CN113092632B - Method for detecting content of dehydroandrographolide in Chuanwang anti-inflammatory tablet - Google Patents
Method for detecting content of dehydroandrographolide in Chuanwang anti-inflammatory tablet Download PDFInfo
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Abstract
The invention provides a method for detecting the content of dehydroandrographolide in a Chuanwang anti-inflammatory tablet, and relates to the content determination of a pharmaceutical composition. The method comprises the following steps: (1) preparation of control solutions: preparing reference substance solution from dehydroandrographolide and methanol; (2) preparation of a test solution: accurately weighing a Chuanwang anti-inflammatory tablet sample, and adding methanol to dilute the Chuanwang anti-inflammatory tablet sample to prepare a test solution; (3) respectively sucking the reference solution and the test solution, injecting into a liquid chromatograph, and simultaneously measuring to obtain the content of dehydroandrographolide; the test solution in the step (2) also contains a stabilizer. The detection method provided by the invention has good reproducibility and high stability, and provides a foundation for detecting the content of the active ingredients of the traditional Chinese medicinal material or the pharmaceutical composition containing the dehydroandrographolide.
Description
Technical Field
The invention relates to a content determination method of a pharmaceutical composition, and in particular relates to a content determination method of dehydroandrographolide in a Chuanwang anti-inflammatory tablet.
Background
The traditional Chinese medicine or traditional Chinese medicine preparation contains complex components, and the main chemical components with physiological activity are generally used as quality evaluation and control standards in the quality standard of preparing the medicinal materials and the Chuanwang anti-inflammatory tablet. The method commonly used for measuring the content of the active ingredients of the traditional Chinese medicine comprises an ultraviolet-visible spectrophotometry method, a high performance liquid chromatography method, a liquid-mass combination method, an infrared spectroscopy method, a gas chromatography method, a gas-mass combination method, a thin layer chromatography scanning method, a nuclear magnetic resonance spectroscopy method and the like, the accuracy of the measurement result of the content of the active ingredients is a key part in the quality control of the traditional Chinese medicine, and different content measurement methods have advantages and disadvantages and need to select different detection methods according to different detection standards. The high performance liquid chromatography has the advantages of high separation efficiency, good selectivity, high analysis speed, high detection sensitivity, wide application range and the like, can be used for analyzing the active ingredients with high boiling point, thermal instability and large molecular weight in a sample, and is an analysis method which is widely applied in the content measurement of the active ingredients in the traditional Chinese medicine.
Chinese patent application 201711369659.4 discloses a method for measuring the content of dehydroandrographolide in andrographis paniculata tablets by HPLC, which comprises the following steps: (1) preparation of control solutions: preparing reference solution from dehydroandrographolide and ethanol; (2) preparing a sample solution to be tested: accurately taking an andrographis tablet sample, adding ethanol for dilution, and preparing a sample solution to be detected; (3) measuring with high performance liquid chromatograph, and establishing standard curve of dehydroandrographolide; (4) injecting the sample solution to be measured into a high performance liquid chromatograph for determination to obtain the peak area of the dehydroandrographolide, so as to obtain the content of the dehydroandrographolide according to a standard curve; the chromatographic conditions of the steps (3) and (4): 4.6mm × 150mm, 5 μm, Agilent Eclipse XDB-C18 column; mobile phase: ethanol-water 65: 35; flow rate: 1.0mL.min-1(ii) a Detecting the double wavelength: 254nm, sample size: 10 μ L, column temperature: room temperature, degree of separation: 1.6, the number of theoretical plates is more than 2000 in terms of dehydroandrographolide, but the content detection method provided by the application does not pay attention to detection repeatability.
Chinese patent application 201810593637.4 discloses a rapid detection method capable of simultaneously quantifying gallic acid, andrographolide and dehydroandrographolide in compound andrographis tablet, comprising 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 method adopts supercritical chromatography for detection, the separation capacity of the supercritical chromatography is strong, only extraction is needed when a 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 labor and the material resources are saved, meanwhile, the contents of gallic acid, andrographolide and dehydroandrographolide can be accurately quantified, but the detection method does not pay attention to the detection reproducibility.
The existing detection method mostly pays attention to the high efficiency of detection, aims to shorten the detection time, does not pay attention to the reproducibility and precision of detection, and therefore, a method for providing the content of the dehydroandrographolide, which has high accuracy, good reproducibility and high precision, is needed.
Disclosure of Invention
The invention aims to provide a method for detecting the content of dehydroandrographolide in a Chuanwang anti-inflammatory tablet, which aims to solve the problems of inaccurate detection and poor reproducibility of the content of the existing medicines.
