CN113655166A - High performance liquid detection method for 14 components in golden flower refreshing granules - Google Patents
High performance liquid detection method for 14 components in golden flower refreshing granules Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Abstract
The invention provides a high performance liquid detection method for simultaneously determining 14 components in Jinhuaqinggan granules, which adopts high performance liquid chromatography to simultaneously determine the content of 14 components in the Xinjiang chlorogenic acid, loganin, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonoside, baicalein, chrysin and oroxylin in the Jinhuaqinggan granules, and comprehensively evaluates the quality of the Jinhuaqinggan granules by taking multiple components as indexes, and specifically comprises the following steps: preparing a test solution, preparing a mixed reference solution and detecting conditions by high performance liquid chromatography. The method is simple, convenient and quick, has stable and reliable detection results, and can be applied to quality control evaluation of the golden flower refreshing particles.
Description
One, the technical field
The invention relates to a detection method for simultaneously determining 14 components in Jinhuaqinggan granules by high performance liquid chromatography, belonging to the technical field of quality control detection methods of traditional Chinese medicines.
Second, background Art
The golden flower influenza-clearing formula is prepared by rapidly establishing an expert committee for preventing and treating influenza A H1N1 by the traditional Chinese medicine administration in Beijing City after a confirmed cases of influenza A appear in 2009 in China, determining the anti-influenza activity traditional Chinese medicine compound golden flower influenza-clearing formula according to the characteristics of the influenza A in China through the practical results of expert experience, activity screening and clinical feedback, wherein the golden flower influenza-clearing formula is derived from Maxingshigan decoction and Yinqiao powder and is transferred to a museum of traditional Chinese medicine in Beijing Imperial Tang in 2010. The Jinhuaqinggan granule, namely the Jinhuaqinggan prescription, obtains a national new drug certificate and a license on the market in 2016, is produced by Fuchang pharmaceutical Co., Ltd at present and consists of 12 traditional Chinese medicines of mix-fried ephedra herb, rhizoma anemarrhenae, sweet wormwood herb, gypsum, honeysuckle flower, baical skullcap root, fried bitter almond, weeping forsythia, mint, thunberg fritillary bulb, great burdock achene and liquorice. The golden flower cold-clearing granules have the effects of dispelling wind, dispersing lung qi, clearing heat and removing toxicity, are used for fever caused by exogenous seasonal pathogens, light or no aversion to cold, sore throat, nasal obstruction, watery nasal discharge, thirst, red tongue, thin and yellow fur and rapid pulse, are clinically used for the patients with the symptoms caused by various influenza including influenza A H1N1, and are recommended to be used for patients in medical observation period in the diagnosis and treatment scheme of new coronary pneumonia.
At present, related researches on Jinhuaqinggan granules mainly focus on clinical evaluation of the Jinhuaqinggan granules for treating influenza and new coronary pneumonia, and main chemical components and related mechanisms of the Jinhuaqinggan granules for exerting the drug effects are analyzed through network pharmacology, and the researches on quality control are rarely reported. The quality of the medicine is directly related to the safety and the effectiveness of the medicine, and research on a quality control detection method technology of the Jinhuaqinggan granules is necessary.
Third, the invention
The invention aims to solve the technical problem of providing a high-efficiency liquid phase detection method for simultaneously detecting 14 components in golden flower refreshing granules, and overcomes the defects of the prior art so that the detection result is more comprehensive, accurate and reliable.
In order to solve the technical problem, the invention provides a high performance liquid detection method for simultaneously detecting 14 components in Jinhuaqinggan granules, which is used for simultaneously measuring the content of 14 components in the Jinhuaqinggan granules, namely neochlorogenic acid, loganin, caffeic acid, swertiamarin, forsythiaside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonoside, baicalin, chrysin and oroxylin.
