CN110780007A - Method for evaluating 6 component contents of mango cough relieving tablet by HPLC (high performance liquid chromatography) method - Google Patents
Method for evaluating 6 component contents of mango cough relieving tablet by HPLC (high performance liquid chromatography) method Download PDFInfo
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- 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
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- 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
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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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Abstract
The invention discloses a method for evaluating the content of 6 components of mango cough relieving tablets by HPLC (high performance liquid chromatography), which adopts an HPLC method, wherein a chromatographic column is a Phenomenex Gemini C18 column (4.6mm multiplied by 250mm, 5 mu m); the mobile phase is acetonitrile (B) -0.1 percent phosphoric acid water solution (A); gradient elution; the flow rate is 1.0 mL/min; detection wavelength: 258nm, and the column temperature is 30 ℃; the method comprises the steps of taking gallic acid as an internal reference, respectively establishing Relative Correction Factors (RCF) of protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin, calculating the content of the RCF, and carrying out comparative analysis on the measured value and the measured result of an external standard method.
Description
Technical Field
The invention relates to the technical field of Chinese patent medicine compound preparation component detection, in particular to a method for measuring the content of 6 components of a multi-evaluation mango cough relieving tablet by an HPLC (high performance liquid chromatography) method.
Background
The mango cough relieving tablet is a Chinese and western medicine compound preparation prepared from mango leaf dry extract, sodium houttuyfonate and chlorphenamine maleate, has the effects of freeing lung, reducing phlegm, relieving cough and asthma, and is clinically used for treating cough, asthma, excessive phlegm and the like. Mango cough-relieving tablets have been widely used for a long time and show good clinical efficacy. The current quality standard is carried out by referring to national drug standard (2014) national standard No. 0154 issued by the State food and drug administration, which has only a simple physicochemical identification method and obviously cannot effectively control the quality of the preparation. Although some literature reports that mangiferin, sodium houttuyfonate and chlorphenamine maleate in mango cough tablets are separately measured by high performance liquid chromatography in the last decade exist, the components of a Chinese patent medicine compound preparation are complex, the quality and curative effect consistency of a single component are difficult to comprehensively control by quantitative measurement of the single component, and the measurement of multiple index components becomes the development trend of related quality evaluation; modern pharmacological research shows that flavonoids and phenolic acid compounds contained in mango leaves have obvious activities of relieving asthma, relieving cough, eliminating phlegm, immunizing, resisting tumor and the like, and are related components of the preparation in efficacy; therefore, the invention adopts the high performance liquid chromatography to research and establish a content determination method for a plurality of components such as protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin in the mango cough relieving tablet, and on the basis, a one-test multi-evaluation determination method for the 6 components is researched and established aiming at the problems that the contrast products such as mangiferin and homomangiferin in the preparation are high in price and high in detection cost.
Disclosure of Invention
The invention aims to provide a method for measuring and evaluating the contents of 6 components of mango cough relieving tablets by an HPLC (high performance liquid chromatography) method. The high performance liquid chromatography one-test-multiple evaluation method established by the invention is used for measuring the content of 6 components of protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin in mango cough tablets, is accurate and reliable, has good repeatability, has no obvious difference (P is more than 0.05) with the measurement result of an external standard method, and can provide reference for the quality control of the mango cough tablets.