The content method of the dehydroandrographolide adopts high performance liquid chromatography.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for content of dehydroandrographolide in Chuanwang anti-inflammatory tablet comprises the following steps:
(1) preparation of control solutions: preparing reference substance solution from dehydroandrographolide and methanol;
(2) preparation of a test solution: accurately weighing a Chuanwang anti-inflammatory tablet sample, and adding methanol to dilute the Chuanwang anti-inflammatory tablet sample to prepare a test solution;
(3) respectively sucking the reference solution and the test solution, injecting into a liquid chromatograph, and simultaneously measuring to obtain the content of dehydroandrographolide.
The chromatographic conditions of the step (3) are as follows: octadecylsilane chemically bonded silica is used as a filling agent; eluting with methanol-water (55: 45) as mobile phase; column temperature: 35 ℃; flow rate: 1.0 mL/min; the sample volume is 20 mu L; the detection wavelength was 250 nm. The number of theoretical plates is not less than 4000 in terms of dehydroandrographolide peak.
In the step (1), a proper amount of dehydroandrographolide reference substances are precisely weighed, and 60% methanol is added to prepare reference substance solution containing 15 microgram of dehydroandrographolide per 1 ml.
In the step (2), 0.5g of inflammation-diminishing, detoxifying and queen-crossing inflammation-diminishing tablet sample is precisely weighed, placed in a 100mL measuring flask, added with 90mL of 60% methanol, ultrasonically treated for 30min, cooled, added with 60% methanol to the scale, and filtered by a microporous filter membrane (0.45 mu m), and the obtained filtrate is the sample solution.
Wherein the ultrasonic conditions are as follows: the ultrasonic power is 250W, and the ultrasonic frequency is 33 kHz.
In some preferred embodiments, the test solution further comprises a stabilizer, wherein the stabilizer is a mixture of arginine and vitamin C;
the mass ratio of the addition amount of the stabilizer to the Chuanwang anti-inflammatory tablet sample is 0.5-1: 100;
preferably, the mass ratio of the added stabilizer to the Chuanwang anti-inflammatory tablet sample is 0.8-1: 100;
still preferably, the mass ratio of the added stabilizer to the Chuanwang anti-inflammatory tablet sample is 0.8: 100.
The mass ratio of the arginine to the vitamin C is 2-3: 1;
preferably, the mass ratio of the arginine to the vitamin C is 2.5-3: 1;
still preferably, the mass ratio of arginine to vitamin C is 3: 1.
Specifically, the invention provides a method for content of dehydroandrographolide in anti-inflammatory and detoxifying Chuanwang anti-inflammatory tablets, which comprises the following steps:
(1) preparation of control solutions: precisely weighing appropriate amount of dehydroandrographolide control, and adding 60% methanol to obtain control solution containing dehydroandrographolide 15 μ g per 1 ml;
(2) preparation of a test solution: taking 0.5g of Chuanwang anti-inflammatory tablet sample and a stabilizer (2.5-5mg), precisely weighing, placing in a 100mL measuring flask, adding 90mL of 60% methanol, carrying out ultrasonic treatment for 30min under the conditions of 250W power and 33kHz frequency, cooling, adding 60% methanol to the scale, and filtering with a microporous filter membrane (0.45 mu m), wherein the obtained filtrate is a sample solution;
(3) respectively sucking 20 μ L of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring to obtain dehydroandrographolide content.
The chromatographic conditions of the step (3) are as follows: octadecylsilane chemically bonded silica is used as a filling agent; eluting with methanol-water (55: 45) as mobile phase; column temperature: 35 ℃; flow rate: 1.0 mL/min; the sample volume is 20 mu L; the detection wavelength was 250 nm. The number of theoretical plates is not less than 4000 in terms of dehydroandrographolide peak.
Compared with the prior art, the invention has the beneficial effects that:
the detection method provided by the invention has good reproducibility and high stability, and provides a foundation for detecting the content of the active ingredients of the traditional Chinese medicinal material or the pharmaceutical composition containing the dehydroandrographolide.
Drawings
FIG. 1 is an HPLC chromatogram of a test solution of example 1 of the present invention;
FIG. 2 is an HPLC chromatogram of a test solution of example 2 of the present invention;
FIG. 3 HPLC chromatogram of the test solution of example 3 of the present invention.
Detailed Description
The following examples are included to provide further detailed descriptions of specific embodiments of the invention and to enable those skilled in the art to better understand the invention and practice it. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The liquid chromatograph used in the invention is Agilent 1260, and the specification of the chromatographic column is 250 multiplied by 4.6 mm.