Preferably, the high performance liquid detection method for simultaneously detecting 14 components in the golden flower refreshing granules comprises the following steps:
(1) preparation of a test solution: taking golden flower refreshing particles, crushing, precisely weighing golden flower refreshing particle powder, placing the golden flower refreshing particle powder into a conical flask with a plug, precisely adding an alcohol solution, weighing the total weight of the golden flower refreshing particles and the alcohol solution added into the conical flask with the plug, carrying out ultrasonic extraction for 30-60 min, cooling to 25 ℃, weighing again, adding the alcohol solution to supplement the loss amount of the golden flower refreshing particles, shaking uniformly, taking supernate, passing through a 0.22 mu m microporous filter membrane, and placing subsequent filtrate into a brown liquid phase sample feeding bottle to obtain a golden flower refreshing particle sample solution; wherein the ratio of the mass of the golden flower refreshing granular powder to the volume of the alcoholic solution is 1 g: 100ml-900 ml;
(2) preparation of 14 mixed control solutions: precisely weighing chlorogenic acid, loganin, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonoside, baicalein, chrysin and oroxylin reference substances respectively, and adding methanol to obtain single reference substance stock solution containing 14 components; precisely sucking the 14 stock solutions in sequence, placing in a 50mL volumetric flask, adding methanol to constant volume, shaking to obtain neochlorogenic acid, loganin, chlorogenic acid, caffeic acid, sweroside, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonoside, baicalein, chrysin, oroxylin with reference quality concentrations of 20.50, 14.76, 34.66, 10.01, 50.15, 50.11, 28.09, 194.45, 20.17, 178.13, 64.79, 10.14, 10.45 and 10.45 μ g/mL respectively-1Mixed reference solution of (1);
(3) color(s)Testing the spectrum condition and the system applicability: the chromatographic column adopts C taking octadecylsilane chemically bonded silica as a filler18A column; the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid aqueous solution, gradient elution is adopted, the flow rate is 1.0mL/min, the column temperature is 25-35 ℃, the sample injection amount is 10 mu L, the detection wavelength is 220-240 nm, the chromatographic analysis time is 95min, the theoretical plate number is not less than 3000 according to the calculation of respective reference peak, and the gradient elution procedure is as follows:
0-5 min, wherein the mobile phase A is 8% → 8%, and the mobile phase B is 92% → 92%;
5-30 min, wherein the mobile phase A is 8% → 18%, and the mobile phase B is 92% → 82%;
30-40 min, wherein the mobile phase A is 18% → 20%, and the mobile phase B is 82% → 80%;
40-60 min, 20% → 22% of mobile phase A and 80% → 78% of mobile phase B;
60-75 min, wherein the mobile phase A is 22% → 35%, and the mobile phase B is 78% → 65%;
75-90 min, 35% → 45% of mobile phase A and 65% → 55% of mobile phase B;
90-95 min, wherein the mobile phase A is 45% → 47%, and the mobile phase B is 55% → 53%;
wherein the proportions of the mobile phases A and B are volume percent;
(4) and (3) determination: respectively absorbing 10 mu L of the golden flower refreshing particle sample solution in the step (1) and 10 mu L of the mixed reference substance solution in the step (2), injecting the mixture into a high performance liquid chromatograph, operating according to the chromatographic conditions in the step (3), measuring respective chromatograms, calculating peak areas, measuring 14 components of neochlorogenic acid, loganin, chlorogenic acid, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonin, baicalein, chrysin and oroxylin in the golden flower refreshing particle sample solution according to an external standard one-point method, and calculating the contents of the neochlorogenic acid, the loganin, the chlorogenic acid, caffeic acid, the swertiamarin, the forsythoside A, the apigenin-7-O-beta-D-glucopyranoside and the like in the golden flower refreshing particle, Baicalin, phillyrin, arctiin, wogonoside, baicalein, chrysin and oroxylin.
Preferably, the alcoholic solution used for ultrasonic extraction of the golden flower refreshing particles in the step (1) is a methanol solution with the volume percentage concentration of 50-70%, and the ultrasonic extraction time of the golden flower refreshing particles is 30 min.
Preferably, the ratio of the mass of the golden flower refreshing feeling particle powder to the volume of the alcoholic solution in the step (1) is 1 g: 200ml of
Preferably, the concentration of the mobile phase B phosphoric acid aqueous solution in the step (3) is 0.05-0.3% by volume.
Preferably, the concentration of the mobile phase B phosphoric acid aqueous solution in the step (3) is 0.1 percent by volume.
Preferably, the column temperature in the step (3) is 30 ℃, and the detection wavelength is 230 nm.
The beneficial effects of the invention at least comprise:
1. the high performance liquid chromatography method for detecting 14 components in the golden flower refreshing granules adopts the high performance liquid chromatography method to detect 3 organic acid components in the golden flower refreshing granules: chlorogenic acid, caffeic acid; 2 iridoid components: loganin, swertiamarin; 3 lignan components: forsythoside A, phillyrin and arctiin; 6 flavonoid components: 14 components of 4 major components, namely apigenin-7-O-beta-D-glucopyranoside, baicalin, wogonoside, baicalein, chrysin and oroxylin, are subjected to content measurement simultaneously, and the internal quality of the golden flower refreshing granules can be comprehensively evaluated on the whole by taking multiple components as indexes;
2. the method for determining the content of 14 components in the golden flower refreshing granules by using the high performance liquid chromatography is simple and convenient to operate, high in sensitivity, stable and reliable, and can be used as a conventional method for detecting the quality of the golden flower refreshing granules;
3. the quality detection method for the golden flower refreshing granules can be used for evaluating the quality of a final product or an intermediate, and has important significance for monitoring and managing the production process.