In order to realize the purpose, the invention is realized by the following technical scheme:
a preparation method of the floral type camellia seed oil shower gel comprises the following steps:
a method for measuring and evaluating the contents of 6 components of mango cough relieving tablets by an HPLC method comprises the following steps:
(1) preparation of control solutions: respectively taking appropriate amount of gallic acid, protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin as reference substances, precisely weighing, adding 70% methanol for dissolving, and making into mixed reference substance solutions containing gallic acid, protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin with mass concentrations of 1.830mg/mL, 0.1010mg/mL, 2.613mg/mL, 0.3623mg/mL, 0.1554mg/mL and 0.07480mg/mL respectively for use;
(2) preparation of a test solution: taking 20 mango cough relieving tablets, removing sugar coating, precisely weighing the total tablet weight, grinding, taking 0.3g of powder, precisely weighing, precisely adding 25mL of 70% methanol, performing ultrasonic treatment for 40min, filtering, precisely adding 25mL of 70% methanol into residues, performing ultrasonic treatment for 40min, filtering, combining filtrates, volatilizing in water bath, dissolving the residues in 70% methanol, fixing the volume to a 10mL volumetric flask, shaking uniformly, centrifuging at 13000r/min for 10min, and taking supernatant as a sample solution;
(3) preparation of negative test solution: preparing a mango leaf-lacking negative sample according to a prescription process, taking a proper mango leaf negative sample, and preparing a mango leaf negative control solution according to the step (2);
(4) and (3) system adaptability test: respectively taking a mixed reference substance solution, a mango cough relieving tablet test sample solution and a mango leaf negative reference solution, and carrying out sample injection analysis and determination according to certain chromatographic conditions, wherein the number of theoretical plates is not less than 120000 calculated according to the hyperin peak;
(5) drawing a standard curve: precisely sucking a proper amount of the mixed reference substance solution prepared in the step (1), respectively placing the mixed reference substance solution in a 5mL volumetric flask, adding 70% methanol to dilute the mixed reference substance solution to a scale, shaking the mixed reference substance solution uniformly, respectively preparing 6 mixed reference substance solutions with different concentrations, taking the reference substance solution, carrying out sample injection of 5 mu L for analysis and determination according to a certain chromatographic condition, recording a peak area, and drawing a standard curve by taking the mass concentration (mu g/mL) as a horizontal coordinate (X) and the peak area as a vertical coordinate (Y);
(6) and (3) precision test: precisely sucking 1.25mL of the mixed reference solution prepared in the step (1) respectively, placing the mixed reference solution in a 5mL volumetric flask, diluting with 70% methanol to a scale, and shaking up; injecting sample according to certain chromatographic condition, continuously injecting sample for 6 times, and recording peak area and retention time;
(7) and (3) stability test: precisely weighing 0.3g of mango cough relieving tablet sample, preparing a sample solution according to the method in the step (2), carrying out sample injection analysis according to certain chromatographic conditions, carrying out sample injection respectively for 0h, 2h, 4h, 8h, 12h and 24h, and recording peak area and retention time;
(8) and (3) repeatability test: precisely weighing 6 parts of mango cough relieving tablet powder, wherein each part is 0.3g, preparing 6 parts of test solution according to the method in the step (2), injecting 5 mu L of sample according to certain chromatographic conditions, measuring and analyzing, and recording corresponding peak areas;
(9) sample adding and recovering test: precisely weighing 0.15g of mango cough relieving tablet powder with known component content, respectively precisely adding 3.00ml of mixed reference solution containing gallic acid, protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin, preparing the test solution according to the method in the step (2), and carrying out sample injection of 5 muL for analysis under certain chromatographic conditions.
Preferably, the chromatographic conditions in steps (4) to (9) are: a chromatographic column: phenomenex GeminiC18 column (4.6 mm. times.250 mm, 5 μm); acetonitrile is taken as a mobile phase B, and 0.1% phosphoric acid aqueous solution is taken as a mobile phase A; gradient elution (0-10 min, 5-7% of B, 10-20 min, 7-11% of B, 20-25 min, 11-13% of B, 25-40 min, 13-16% of B, 40-50 min, 16-20% of B, 50-60 min, 20-25% of B, 60-70 min, 25-35% of B, 70-75 min, 35% of B); the flow rate is 1.0 mL/min; the column temperature is 30 ℃; the sample injection amount is 5 mu L; detection wavelength: 258 nm.
Preferably, the mass concentrations of gallic acid, protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin in the mixed control solution in the step (9) are respectively as follows: 0.9536mg/mL, 0.0490mg/mL, 2.187mg/mL, 0.1300mg/mL, 0.0700mg/mL, 0.0500 mg/mL.
The invention has the beneficial effects that:
the invention adopts an HPLC method, and a chromatographic column is a Phenomenex Gemini C18 column (4.6mm multiplied by 250mm, 5 mu m); the mobile phase is acetonitrile (B) -0.1 percent phosphoric acid water solution (A); gradient elution; the flow rate is 1.0 mL/min; detection wavelength: 258nm, and the column temperature is 30 ℃; the method comprises the steps of taking gallic acid as an internal reference, respectively establishing Relative Correction Factors (RCF) of protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin, calculating the content of the RCF, and carrying out comparative analysis on the measured value and the measured result of an external standard method.
