a pretreatment method is too complicated and too long, an experimenter with incomplete transfer and shallow experience is difficult to grasp the experimental pharmacopoeia, the measurement result of andrographolide and dehydroandrographolide is inaccurate; in each of the Chinese medicine standards, the standard control of Arisaema cum bile is not recorded. The characteristic component of the Chinese lobelia herb is gallic acid, and the component is not used as a control component of the compound common andrographis herb tablet in the existing secondary detection method. The existing methods are mostly thin-layer identification and common high-performance liquid chromatography, are all one-component methods, cannot embody advantages in experiment time, extraction efficiency, solvent loss and experiment hand degree, and need a method for simultaneously determining the content of common andrographis herb and wayside herb, which is established by the characteristics of environmental protection, rapidness and strong separation capability.
Disclosure of Invention
The invention aims to provide a rapid detection method capable of simultaneously quantifying gallic acid, andrographolide and dehydroandrographolide in compound andrographis tablet.
The purpose of the invention is realized by the following technical scheme: a rapid detection method capable of simultaneously quantifying gallic acid, andrographolide and dehydroandrographolide in compound andrographis tablet comprises the following steps:
(1) preparing a standard solution: taking gallic acid standard, andrographolide standard and dehydroandrographolide standard, and adding methanol to obtain standard solution;
(2) preparing a test solution: grinding compound herba Andrographitis tablet sample into powder, and extracting with methanol to obtain test solution;
(3) detecting the standard solution and the sample solution with supercritical chromatograph, and simultaneously obtaining the contents of gallic acid, andrographolide and dehydroandrographolide in the sample solution; wherein, the chromatographic conditions are as follows: the chromatographic column is silica gel column specification 3.0 × 150mm, 3 μm; with CO2-0.1% phosphoric acid solution as mobile phase; the flow rate is 2.5ml/min, the detector is a diode array detector, the detection wavelength of andrographolide is 225nm, the detection wavelength of dehydroandrographolide is 254nm, the detection wavelength of gallic acid is 273nm, and the sample injection amount is 5 μ l.
In the step (1), the concentrations of gallic acid, andrographolide and dehydroandrographolide in the standard solution are all 0.1 mg/ml.
In the step (2), 1.0g of compound andrographis tablet sample powder is taken and placed in a container, methanol is added for ultrasonic extraction, then the mixture is cooled to room temperature, methanol is added for holding 10ml, and the solution is filtered by a filter membrane to obtain a sample solution. Specifically, the ultrasonic extraction time is 15-25 min.
In the step (3), CO2The volume ratio of the-0.1% phosphoric acid solution is 85-95: 5-15.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention adopts supercritical chromatography for detection, the separation capability of the supercritical chromatography is strong, only extraction is needed when the test solution is prepared, the step of column chromatography purification is omitted, the experimental time is shortened from 4-5 hours to 40-50 minutes, the working efficiency is greatly improved, the time, the manpower and the material resources are saved, and simultaneously, the contents of gallic acid, andrographolide and dehydroandrographolide can be accurately quantified.
2. The invention takes carbon dioxide as a mobile phase to replace acetonitrile and methanol required by a common liquid phase, and has outstanding advantages in use cost and environmental protection.
Drawings
FIG. 1 is a supercritical chromatogram of a standard solution of an example of the invention.
FIG. 2 is a supercritical chromatogram of a test solution according to an embodiment of the present invention.
Detailed Description
Example 1
(1) Preparing a standard solution: taking gallic acid standard, andrographolide standard and dehydroandrographolide standard, and adding 50% methanol to obtain standard solution; the concentrations of gallic acid, andrographolide and dehydroandrographolide in the standard solution were all 0.1 mg/ml.
(2) Preparing a test solution: taking 20 tablets of the compound common andrographis tablet according to the weight difference, grinding, precisely weighing 1.0g of compound common andrographis tablet sample powder, placing the powder in a 10ml volumetric flask, adding 8ml of methanol, carrying out ultrasonic treatment for 20min, cooling to room temperature, adding methanol to reach a constant volume of 10ml, and filtering with a filter membrane to obtain a sample solution.
(3) Performing supercritical chromatography detection on the standard solution and the test solution to obtain the contents of gallic acid, andrographolide and dehydroandrographolide in the test solution; wherein, the chromatographic conditions are as follows: the chromatographic column is silica gel column specification 3.0 × 150mm, 3 μm; with CO2-a mobile phase of 90:10 in 0.1% phosphoric acid solution; the flow rate is 2.5ml/min, the detector is a diode array detector, the detection wavelength of andrographolide is 225nm, the detection wavelength of dehydroandrographolide is 254nm, the detection wavelength of gallic acid is 273nm, and the sample injection amount is 5 μ l. The results are shown in FIGS. 1-2.
Taking 10 batches of compound andrographis tablet samples to do 3 times of reproducibility respectively: the gallic acid RSD is 1.4-2.6%, and the average recovery rate is 98.7%; the andrographolide RSD is 1.5-2.3%, and the average recovery rate is 97.3%; the RSD of the dehydroandrographolide is 1.0-1.7%, and the average recovery rate is 98.2%.