Drawings
FIG. 1 is a high performance liquid chromatogram of a mixed control of 14 kinds of neochlorogenic acid, loganin, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, forsythin, arctiin, wogonoside, baicalin, chrysin and oroxylin in example 1;
FIG. 2 is a high performance liquid chromatogram of a test solution of the Gossypium aurantiacum particles in example 1;
FIG. 3 is a high performance liquid chromatogram of an air-white solvent in example 1;
FIG. 4 is a high performance liquid chromatogram of the mixed control detection wavelength screen of example 3;
FIG. 5 is a high performance liquid chromatogram from a 60min gradient elution procedure for test set 1 in example 4;
FIG. 6 is a high performance liquid chromatogram of the 65min gradient elution procedure of test set 2 in example 4;
FIG. 7 is a high performance liquid chromatogram of the 70min gradient elution procedure of test set 3 in example 4;
FIG. 8 is a high performance liquid chromatogram of the 75min gradient elution procedure of test set 4 in example 4;
FIG. 9 is a high performance liquid chromatogram of the 80min gradient elution procedure of test set 5 in example 4;
FIG. 10 is a high performance liquid chromatogram of the 85min gradient elution procedure of test set 6 in example 4;
FIG. 11 is a high performance liquid chromatogram of the 90min gradient elution procedure of test group 7 of example 4;
FIG. 12 is a high performance liquid chromatogram of the 110min gradient elution procedure of test group 8 of example 4;
FIG. 13 is a high performance liquid chromatogram of ephedrine hydrochloride reference at a detection wavelength of 207 nm;
FIG. 14 is a high performance liquid chromatogram of a pseudoephedrine hydrochloride control at a detection wavelength of 207 nm;
FIG. 15 is a high performance liquid chromatogram measured at a detection wavelength of 207nm of the test sample of the Gossypium aureus Rongqi particles in example 1;
FIG. 16 is a high performance liquid chromatogram of ephedrine hydrochloride reference at 257nm wavelength;
FIG. 17 is a high performance liquid chromatogram of pseudoephedrine hydrochloride control at 257nm wavelength;
FIG. 18 is a high performance liquid chromatogram measured at 257nm of the detection wavelength of the sample of the phlogistic particles in example 1.
Fourth, detailed description of the invention
The following describes the quality detection method of the golden flower refreshing granules in detail with reference to specific examples, but the scope of the invention is not limited thereto.
First, analysis of detection method
Example 1
The embodiment of the quality detection method of the golden flower refreshing particles comprises the following specific steps:
experimental materials and reagents: golden flower refreshing granules (specification: 5 g/bag) 10 batches with the batch numbers of 20200106, 20200114, 20200239, 20200601, 20200909, 20201005, 20201008 and 20201101 in sequence, which are all purchased from Fuchang (Beijing) pharmaceutical Co Ltd (manufacturing enterprise); the batches are 200501 and 200504 in sequence, are purchased from Tianshili pharmaceutical group, Inc. (manufacturing enterprise), and 10 batches of golden flower refreshing granules are respectively numbered as J1-J10. Loganin acid reference (purity 97.5%, batch No. 111865-202005), chlorogenic acid reference (purity 96.1%, batch No. 110753-202018), caffeic acid reference (purity 99.7%, batch No. 110885-201703), forsythoside A (purity 97.2%, batch No. 111810-201707), forsythin reference (purity 94.9%, batch No. 110821-202117), wogonoside reference (purity 98.5%, batch No. 112002-201702), baicalein reference (purity 97.9%, batch No. 111595-201808), chrysin reference (purity 98%, batch No. 111701-200501), ephedrine hydrochloride (purity 100%, batch No. 171241-201809), pseudoephedrine hydrochloride (purity 99.80%, batch No. 1712015237 201510) were purchased from the Chinese drug research institute. Neochlorogenic acid (purity: 98% or more, batch No. M07GB140938), swertiamarin (purity: 98% or more, batch No. P25O10F101344), apigenin-7-O- β -D-glucopyranoside (purity: 98% or more, batch No. Y29M10H84489), baicalin (purity: 98% or more, batch No. P20A9F59353), arctiin (purity: 98% or more, batch No. R12O8F45507), oroxylin (purity: 98% or more, batch No. Y03M11H109208) were purchased from shanghai source leaf biotechnology limited. Methanol, acetonitrile (chromatographic grade, Fisher corporation, usa); phosphoric acid (super grade pure GR, chemical reagents of national drug group, Inc.); isopropanol (chromatographically pure, Shandong Yuwang and Tianxia New materials Co., Ltd.); the ultrapure water is self-made in a laboratory (a series of ultra pure water machines of Yopu).