Drawings
FIG. 1 is an HPLC chromatogram of a mixed control solution (A), a mango cough relieving tablet test solution (B) and a mango leaf negative control solution (C), wherein 1, gallic acid; 2. protocatechuic acid; 3. mangiferin; 4. homomangiferin; 5. hyperin; 6. isoquercitrin.
Detailed Description
1. Experimental Material
1.1 Experimental reagent, medicine and medicinal material
The 11 batches of mango cough relievers were provided by the pharmaceutical industry of the university of traditional Chinese medicine of Guangxi (batch No.: 20161201, 20161202, 20170304, 20170401, 20170501, 20170901, 20180601, 20180602, 20180701, 20180702, 20181001). Gallic acid (batch: 110831-201605, the mass fraction is 90.8%), protocatechuic acid (batch: 110809-201205, the mass fraction is 99.9%) were purchased from China institute for testing and determining food and drug; homomangiferin (batch No. ST216701, purity not less than 98%), hyperoside (batch No. ST008801, purity not less than 98%), isoquercitrin (batch No. ST114401, purity not less than 98%) were purchased from Shanghai Shidande Standard technology Limited; mangiferin (batch No. wkq18050201, HPLC ≥ 98%) was purchased from Szechwan Vickqi Biotech limited. The mango leaf reference drug (batch number: 121507-. Acetonitrile (chromatographically pure, Fisher corporation); ultrapure water is self-made by a laboratory; methanol (analytically pure, metropolis chemicals, ltd.).
1.2 instruments
Agilent-1260 high performance liquid chromatograph including UV type ultraviolet detector, Agilent workstation (Agilent corporation, USA); waters e2695 hplc; model KQ5200B ultrasonic cleaner (kunshan ultrasonic instruments ltd); a Misibo super water purifier; electronic analytical balance (one hundred thousand, SQP, sydows scientific instruments ltd); TGL-16G centrifuge (Shanghai' an pavilion scientific instruments factory); HWS-26 model electric heating constant temperature water bath (Shanghai-Hengscientific instruments Co., Ltd.).
2. Method and results
2.1 chromatographic conditions
A chromatographic column: phenomenex Gemini C18 column (4.6 mm. times.250 mm, 5 μm); acetonitrile is taken as a mobile phase B, and 0.1% phosphoric acid aqueous solution is taken as a mobile phase A; gradient elution (0-10 min, 5-7% of B, 10-20 min, 7-11% of B, 20-25 min, 11-13% of B, 25-40 min, 13-16% of B, 40-50 min, 16-20% of B, 50-60 min, 20-25% of B, 60-70 min, 25-35% of B, 70-75 min, 35% of B); the flow rate is 1.0 mL/min; the column temperature is 30 ℃; the sample injection amount is 5 mu L; detection wavelength: 258 nm.
2.2 methodological observations and results
2.2.1 preparation of control solutions
Taking appropriate amount of gallic acid, protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin reference substances respectively, precisely weighing, adding 70% methanol for dissolving, and making into mixed reference substance solutions containing gallic acid, protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin with mass concentrations of 1.830mg/mL, 0.1010mg/mL, 2.613mg/mL, 0.3623mg/mL, 0.1554mg/mL and 0.07480mg/mL respectively for use.
2.2.2 preparation of test solutions
Taking 20 mango cough relieving tablets, removing sugar coating, precisely weighing the total tablet weight, grinding, taking 0.3g of powder, precisely weighing, precisely adding 25mL of 70% methanol, performing ultrasonic treatment for 40min, filtering, precisely adding 25mL of 70% methanol into residues, performing ultrasonic treatment for 40min, filtering, combining filtrates, volatilizing in water bath, dissolving the residues in 70% methanol, fixing the volume to a 10mL volumetric flask, shaking uniformly, centrifuging at 13000r/min for 10min, and taking supernatant as a sample solution.
2.2.3 preparation of negative test solutions
Preparing a mango leaf-lacking negative sample according to a prescription process, taking a proper mango leaf negative sample, and preparing a mango leaf negative control solution according to the item of 2.2.2.