Example 1 method for content of dehydroandrographolide in Chuanwang anti-inflammatory tablet
The method comprises the following steps:
(1) preparation of control solutions: precisely weighing appropriate amount of dehydroandrographolide control, and adding 60% methanol to obtain control solution containing dehydroandrographolide 15 μ g per 1 ml;
(2) preparation of a test solution: taking 0.5g of Chuanwang anti-inflammatory tablet sample, precisely weighing, placing in a 100mL measuring flask, adding 90mL of 60% methanol, carrying out ultrasonic treatment for 30min under the conditions of 250W power and 33kHz frequency, cooling, adding 60% methanol to the scale, and filtering with a microporous filter membrane (0.45 mu m) to obtain a filtrate, namely a test solution;
(3) respectively sucking 20 μ L of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring to obtain dehydroandrographolide content.
The chromatographic conditions of the step (3) are as follows: octadecylsilane chemically bonded silica is used as a filling agent; eluting with methanol-water (55: 45) as mobile phase; column temperature: 35 ℃; flow rate: 1.0 mL/min; the sample volume is 20 mu L; the detection wavelength was 250 nm. The number of theoretical plates is not less than 4000 in terms of dehydroandrographolide peak.
Test example 1 reproducibility test
A sample of 0.5g of Chuanwang anti-inflammatory tablet was prepared in 5 portions according to the test method of example 1, and the content of dehydroandrographolide in each portion was calculated according to the measurement method of example 1, and the results are shown in Table 1.
TABLE 1 reproducibility test
According to the detection results in the table 1, the detection method provided by the invention has better reproducibility.
Test example 2 sample stability test
Taking 0.5g of Chuanwang anti-inflammatory tablet sample, preparing sample solution according to the preparation method of the test solution, respectively placing for 0 hour, 4 hours, 8 hours, 12 hours and 24 hours, injecting into a liquid chromatograph, recording the content of dehydroandrographolide in the sample, calculating the RSD value, observing the stability of the sample, and the result is shown in table 2.
TABLE 2 sample stability test
According to the detection results in the table 2, the detection method provided by the invention has better sample stability.
Example 2 method for content of dehydroandrographolide in Chuanwang anti-inflammatory tablet
The method comprises the following steps:
(1) preparation of control solutions: precisely weighing appropriate amount of dehydroandrographolide control, and adding 60% methanol to obtain control solution containing dehydroandrographolide 15 μ g per 1 ml;
(2) preparation of a test solution: taking 0.5g of Chuanwang anti-inflammatory tablet sample and 4.0mg of stabilizer (arginine 3.0mg and vitamin C1.0mg), precisely weighing, placing in a 100mL measuring flask, adding 90mL of 60% methanol, carrying out ultrasonic treatment for 30min under the conditions of power of 250W and frequency of 33kHz, cooling, adding 60% methanol to the scale, and filtering with a microporous membrane (0.45 mu m), wherein the obtained filtrate is a sample solution;
(3) respectively sucking 20 μ L of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring to obtain dehydroandrographolide content.
The chromatographic conditions of the step (3) are as follows: octadecylsilane chemically bonded silica is used as a filling agent; eluting with methanol-water (55: 45) as mobile phase; column temperature: 35 ℃; flow rate: 1.0 mL/min; the sample volume is 20 mu L; the detection wavelength was 250 nm. The number of theoretical plates is not less than 4000 in terms of dehydroandrographolide peak.
Test example 3 reproducibility test
A sample of 0.5g of Chuanwang anti-inflammatory tablet was prepared in 5 portions according to the test method of example 2, and the content of dehydroandrographolide in each portion was calculated according to the measurement method of example 2, and the results are shown in Table 3.
TABLE 3 reproducibility test
According to the detection results in the above table 3, the detection method provided by the invention has good reproducibility.
Test example 4 sample stability test
Taking 0.5g of Chuanwang anti-inflammatory tablet sample, preparing sample solution according to the preparation method of the test solution, respectively placing for 0 hour, 4 hours, 8 hours, 12 hours and 24 hours, injecting into a liquid chromatograph, recording the content of dehydroandrographolide in the sample, calculating the RSD value, observing the stability of the sample, and the result is shown in Table 4.
TABLE 4 sample stability test
As can be seen from the test results in Table 4 above, the test method provided by the present invention is better in the sample stability test example 1.