Preparing a test solution: taking honeysuckle clear-feeling particles, crushing, taking about 0.10g of honeysuckle clear-feeling particle powder, precisely weighing, placing in a conical flask with a plug, adding 20mL of 50% methanol, weighing, carrying out ultrasonic treatment for 30min to dissolve the particles, cooling, weighing again, adding 50% methanol to complement the loss amount, shaking uniformly, filtering by using a 0.22 mu m microporous membrane, and placing in a brown liquid phase sample bottle to obtain the honeysuckle clear-feeling particle.
Preparation of 14 mixed control solutions: precisely weighing chlorogenic acid, loganin, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonoside, baicalein, chrysin and oroxylin reference substances respectively, and dissolving with methanol to obtain single reference substance stock solution containing 14 components; precisely sucking the 14 stock solutions in sequence, placing in a 50mL volumetric flask, adding methanol to constant volume, shaking to obtain neochlorogenic acid, loganin, chlorogenic acid, caffeic acid, sweroside, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonoside, baicalein, chrysin, oroxylin with reference quality concentrations of 20.50, 14.76, 34.66, 10.01, 50.15, 50.11, 28.09, 194.45, 20.17, 178.13, 64.79, 10.14, 10.45 and 10.45 μ g/mL respectively-1Mixed control solution of (4).
Chromatographic conditions and system applicability test: the chromatographic column adopts Agilent-Eclipse XDB-C18Chromatography column (4.6 mm. times.250 mm,5 μm); acetonitrile (A) -0.1% phosphoric acid water (B) is used as a mobile phase, gradient elution is carried out, the flow rate is 1.0mL/min, the column temperature is 30 ℃, the sample injection amount is 10 mu L, the detection wavelength is 230nm, the chromatographic analysis time is 95min, and the number of theoretical plates is not less than 3000 according to the calculation of respective control peak.
And (3) determination: precisely absorbing 10 mu L of each of the test solution and the mixed reference solution, respectively, injecting into a liquid chromatograph, and detecting according to the gradient elution conditions in Table 1 to obtain chromatograms shown in figures 1-3, wherein figure 1 is a mixed reference high-performance liquid chromatogram comprising 14 components of neochlorogenic acid, loganin, chlorogenic acid, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonoside, baicalein, chrysin and oroxylin, figure 2 is a high-performance liquid chromatogram of the test sample of the Jinhuaqinggan granule, and figure 3 is a high-performance liquid chromatogram of a blank solvent.
TABLE 1 gradient elution schedule
Example 2
The method for extracting the golden flower refreshing particles adopts an ultrasonic extraction method, inspects the influence of three factors of solvent types, solvent volumes and ultrasonic time on the extraction efficiency of 14 chemical components, measures according to the chromatographic conditions of the example 1, and records the peak area.
Inspection of five extraction solvents: respectively and precisely weighing five parts of golden flower refreshing granules, namely 0.0989g, 0.1051g, 0.1080g, 0.0989g and 0.0995g, respectively adding pure water, 30% methanol, 50% methanol, 70% methanol and 50mL of pure methanol, respectively, carrying out ultrasonic treatment for 30min to dissolve the granules, filtering the granules by using a 0.22 mu m microporous filter membrane, respectively injecting 10 mu L of the granules into a liquid chromatograph, and detecting according to the gradient elution conditions in the table 1 to obtain the total content of 14 components, wherein the result is shown in the table 2.
TABLE 2 test results of five extraction solvents (mg. g)-1)
Investigation of the volume of seven extraction solvents: respectively and precisely weighing 0.1018g, 0.1009g, 0.1028g, 0.1022g, 0.1022g, 0.1024g and 0.1023g of the golden flower refreshing granules, respectively adding 10mL, 15mL, 20mL, 30mL, 50mL, 70mL and 90mL of 50% methanol, respectively, carrying out ultrasonic treatment for 30min to dissolve the granules, filtering the granules by using a 0.22 mu m microporous filter membrane, respectively injecting 10 mu L of the granules into a liquid chromatograph, and detecting according to the gradient elution conditions in the table 1 to obtain the total content of 14 components, wherein the result is shown in the table 3.
TABLE 3 volume test results (mg. g) for seven extraction solvents-1)
Investigation of five different ultrasonic times: respectively and precisely weighing 0.1002g, 0.1009g, 0.1008g, 0.1009g and 0.1003g of golden flower refreshing granules, adding 30mL of 50% methanol, performing ultrasonic treatment for 20min, 30min, 45min, 60min and 90min respectively, filtering by using a 0.22-micron microporous filter membrane, and injecting 10 mu L of the mixture into a liquid chromatograph to perform detection according to the gradient elution conditions in the table 1 to obtain the total content of 14 components, wherein the result is shown in the table 4.