2.2.4 systematic adaptability test
And respectively taking the mixed reference substance solution, the mango cough relieving tablet test sample solution and the mango leaf negative reference solution, and carrying out sample injection analysis and determination according to the chromatographic condition under the item of 2.1, wherein the experimental result shows that the mixed reference substance chromatogram and the sample chromatogram have corresponding chromatographic peaks at the same retention time, and the mango leaf negative reference solution has no chromatographic peak at the corresponding retention time. The theoretical plate number calculated by hyperin peak should be not less than 120000, and the result is shown in FIG. 1.
2.2.5 drawing of Standard Curve
Precisely sucking a proper amount of the mixed reference substance solution under the item of 2.2.1, respectively placing in 5mL volumetric flasks, adding 70% methanol for dilution to scale, shaking up, and respectively preparing 6 mixed reference substance solutions with different concentrations; taking the above control solution, injecting 5 μ L of the sample under the chromatographic condition of "2.1" for analysis and determination, and recording the peak area. The linear relationship of gallic acid, protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin obtained by drawing a standard curve with the mass concentration (μ g/mL) as abscissa (X) and the peak area as ordinate (Y) is shown in Table 1.
TABLE 1 Linear relationship of the components
2.2.6 precision test
Precisely sucking 1.25mL of the mixed reference solution under the item "2.2.1" respectively, placing in a 5mL volumetric flask, diluting with 70% methanol to scale, and shaking up. Sample introduction is carried out according to the chromatographic condition under the item of 2.1, sample introduction is carried out for 6 times continuously, peak areas and retention time are recorded, the calculated peak area RSD values of all the peaks are respectively 0.17%, 0.18%, 0.19%, 0.22%, 0.17% and 0.27%, and the RSD values of the retention time are respectively 0.90%, 1.0%, 0.58%, 0.49%, 0.55% and 0.50%, and the result shows that the precision of the instrument is good.
2.2.7 stability test
Precisely weighing 0.3g of mango cough relieving tablet sample (batch number: 20180601), preparing a sample solution according to the method under the item '2.2.2', carrying out sample injection analysis according to the chromatographic condition under the item '2.1', carrying out sample injection at 0h, 2h, 4h, 8h, 12h and 24h respectively, recording peak areas and retention time, and calculating to obtain the RSD values of the peak areas of 0.29%, 0.76%, 0.32%, 0.48%, 0.70% and 2.0% respectively, and the RSD values of the retention time are 0.27%, 0.34%, 0.31%, 0.34%, 0.32% and 0.29% respectively, wherein the result shows that the sample solution is stable within 24 h.
2.2.8 repeatability test
6 parts of mango cough relieving tablet (batch number: 20180601) powder is precisely weighed, 0.3g of each part is prepared into 6 parts of test solution according to the method under the item '2.2.2', 5 microliter of sample introduction is measured and analyzed according to the chromatographic condition under the item '2.1', corresponding peak areas are recorded, the average contents of gallic acid, protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin in 6 parts of samples are calculated to be 4.823 mg/tablet, 0.2269 mg/tablet, 10.89 mg/tablet, 1.198 mg/tablet, 0.3813 mg/tablet and 0.1836 mg/tablet respectively, the RSD values of the contents of the components measured in 6 parts of samples are respectively 0.39%, 0.33%, 0.59%, 0.50%, 1.7% and 2.3%, and the result shows that the experimental method has good repeatability.
2.2.9 sample recovery test
Precisely weighing 0.15g of mango cough relieving tablet powder (batch No. 20180601) with known component content, respectively precisely adding 6 parts, respectively precisely adding 3.00mL of mixed reference solution containing gallic acid (0.9536mg/mL), protocatechuic acid (0.0490mg/mL), mangiferin (2.187mg/mL), homomangiferin (0.1300mg/mL), hyperoside (0.0700mg/mL) and isoquercitrin (0.0500mg/mL), preparing a test solution according to the method under the '2.2' item, injecting 5 muL under the chromatographic condition of the '2.1' item, analyzing the sample, and obtaining the result shown in the table 2.