Example 3 method for content of dehydroandrographolide in Chuanwang anti-inflammatory tablet
The method comprises the following steps:
(1) preparation of control solutions: precisely weighing appropriate amount of dehydroandrographolide control, and adding 60% methanol to obtain control solution containing dehydroandrographolide 15 μ g per 1 ml;
(2) preparation of a test solution: 0.5g of a sample of the inflammation-diminishing, detoxifying and queen-bee-cross anti-inflammatory tablet and 4.8mg of a stabilizer (arginine 3.2mg and vitamin C1.6mg) are precisely weighed, placed in a 100mL measuring flask, added with 90mL of 60% methanol, ultrasonically treated for 30min under the conditions of power of 250W and frequency of 33kHz, cooled, added with 60% methanol to the scale, and filtered by a microporous filter membrane (0.45 mu m), and the obtained filtrate is a sample solution;
(3) respectively sucking 20 μ L of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring to obtain dehydroandrographolide content.
The chromatographic conditions of the step (3) are as follows: octadecylsilane chemically bonded silica is used as a filling agent; eluting with methanol-water (55: 45) as mobile phase; column temperature: 35 ℃; flow rate: 1.0 mL/min; the sample volume is 20 mu L; the detection wavelength was 250 nm. The number of theoretical plates is not less than 4000 in terms of dehydroandrographolide peak.
Test example 3 reproducibility test
A sample of 0.5g of Chuanwang anti-inflammatory tablet was prepared in 5 portions according to the test method of example 3, and the content of dehydroandrographolide in each portion was calculated by the measurement method of example 3, and the results are shown in Table 5.
TABLE 5 reproducibility test
From the results of table 5 above, it can be seen that the reproducibility of the detection method provided by the present invention is better than that of example 1, but worse than that of example 2.
Test example 4 sample stability test
Taking 0.5g of Chuanwang anti-inflammatory tablet sample, preparing sample solution according to the preparation method of the test solution, respectively placing for 0 hour, 4 hours, 8 hours, 12 hours and 24 hours, injecting into a liquid chromatograph, recording the content of dehydroandrographolide in the sample, calculating the RSD value, observing the stability of the sample, and the result is shown in Table 6.
TABLE 6 sample stability test
From the results of table 6 above, it can be seen that the reproducibility of the detection method provided by the present invention is better than that of example 1, but worse than that of example 2.
Therefore, arginine and vitamin C which are used as stabilizing agents and have the mass ratio of 3:1 are added into the test solution, and the mass ratio of the added amount to the sample of the anti-inflammatory tablet for Chuanwang is 0.8:100, so that the stability of the sample can be improved, and the detection accuracy can be improved.
In conclusion, the detection method provided by the invention has good reproducibility and high stability, and provides a basis for detecting the content of the active ingredients of the traditional Chinese medicinal material or the pharmaceutical composition containing the dehydroandrographolide.
The present invention has been further described with reference to specific embodiments, which are only exemplary and do not limit the scope of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (4)
1. A method for detecting the content of dehydroandrographolide in Chuanwang anti-inflammatory tablets is characterized by comprising the following steps: the method comprises the following steps:
(1) preparation of control solutions: precisely weighing appropriate amount of dehydroandrographolide control, and adding 60% methanol to obtain control solution containing dehydroandrographolide 15 μ g per 1 ml;
(2) preparation of a test solution: taking 0.5g of Chuanwang anti-inflammatory tablet sample, precisely weighing, placing in a 100mL measuring flask, adding 90mL of 60% methanol, performing ultrasonic treatment for 30min, cooling, adding 60% methanol to scale, and filtering with a microporous membrane of 0.45 μm to obtain filtrate as sample solution;
(3) respectively sucking the reference solution and the test solution, injecting into a liquid chromatograph, and simultaneously measuring to obtain the content of dehydroandrographolide;
the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; and (2) adding the following components in a ratio of 55: 45 methanol-water is used as a mobile phase for elution; column temperature: 35 ℃; flow rate: 1.0 mL/min; the sample volume is 20 mu L; the detection wavelength is 250 nm; the number of theoretical plates is not less than 4000 calculated according to the peak of dehydroandrographolide;
the test solution in the step (2) also contains a stabilizer;
the mass ratio of the addition amount of the stabilizer to the Chuanwang anti-inflammatory tablet sample is 0.5-1: 100;
the stabilizer is a mixture of arginine and vitamin C in a mass ratio of 2-3: 1.
2. The content detection method according to claim 1, characterized in that: the ultrasonic conditions are as follows: the ultrasonic power is 250W, and the ultrasonic frequency is 33 kHz.
3. The content detection method according to claim 1, characterized in that: the stabilizer is a mixture of arginine and vitamin C in a mass ratio of 3: 1.
4. The content detection method according to claim 1, characterized in that: the Chuanwang antiphlogistic tablet comprises common andrographis herb.
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| CN103371972A (en) * | 2012-04-21 | 2013-10-30 | 苏州雷纳药物研发有限公司 | Andrographolide suspension and preparation method and medical application thereof |
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