TABLE 4 five ultrasonic time test results (mg. g)-1)
In conclusion, according to the investigation results of different factors influencing the extraction efficiency of the golden flower refreshing granules, the finally determined sample extraction method is to add 20mL of 50% methanol into 0.10g of the golden flower refreshing granules, perform ultrasonic extraction for 30min, and filter to obtain the golden flower refreshing granules.
Example 3
Scanning a control solution at full wavelength of an ultraviolet detector, comprehensively considering ultraviolet absorption wavelengths of neochlorogenic acid, loganin, chlorogenic acid, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, forsythin, arctiin, wogonin, baicalein, chrysin and oroxylin, and comparing the numbers of chromatographic peaks and peak areas at 230nm, 246nm, 280nm and 330nm respectively, the result shows that the control solution has good absorption at 230nm and a stable baseline, so 230nm is selected as the detection wavelength, and the corresponding chromatogram is shown in figure 4.
Example 4
Test objects: the golden flower refreshing particle sample solution (as a control group) is injected into a liquid chromatograph, then different gradient elution programs are adopted, and other steps and specific parameters are the same as those in the example 1:
table 5 test group 1 gradient elution schedule
table 6 test group 2 gradient elution schedule
Test group 3 differs from example 1 in that: the sample of JINHUAQINGGAN granule is 0.30g, the gradient elution procedure is shown in Table 7, and the corresponding chromatogram is shown in FIG. 7;
table 7 test group 3 gradient elution schedule
Test group 4 differs from example 1 in that: the sample of JINHUAQINGGAN granule is 0.30g, the gradient elution procedure is shown in Table 8, and the corresponding chromatogram is shown in FIG. 8;
table 8 test group 4 gradient elution schedule
TABLE 9 test group 5 gradient elution schedule
Test group 6 differs from example 1 in that: the sample of JINHUAQINGGAN granule is 0.30g, the gradient elution procedure is shown in Table 10, and the corresponding chromatogram is shown in FIG. 10;
TABLE 10 test group 6 gradient elution schedule
Test group 7 differs from example 1 in that: the sample of JINHUAQINGGAN granule is 0.30g, the gradient elution procedure is shown in Table 11, and the corresponding chromatogram is shown in FIG. 11;
table 11 test set 7 gradient elution schedule
table 12 test set 8 gradient elution schedule
Wherein, the proportion of A and B is volume percentage.
The test method comprises the following steps: the test was carried out according to the test method of example 1;
and (3) testing results: compared with fig. 2, the arctiin peak areas in fig. 6, fig. 7, fig. 8, fig. 10 and fig. 11 are all higher than the baicalin peak area in the same figure, probably because of the poor separation effect thereof, and the peak areas are increased due to the overlapping of the multiple peaks. In short, the chromatographic peak separation degree of the corresponding index component in each test group of fig. 5 to 12 is not satisfactory. Therefore, a large number of tests show that the detection method of the example 1 is the best quality detection method of the golden flower refreshing particles.
Second, methodology verification
(I) specificity test
According to the detection method of example 1, respectively and precisely absorbing 10 μ L of each of the test solution and the mixed reference solution, injecting the test solution into a liquid chromatograph, and detecting the test solution according to the gradient elution conditions in table 1 to obtain chromatograms shown in fig. 1-3, wherein fig. 1 is a mixed reference high-performance liquid chromatogram of 14 components including neochlorogenic acid, loganin, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonoside, baicalin, chrysin and oroxylin, fig. 2 is a high-performance liquid chromatogram of the test sample of the golden flower Qinggan granules, and fig. 3 is a high-performance liquid chromatogram of a solvent. As can be seen from the figure, corresponding chromatographic peaks are arranged at the same retention time of the reference chromatogram and the test chromatogram, each component to be measured and adjacent components can achieve baseline separation, and the negative sample solution is free of interference, which indicates that the method has good specificity and meets the content measurement requirement.
(II) examination of Linear relationship
According to the detection method of the embodiment 1, a proper amount of a mixed reference substance is precisely absorbed, methanol is added for dilution to obtain a mixed reference substance solution with a series of gradient concentrations, a chromatogram is measured, and the peak area is recorded. Linear regression was performed with the mass concentration of the control solution as abscissa (X) and the peak area as ordinate (Y) to obtain linear regression equations for 14 components, as shown in table 13. As a result, it was found that the components were in good linear relationship in the respective concentration ranges.
(III) examination of detection and quantitation limits
According to the detection method of the embodiment 1, a proper amount of mixed reference substance solution is precisely absorbed, methanol is added for equal-time gradual dilution, 6 times of sample injection detection are respectively carried out under different dilution times, the detection limit of each component is obtained by calculating according to the signal-to-noise ratio S/N which is 3, and the quantification limit of each component is obtained by calculating according to the signal-to-noise ratio S/N which is 10. The results are shown in Table 13.