Table 2 recovery test results for each component (n ═ 6)
2.3 one-test-multiple-evaluation method
2.3.1 determination of the relative correction factors for different concentrations
Taking 0.5mL, 1.0, 2.0, 3.0 and 4.0mL of the mixed reference substance solution under the item of '2.2.1' to be respectively placed in a 5mL volumetric flask, diluting the mixed reference substance solution to a scale with 70% methanol, shaking up, adding the mixed reference substance solution under the item of '2.2.1' to obtain mixed reference substance solutions with 6 series of concentrations, carrying out analysis and determination according to the sample introduction condition of 5 mu L under the item of '2.1', and recording the peak area. Taking the gallic acid chromatographic peak as an internal standard peak (S), respectively calculating the relative correction factors of the gallic acid relative to protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin, and calculating that the RSD of each component relative to the correction factors is less than 2.5% under different concentrations, wherein the results are shown in Table 3.
Table 3 relative calibration factor determination for each component at different concentrations (n ═ 6)
2.3.2 determination of relative correction factors for different chromatography columns
Shim-pack CLC-ODS C18(6.0mmID 15cm) and Waters SunFire were examined by Agilent 1260 type HPLC
RC18 (4.6X 250mm, 5.0 μm), Phenomenex Gemini C18(4.6mm X250 mm, 5.0 μm)3 different pairs of columnsThe relative correction factors of other components are respectively calculated by taking the gallic acid chromatographic peak as an internal standard peak (S), and the RSD values of the components relative to the correction factors are less than 1.5 percent, and the results are detailed in Table 4. The measurement result shows that the relative correction factors measured by different chromatographic columns have no significant difference.
Table 4 results of relative calibration factor determination for different columns (n ═ 3)
2.3.3 determination of relative correction factors for different instruments
The experiment examined Shimadzu-pack CLC-ODS C18(6.0 mmID. times.15 cm) and WatersSunFire respectively
RInfluence of C18(4.6 × 250mm, 5.0 μm), Phenomenex Gemini C18(4.6mm × 250mm, 5.0 μm)3 different chromatographic columns on relative correction factors of each component on two different experimental instruments of Agilent 1260 type and Waters e2695 type high performance liquid chromatographs; the relative correction factors of other components were calculated using the gallic acid chromatogram peak as an internal standard peak (S), and the relative correction factor RSD values of the components were less than 3.0%, as detailed in Table 5. The experimental results show that the relative correction factors of 3 different chromatographic columns measured on two different instruments have no significant difference.
TABLE 5 relative calibration factor determination of various components for different columns and different instruments (n ═ 3)
2.3.4 calculation of relative correction factors for different column temperatures and flow rates
The experiment investigated the effect of different column temperatures (25, 30, 35 ℃) and different flow rates (0.80, 1.0, 1.2mL/min) on the relative correction factor, and the results showed that the corresponding RSD values of the RCFs of the components to be measured were all less than 1.5%, and the results are detailed in Table 6. The experimental result shows that the relative correction factors of all the components have no significant difference when measured at different chromatographic column temperatures and different flow rates.
TABLE 6 relative calibration factor measurements for different column temperatures and different flow rates (n ═ 3)
2.3.5 localization of chromatographic peaks
The premise that a one-test-multiple-evaluation method (QAMS) is used for simultaneously quantifying multiple index components is to accurately position chromatographic peaks of the components to be measured. The methods adopted in the prior documents [18-20] are the relative retention time positioning method or the retention time difference positioning method. In the experiment, the reproducibility of the relative retention time and the retention time difference of chromatographic peaks of components to be detected is respectively inspected by using 3 different chromatographic columns on Agilent 1260 type and Waters e2695 high performance liquid chromatographs; the experimental result shows that under the same chromatographic condition, the RSD values of the mangiferin, homomangiferin, hyperoside and isoquercitrin which are positioned by adopting the relative retention time and the retention time difference are all less than 5 percent, but the RSD values of the protocatechuic acid which is positioned by adopting the relative retention time and the retention time difference are all more than 5 percent, so that the positioning of the protocatechuic acid can only be positioned by adopting a qualitative comparison product, and the result is shown in tables 7 and 8.