And (3) recovery rate determination: taking 10mg of gallic acid standard, 10mg of andrographolide standard and 50mg of dehydroandrographolide standard, respectively placing in 10ml, 10ml and 5ml volumetric flasks, adding 50% methanol for dissolving, diluting to scale, shaking up, and respectively taking appropriate amount of each solution to prepare a mixed control solution containing 50 microgram/ml of gallic acid, 0.5mg/ml of andrographolide and 5mg/ml of dehydroandrographolide. Accurately weighing 1.0g of andrographis tablet sample powder, putting 1ml of mixed control solution into a 10ml volumetric flask, adding 8ml of methanol, performing ultrasonic treatment for 20min, cooling to room temperature, adding methanol to fix the volume to 10ml, filtering with a filter membrane, detecting the solution with a supercritical chromatograph under the detection conditions shown in step (3), synchronously measuring the content of the sample added with the standard, deducting the background content of the sample, and calculating the recovery rate.
The results of the measurements are shown in the following table:
| batches of | Gallic acid microgram/tablet | Andrographolide mg/tablet | Mg/tablet of dehydroandrographolide |
| 1 | 15 | 0.062 | 2.053 |
| 2 | 9 | 0.098 | 1.833 |
| 3 | 6 | 0.051 | 1.331 |
| 4 | 12 | 0.032 | 1.512 |
| 5 | 10 | 0.054 | 0.986 |
| 6 | 7 | 0.064 | 0.876 |
| 7 | 5 | 0.087 | 1.834 |
| 8 | 9 | 0.091 | 1.762 |
| 9 | 11 | 0.037 | 1.567 |
| 10 | 15 | 0.045 | 1.054 |
The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The above-described embodiments of the present invention are to be considered as illustrative and not restrictive, and therefore all slight modifications, equivalent changes and modifications made to the above examples in accordance with the spirit of the present invention are within the scope of the present invention.
Claims (3)
1. A rapid detection method capable of simultaneously quantifying gallic acid, andrographolide and dehydroandrographolide in compound andrographis paniculata tablets is characterized by comprising the following steps:
(1) preparing a standard solution: taking gallic acid standard, andrographolide standard and dehydroandrographolide standard, and adding methanol to obtain standard solution;
(2) preparing a test solution: grinding compound herba Andrographitis tablet sample into powder, and extracting with methanol to obtain test solution;
(3) detecting the standard solution and the sample solution with supercritical chromatograph, and simultaneously obtaining the contents of gallic acid, andrographolide and dehydroandrographolide in the sample solution;
wherein, the chromatographic conditions are as follows: the chromatographic column is a silica gel column: the specification is 3.0 multiplied by 150mm, 3 μm; with CO2-0.1% phosphoric acid solution as mobile phase; the flow rate is 2.5ml/min, the detector is a diode array detector, the detection wavelength of andrographolide is 225nm, the detection wavelength of dehydroandrographolide is 254nm, the detection wavelength of gallic acid is 273nm, and the sample injection amount is 5 mul;
in the step (3), CO2The volume ratio of the-0.1% phosphoric acid solution is 85-95: 5-15.
2. The method for simultaneously quantifying gallic acid, andrographolide and dehydroandrographolide in compound andrographis tablet according to claim 1, wherein in step (1), the concentration of gallic acid, andrographolide and dehydroandrographolide in the standard solution is 0.1 mg/ml.
3. The method for simultaneously and quantitatively detecting gallic acid, andrographolide and dehydroandrographolide in compound andrographis tablet as claimed in claim 1, wherein in step (2), 1.0g of compound andrographis tablet sample powder is taken and placed in a container, methanol is added for ultrasonic extraction, then the mixture is cooled to room temperature, and then the mixture is added with methanol for being accommodated in 10ml, and the solution is filtered by a filter membrane to obtain the sample solution.
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| CN1318398A (en) * | 2000-04-19 | 2001-10-24 | 广州市轻工研究所 | Method of extracting effective component of Herba Andrographitis |
| US7604823B2 (en) * | 2006-09-07 | 2009-10-20 | Jose Angel Olalde Rangel | Synergistic HIV/AIDS and/or immune disease phyto-nutraceutical composition |
| CN101559088A (en) * | 2009-05-21 | 2009-10-21 | 雷允上药业有限公司 | Production technique of andrographolide and neoandrographolide, dehydroanddrographolide, oxyandrographolide |
| CN103435578A (en) * | 2013-09-17 | 2013-12-11 | 南京通泽农业科技有限公司 | Method for extracting andrographolide from andrographis paniculata |
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| CN105884722A (en) * | 2016-04-27 | 2016-08-24 | 聊城大学 | Method of separating and purifying andrographolide and dehydrated andrographolide from Herba andrographitis |
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| US7604823B2 (en) * | 2006-09-07 | 2009-10-20 | Jose Angel Olalde Rangel | Synergistic HIV/AIDS and/or immune disease phyto-nutraceutical composition |
| CN101559088A (en) * | 2009-05-21 | 2009-10-21 | 雷允上药业有限公司 | Production technique of andrographolide and neoandrographolide, dehydroanddrographolide, oxyandrographolide |
| CN101559088B (en) * | 2009-05-21 | 2012-07-25 | 雷允上药业有限公司 | Production technique of andrographolide and neoandrographolide, dehydroanddrographolide, oxyandrographolide |
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Application publication date: 20181016 Assignee: GUANGXI GUILIN RUIDE DETECTION AUTHENTICATION TECHNOLOGY Co.,Ltd. Assignor: WUZHOU FOOD AND DRUG INSPECTION INSTITUTE Contract record no.: X2022450000291 Denomination of invention: A Rapid Determination Method for Gallic Acid, Andrographolide and Dehydrated Andrographolide in Compound Andrographolide Tablets Granted publication date: 20210416 License type: Common License Record date: 20221214 |