TABLE 1314 Linear relationship examination, detection limits and quantitation limits for the ingredients
(IV) precision test
According to the detection method of example 1, 10. mu.L of the mixed control solution is precisely sucked, and the peak area is recorded after 6 times of continuous sample injection. The RSD values of the peak areas of the neochlorogenic acid, the loganin, the chlorogenic acid, the caffeic acid, the swertiamarin, the forsythoside A, the apigenin-7-O-beta-D-glucopyranoside, the baicalin, the forsythin, the arctiin, the wogonoside, the baicalein, the chrysin and the oroxylin are respectively 0.43 percent, 0.38 percent, 0.42 percent, 0.31 percent, 0.52 percent, 0.21 percent, 0.20 percent, 0.30 percent, 0.24 percent, 0.20 percent, 0.22 percent, 0.29 percent and 0.48 percent which are all less than 3 percent, and the instrument precision is good.
(V) stability test
According to the detection method of example 1, about 0.10g of golden flower refreshing granules (batch number: 20200106) powder is precisely weighed, sample solutions are prepared, sample injection measurement is carried out for 0, 2, 4, 8, 12 and 24 hours respectively, and peak areas are recorded. The results show that the RSD values of the peak areas of the neochlorogenic acid, the loganin, the chlorogenic acid, the caffeic acid, the swertiamarin, the forsythin A, the apigenin-7-O-beta-D-glucopyranoside, the baicalin, the phillyrin, the arctiin, the wogonoside, the baicalein, the chrysin and the oroxylin are respectively 0.87%, 1.69%, 0.08%, 2.26%, 0.40%, 0.21%, 0.42%, 0.10%, 2.18%, 0.05%, 0.19%, 2.57%, 2.09% and 2.74%, and the RSD values are all less than 3%, which indicates that the stability of the test solution is good within 24 hours at room temperature.
(VI) repeatability test
According to the detection method of example 1, about 0.10g of golden flower refreshing granules (lot: 20200106) powder is precisely weighed, 6 parts of test solution is prepared in parallel, and the sample injection determination and peak area recording are carried out. The results show that the RSD values of the contents of neochlorogenic acid, loganin, chlorogenic acid, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonoside, baicalein, chrysin and oroxylin are respectively 0.48%, 1.65%, 0.13%, 2.28%, 0.59%, 0.48%, 0.50%, 0.26%, 1.99%, 0.15%, 0.36%, 1.72%, 1.95% and 2.15%, which indicates that the method has good repeatability.
(VII) sample application recovery test
According to the detection method of example 1, about 0.05g of powder of known content of JINHUAQINGGAN granule (lot: 20200106) is precisely weighed, 9 parts of test solution are prepared in parallel, and 16.40 μ g/mL of neochlorogenic acid is added-13.41. mu.g/mL of loganine acid-1Chlorogenic acid 8.52. mu.g/mL-1Caffeic acid 2.34. mu.g/mL-1Swertiamarin 22.36 mu g/mL-1Forsythoside A25.01 μ g/mL-1apigenin-7-O-beta-D-glucopyranoside 12.72 mu g/mL-1152.10 mug/mL of baicalin-1Forsythiaside 4.18. mu.g/mL-1And 202.00 mug/mL of arctiin-129.68 μ g/mL wogonoside-13.90. mu.g/mL of baicalein-12.20. mu.g/mL of chrysin-1Oroxylin 2.09. mu.g/mL-1The mixed reference substance solution of (1) is prepared into a test solution, the sample injection is measured, the peak area is recorded, the sample injection recovery rate and the RSD value are calculated, and the calculation result is shown in a table 14.
TABLE 14 sample recovery test for 14 ingredients in JINHUAQINGGAN granule (n ═ 9)
(VIII) determination of sample content
Golden flower refreshing granules (specification: 5 g/bag) 10 batches with the batch numbers of 20200106, 20200114, 20200239, 20200601, 20200909, 20201005, 20201008 and 20201101 in sequence, which are all purchased from Fuchang (Beijing) pharmaceutical Co Ltd (manufacturing enterprise); the batches are 200501 and 200504 in sequence, are purchased from Tianshili pharmaceutical group, Inc. (manufacturing enterprise), and 10 batches of golden flower refreshing granules are respectively numbered as J1-J10. The content of 14 components was measured for 10 batches of the bought golden flower refreshing granules according to the test method of example 1, and the results are shown in table 15.
Table 15 measurement results (mg g) of 14 components in the Jinhua Qinggan granules-1，n＝3)
The content ranges of neochlorogenic acid, loganin, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonoside, baicalein, chrysin and oroxylin in 10 batches of the golden flower refreshing granules are respectively 1.19-1.42、0.20～0.37、6.51～7.72、0.20～0.25、2.12～2.80、1.75～2.46、0.89～1.14、14.41～16.58、0.41～0.64、15.85～20.01、2.80～3.44、0.38～0.53、0.02～0.03、0.07～0.11mg·g-1In the method, the HPLC chromatograms of 10 batches of samples have high similarity, 14 components such as neochlorogenic acid and loganine acid are common components of different batches of golden flower refreshing granules, and the content of the component to be detected in each batch of samples has small difference and can be related to the source of medicinal materials and the stability of the production process.