TABLE 7 measurement of relative Retention time of Components for different columns and different instruments (n-3)
Table 8 difference in retention time for different components of different apparatus and different columns (n ═ 3)
2.3.6 comparison of results of one-test-multiple-test method and external standard method
11 batches of mango cough relieving tablets with different batches are taken, sample injection is carried out according to the chromatographic condition under the item of '2.1', the retention time and the peak area of each component to be measured are recorded, the measurement results of each component are calculated by an External Standard Method (ESM) and an established one-measurement-multiple-evaluation method (QAMS), the content measurement results of the two methods are compared, the difference of the content measurement results of the two methods is evaluated by a Relative Error (RE), the relative error of the content measurement results of the two methods is less than 4%, and the results are detailed in a table 9. The data measured by the two methods are subjected to independent sample t test analysis by SPSS22.0 software, the difference of the results measured by the two methods has no statistical significance (P is more than 0.05), and the one-test-multiple-evaluation method for simultaneously measuring 6 components in the mango cough relieving tablet established in the experiment is good in accuracy.
Results of 6-component one-test-multiple evaluation method and external standard method in table 911 batch mango cough relieving tablet sample (mg/tablet, n ═ 2)
2.4 discussion
Determination of optimal detection wavelength: the experiment carries out full-wavelength scanning by a Waters PDA detector, the result shows that each component to be detected has better absorption under 258nm, and the maximum absorption wavelength of mangiferin, homomangiferin, hyperoside and isoquercitrin is 258nm, so 258nm is selected as the measurement wavelength.
Determination of the test article preparation method: in the experiment, 7 extraction solvents of water, methanol, ethanol, 40% methanol, 70% methanol, 40% ethanol and 70% ethanol are investigated, and the result shows that the total extraction efficiency of each component by 70% methanol is better, so 70% methanol is selected as the extraction solvent; 3 extraction modes of standing, ultrasonic and refluxing are investigated, the difference of the extraction efficiency of the ultrasonic and refluxing is found to be small, but the ultrasonic extraction operation is simple, so the ultrasonic extraction method is selected in the experiment; examining the extraction time of 20min, 30 min, 40min, 50min and 60min, finding that the extraction efficiency of each component to be detected is not increased when the extraction time is 40min, examining the volume and the extraction times of different extraction solvents, finding that the extraction times are two, and when the volume of the solvent added each time is 25mL, each component is basically and completely extracted.
From the test data, the content of each component in different batches of mango cough relieving tablets is obviously different, wherein the content difference of mangiferin and high mangiferin is especially obvious. The lower content of mangiferin and homomangiferin in the first 5 batches from 2016 may be related to longer storage of the preparation, resulting in reduced content; or 11 bulk preparations are prepared by collecting the mango leaf crude drugs from different production places or in different seasons, so that the content difference between mangiferin and high mangiferin is large [22-23], the detection of the content of relevant index components of the raw materials is enhanced, and the extraction and production process parameters are strictly controlled.
Phenolic acid components and flavonoid components are important active substances of mango leaves, and have the effects of resisting tumors, resisting inflammation, relieving cough and asthma and the like. The gallic acid is a phenolic acid component with relatively high mango leaf content, has high content in mango cough relieving tablets and good stability, and meanwhile, a gallic acid reference product is cheap and easy to obtain. Therefore, the gallic acid is selected as the internal standard substance in the experiment, and the requirement of a one-test-multiple-evaluation method on the internal standard substance is met.
In the experiment, gallic acid is used as an internal standard substance, relative correction factors of protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin are measured, the influence of different column temperatures, flow rates and instrument chromatographic columns on the measurement result is respectively inspected, and the RSD of the result is less than 5%, which indicates that the correction factor established by the one-test-multiple-evaluation method is good in durability. The content determination results of 11 batches of mango cough relieving tablets show that the results measured by the one-test and multi-evaluation method have no significant difference from the results measured by the external standard method; the two groups of data are analyzed by SPSS22.0 software, and the difference has no statistical significance (P is more than 0.05), which indicates that the method has good accuracy, is feasible and has reliable results. The research provides a new method and thought for the quality control of mango cough relieving tablets.