The chemical components of the golden flower refreshing granules mainly comprise flavonoids, saponins, iridoids, lignans, organic acids and volatile oils, and the components interact with each other to jointly exert the activities of sweating, clearing heat, resisting bacteria, resisting viruses and the like. In the early research, the contents of the golden flower refreshing granules are determined and researched by taking main effective components, such as ephedrine hydrochloride, pseudoephedrine hydrochloride, neochlorogenic acid, loganin, chlorogenic acid, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, luteolin, baicalin, forsythiaside, forsythin, arctiin, wogonin, baicalin, chrysin and oroxylin, which have the functions of resisting virus, resisting inflammation and regulating immunity, in the prescription medicine of the golden flower refreshing granules as determination indexes. The results in FIGS. 13-18 show that the ephedrine hydrochloride and pseudoephedrine hydrochloride contents in the samples are lower; the content of the luteolin is low, the separation degree of the forsythiaside under the detection method can not meet the requirement of Chinese pharmacopoeia, the luteolin and the forsythiaside are not suitable to be used as quantitative indexes because the luteolin and the forsythiaside can not be detected in the chromatogram of a test sample or the error of the measurement result is large due to the low content, and a high performance liquid chromatogram of the luteolin and the forsythiaside is not shown. Therefore, through methodology investigation, a high performance liquid detection method for simultaneously measuring 14 components including neochlorogenic acid, loganin, chlorogenic acid, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, forsythin, arctiin, wogonin, baicalein, chrysin and oroxylin is established, and multiple groups are selected as indexes to carry out quantitative quality control, so that the inherent quality of the golden flower refreshing granules can be comprehensively and scientifically evaluated.
The chemical components in the golden flower refreshing granules are complex, 14 components have wide polarity ranges, four acids with different concentrations, namely methanol-water, acetonitrile-water, methanol-0.1% phosphoric acid water, acetonitrile-0.1% phosphoric acid water solution and phosphoric acid, formic acid, acetic acid and trifluoroacetic acid used in the mobile phase B acid water solution are selected through literature analysis and repeated tests and are used as mobile phases for investigation, and the results show that gradient elution is carried out by using the acetonitrile-0.1% phosphoric acid water solution as the mobile phase, 14 components can be simultaneously measured within 95min, the chromatographic peak shape is good, the separation degree is high, and the separation degree is larger than 1.5. According to the result of scanning of the 14 components at the full wavelength of 200-400 nm, the maximum absorption of the neochlorogenic acid and the chlorogenic acid is 330nm, the maximum absorption of the baicalin, the wogonoside and the baicalein is 278nm, the greater absorption of the forsythin and the arctiin is 230nm, and the 230nm is determined as the detection wavelength due to the comprehensive consideration that the good absorption is achieved at 230nm and the base line is relatively stable. The chromatographic conditions of different column temperatures (30 ℃, 35 ℃, 40 ℃ and 45 ℃), the mobile phase and the mobile phase proportion, the detection wavelength and the like are considered, and the optimal chromatographic conditions are obtained.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (9)
1. A high performance liquid detection method for 14 components in Jinhuaqinggan granules is characterized in that the high performance liquid detection method simultaneously measures the content of 14 components of neochlorogenic acid, loganin, chlorogenic acid, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonin, baicalein, chrysin and oroxylin in the Jinhuaqinggan granules by adopting a high performance liquid chromatography, and comprises the following steps:
(1) preparation of a test solution: taking golden flower refreshing particles, crushing, precisely weighing golden flower refreshing particle powder, placing the golden flower refreshing particle powder into a conical flask with a plug, precisely adding an alcohol solution, weighing the total weight of the golden flower refreshing particles and the alcohol solution added into the conical flask with the plug, carrying out ultrasonic extraction for 30-60 min, cooling to 25 ℃, weighing again, adding the alcohol solution to supplement the loss amount of the golden flower refreshing particles, shaking uniformly, taking supernate, passing through a 0.22 mu m microporous filter membrane, and placing subsequent filtrate into a brown liquid phase sample feeding bottle to obtain a golden flower refreshing particle sample solution; wherein the ratio of the mass of the golden flower refreshing granular powder to the volume of the alcoholic solution is 1 g: 100ml-900 ml;