Claims (3)
1. A method for measuring and evaluating the contents of 6 components of mango cough relieving tablets by an HPLC method is characterized by comprising the following steps:
(1) preparation of control solutions: respectively taking appropriate amount of gallic acid, protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin as reference substances, precisely weighing, adding 70% methanol for dissolving, and making into mixed reference substance solutions containing gallic acid, protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin with mass concentrations of 1.830mg/mL, 0.1010mg/mL, 2.613mg/mL, 0.3623mg/mL, 0.1554mg/mL and 0.07480mg/mL respectively for use;
(2) preparation of a test solution: taking 20 mango cough relieving tablets, removing sugar coating, precisely weighing the total tablet weight, grinding, taking 0.3g of powder, precisely weighing, precisely adding 25mL of 70% methanol, performing ultrasonic treatment for 40min, filtering, precisely adding 25mL of 70% methanol into residues, performing ultrasonic treatment for 40min, filtering, combining filtrates, volatilizing in water bath, dissolving the residues in 70% methanol, fixing the volume to a 10mL volumetric flask, shaking uniformly, centrifuging at 13000r/min for 10min, and taking supernatant as a sample solution;
(3) preparation of negative test solution: preparing a mango leaf-lacking negative sample according to a prescription process, taking a proper mango leaf negative sample, and preparing a mango leaf negative control solution according to the step (2);
(4) and (3) system adaptability test: respectively taking a mixed reference substance solution, a mango cough relieving tablet test sample solution and a mango leaf negative reference solution, and carrying out sample injection analysis and determination according to certain chromatographic conditions, wherein the number of theoretical plates is not less than 120000 calculated according to the hyperin peak;
(5) drawing a standard curve: precisely sucking a proper amount of the mixed reference substance solution prepared in the step (1), respectively placing the mixed reference substance solution in a 5mL volumetric flask, adding 70% methanol to dilute the mixed reference substance solution to a scale, shaking the mixed reference substance solution uniformly, respectively preparing 6 mixed reference substance solutions with different concentrations, taking the reference substance solution, carrying out sample injection of 5 mu L for analysis and determination according to a certain chromatographic condition, recording a peak area, and drawing a standard curve by taking the mass concentration (mu g/mL) as a horizontal coordinate (X) and the peak area as a vertical coordinate (Y);
(6) and (3) precision test: precisely sucking 1.25mL of the mixed reference solution prepared in the step (1) respectively, placing the mixed reference solution in a 5mL volumetric flask, diluting with 70% methanol to a scale, and shaking up; injecting sample according to certain chromatographic condition, continuously injecting sample for 6 times, and recording peak area and retention time;
(7) and (3) stability test: precisely weighing 0.3g of mango cough relieving tablet sample, preparing a sample solution according to the method in the step (2), carrying out sample injection analysis according to certain chromatographic conditions, carrying out sample injection respectively for 0h, 2h, 4h, 8h, 12h and 24h, and recording peak area and retention time;
(8) and (3) repeatability test: precisely weighing 6 parts of mango cough relieving tablet powder, wherein each part is 0.3g, preparing 6 parts of test solution according to the method in the step (2), injecting 5 mu L of sample according to certain chromatographic conditions, measuring and analyzing, and recording corresponding peak areas;
(9) sample adding and recovering test: precisely weighing 0.15g of mango cough relieving tablet powder with known component content, respectively precisely adding 3.00ml of mixed reference solution containing gallic acid, protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin, preparing the test solution according to the method in the step (2), and carrying out sample injection of 5 muL for analysis under certain chromatographic conditions.
2. The HPLC method for measuring the content of 6 components in mango cough relieving tablets is characterized in that the chromatographic conditions in the steps (4) to (9) are as follows: a chromatographic column: phenomenex Gemini C18 column (4.6 mm. times.250 mm, 5 μm); acetonitrile is taken as a mobile phase B, and 0.1% phosphoric acid aqueous solution is taken as a mobile phase A; gradient elution (0-10 min, 5-7% of B, 10-20 min, 7-11% of B, 20-25 min, 11-13% of B, 25-40 min, 13-16% of B, 40-50 min, 16-20% of B, 50-60 min, 20-25% of B, 60-70 min, 25-35% of B, 70-75 min, 35% of B); the flow rate is 1.0 mL/min; the column temperature is 30 ℃; the sample injection amount is 5 mu L; detection wavelength: 258 nm.
3. The HPLC method for measuring and evaluating the contents of 6 components in mango cough relieving tablets according to claim 1, wherein the mixed control solution in step (9) contains gallic acid, protocatechuic acid, mangiferin, homomangiferin, hyperoside and isoquercitrin in the following concentrations by mass: 0.9536mg/mL, 0.0490mg/mL, 2.187mg/mL, 0.1300mg/mL, 0.0700mg/mL, 0.0500 mg/mL.
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