(2) preparation of 14 mixed control solutions: precisely weighing chlorogenic acid, loganin, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonoside, baicalein, chrysin and oroxylin reference substances respectively, and dissolving with methanol to obtain single reference substance stock solution containing 14 components; precisely sucking the 14 stock solutions in sequence, placing in a 50mL volumetric flask, adding methanol to constant volume, shaking to obtain neochlorogenic acid, loganin, chlorogenic acid, caffeic acid, sweroside, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonoside, baicalein, chrysin, oroxylin with reference quality concentrations of 20.50, 14.76, 34.66, 10.01, 50.15, 50.11, 28.09, 194.45, 20.17, 178.13, 64.79, 10.14, 10.45 and 10.45 μ g/mL respectively-1Mixed reference solution of (1);
(3) chromatographic conditions and system applicability test: the chromatographic column adopts C taking octadecylsilane chemically bonded silica as a filler18A column; the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid aqueous solution, gradient elution is adopted, the flow rate is 1.0mL/min, the column temperature is 25-35 ℃, the sample injection amount is 10 mu L, the detection wavelength is 220-240 nm, the chromatographic analysis time is 95min, the theoretical plate number is not less than 3000 according to the calculation of respective reference peak, and the gradient elution procedure is as follows:
0-5 min, wherein the mobile phase A is 8% → 8%, and the mobile phase B is 92% → 92%;
5-30 min, wherein the mobile phase A is 8% → 18%, and the mobile phase B is 92% → 82%;
30-40 min, wherein the mobile phase A is 18% → 20%, and the mobile phase B is 82% → 80%;
40-60 min, 20% → 22% of mobile phase A and 80% → 78% of mobile phase B;
60-75 min, wherein the mobile phase A is 22% → 35%, and the mobile phase B is 78% → 65%;
75-90 min, 35% → 45% of mobile phase A and 65% → 55% of mobile phase B;
90-95 min, wherein the mobile phase A is 45% → 47%, and the mobile phase B is 55% → 53%;
wherein the proportions of the mobile phases A and B are volume percent;
(4) and (3) determination: respectively absorbing 10 mu L of the golden flower refreshing particle sample solution in the step (1) and 10 mu L of the mixed reference substance solution in the step (2), injecting the mixture into a high performance liquid chromatograph, operating according to the chromatographic conditions in the step (3), measuring respective chromatograms, calculating peak areas, measuring 14 components of neochlorogenic acid, loganin, chlorogenic acid, caffeic acid, swertiamarin, forsythoside A, apigenin-7-O-beta-D-glucopyranoside, baicalin, phillyrin, arctiin, wogonin, baicalein, chrysin and oroxylin in the golden flower refreshing particle sample solution according to an external standard one-point method, and calculating the contents of the neochlorogenic acid, the loganin, the chlorogenic acid, caffeic acid, the swertiamarin, the forsythoside A, the apigenin-7-O-beta-D-glucopyranoside and the like in the golden flower refreshing particle, Baicalin, phillyrin, arctiin, wogonoside, baicalein, chrysin and oroxylin.
2. The high performance liquid chromatography detection method for 14 components in golden flower refreshing granules according to claim 1, wherein the alcohol solution used for ultrasonic extraction of golden flower refreshing granules in the step (1) is a methanol solution with a volume percentage concentration of 50-70%.
3. The quality detection method according to claim 1, wherein the ultrasonic extraction time for dissolving the whey-like particles in step (1) is 30 min.
4. The high performance liquid chromatography detection method for 14 components in the golden flower refreshing granules according to claim 1, wherein the mass of the golden flower refreshing granule powder weighed in the step (1) is 0.10 g.
5. The high performance liquid chromatography detection method for 14 ingredients in golden flower refreshing granules according to claim 1, wherein the volume ratio of the mass of golden flower refreshing granule powder to the volume of the alcohol solution in the step (1) is 1 g: 200 ml.
6. The high performance liquid chromatography detection method for 14 components in the Jinhua qinggan granules according to claim 1, wherein the volume percentage concentration of the phosphoric acid aqueous solution in the mobile phase B in the step (3) is 0.05-0.3%.
7. The HPLC method for detecting 14 components in JINHUAQINGGAN granule as claimed in claim 1, wherein the concentration of phosphoric acid in mobile phase B in step (3) is 0.1% by volume.
8. The high performance liquid chromatography detection method for 14 components in Jinhuaqing granules according to claim 1, wherein the detection column temperature in step (3) is 30 ℃ and the detection wavelength is 230 nm.
9. The high performance liquid detection method for 14 components in the golden flower refreshing granules according to claim 1, wherein the high performance liquid detection method is used for simultaneously detecting the 14 components in the golden flower refreshing granules or simultaneously detecting any several components in the golden flower refreshing granules.
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| CN115356415B (en) * | 2022-08-26 | 2023-12-26 | 上海和黄药业有限公司 | Method for detecting content of 5 components in manna disinfection pill and application |
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