CN115389654A - Method for constructing fingerprint spectrum of centipeda minima medicinal preparation and content testing method - Google Patents
Method for constructing fingerprint spectrum of centipeda minima medicinal preparation and content testing method Download PDFInfo
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- CN115389654A CN115389654A CN202210932265.XA CN202210932265A CN115389654A CN 115389654 A CN115389654 A CN 115389654A CN 202210932265 A CN202210932265 A CN 202210932265A CN 115389654 A CN115389654 A CN 115389654A
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- peak
- mobile phase
- acid
- centipeda minima
- solution
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- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
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- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- VETNMUXXCNEZFK-OYBMWFINSA-N [(3aR,4S,6S,6aS,9aR,9bR)-6,9-dimethyl-3-methylidene-2,7-dioxo-4,5,6,6a,9a,9b-hexahydro-3aH-azuleno[4,5-b]furan-4-yl] acetate Chemical compound O=C(O[C@@H]1[C@H]2C(=C)C(=O)O[C@@H]2[C@H]2C(C)=CC(=O)[C@H]2[C@@H](C)C1)C VETNMUXXCNEZFK-OYBMWFINSA-N 0.000 description 1
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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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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
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- G—PHYSICS
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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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Abstract
The invention relates to the field of detection of traditional Chinese medicine preparations, and particularly provides a method for constructing a fingerprint of a centipeda minima medicinal preparation and a content test method, wherein a test solution is adopted for ultra-performance liquid chromatography detection, and the chromatographic conditions comprise that an Agilent Infinity LabPoroshell 120SB-Aq chromatographic column is adopted, acetonitrile is taken as a mobile phase A, a 0.2% phosphoric acid solution is taken as a mobile phase B, and elution is carried out according to a program defined in the description. The method can effectively and comprehensively detect the medicinal preparation of the centipeda minima.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine detection, in particular to a method for constructing a fingerprint of a centipeda minima medicinal preparation and a content testing method.
Background
The centipeda minima formula particle is obtained by extracting, concentrating and granulating the traditional Chinese medicine centipeda minima. The herba Centipedae is dry whole plant of Centipeda minima (L.) A.Br.et Aschers. Of Compositae, mainly contains volatile oil, sterols, flavonoids, triterpenes, guaianolides and pseudo-guaianolides, and is mainly used for treating wind-cold headache, cough, traumatic injury, pertussis, malaria, acute and chronic rhinitis, allergic rhinitis, etc.
The quality control of Centipeda minima in 2020 edition in Chinese pharmacopoeia comprises the items of original plant varieties, decoction piece processing, decoction piece properties, physicochemical identification, extract content measurement and the like, and the literature describes the chemical components in Centipeda minima, which comprise compounds containing volatile oil, sterols, flavonoids, triterpenes, guaialactone, pseudo-guaialactone and the like. On the one hand, however, the quality of the centipeda minima formula granules cannot be integrally detected and controlled by measuring or identifying the content of the single component; on the other hand, the identification of the centipeda minima formula particles by combining the content measurement of a single component with the identification of other components wastes time and labor and is difficult to be widely applied to production practice.
However, in the prior art, the quality control of centipeda minima is performed on centipeda minima herbal materials, but not on medicinal preparations of centipeda minima, and the construction method for the centipeda minima herbal materials in the prior art cannot be applied to the medicinal preparations of the centipeda minima. Therefore, the method for comprehensively and quickly detecting the medicinal preparation of the centipeda minima is established, and has important significance for comprehensive quality detection and overall quality control of the centipeda minima.
The laboratory discloses a method for constructing a centipeda minima fingerprint in Chinese patent document CN 1128526A, wherein the fingerprint only has 14 characteristic peaks, and the detection time is as long as 146-155 minutes.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the problems of long detection time and small number of characteristic peaks of the fingerprint construction method of the centipeda minima medicinal preparation disclosed in the prior art, so that the construction method and the content test method of the fingerprint of the centipeda minima medicinal preparation are provided.
A method for constructing fingerprint of herba Centipedae medicinal preparation comprises preparing test solution;
detecting the test solution by adopting ultra-high performance liquid chromatography, wherein the chromatographic conditions are that an Agilent InfinityLab poroschel 120SB-Aq chromatographic column is adopted, acetonitrile is taken as a mobile phase A, a water solution containing phosphoric acid is taken as a mobile phase B, and gradient elution is carried out according to the following procedures:
0-15min, the volume ratio of A to B is 3%:97% → 8%: the content of the active ingredients is 92%,
15-20min, wherein the volume ratio of A to B is 8%:92% → 12%:88 percent of the total weight of the mixture,
20-50min, the volume ratio of A to B is 12%:92 → 20%:80 percent of the total weight of the mixture,
50-60min, wherein the volume ratio of A to B is 20%:88% → 20%:80 percent of the total weight of the mixture,
60-70min, the volume ratio of A to B is 20%:80% → 25%:75 percent of the total weight of the mixture,
70-90min, wherein the volume ratio of A to B is 25%:75% → 30%:70 percent of the total weight of the mixture,
90-100min, the volume ratio of A to B is 30%:70% → 35%:65 percent;
the elution time is less than 55-65 minutes, the detection wavelength is 320-330nm, the elution time is more than or equal to 55-65 minutes, the detection wavelength is 210-230nm, the column temperature is 23-27 ℃, and the flow rate is 0.19-0.25mL/min.
Further, an Agilent InfinityLab Poroshell 120SB-Aq column having a size of 2.1X 150mm,1.9 μm was used; and/or the sample amount of the test solution is 1-3 mu L; and/or the flow rate is 0.20-0.25mL/min; and/or the volume percentage of the phosphoric acid in the phosphoric acid-containing aqueous solution is 0.1-0.4%.
Further, the preparation method of the test solution comprises the following steps: weighing a sample, adding a solvent for extraction to obtain an extracting solution, carrying out solid-liquid separation, and taking a liquid, namely the sample solution.
The preparation method of the test solution also meets any one or more of the following A-E:
A. the ratio of the mass of the sample to the volume of the solvent is 0.3-0.5:10-50 parts of; the relation between the mass and the volume is g/mL;
B. the extraction mode is reflux extraction or ultrasonic extraction;
C. the extraction time is more than or equal to 10min, preferably 15-60min;
D. the solid-liquid separation is selected from centrifugation or filter membrane filtration;
E. the solvent is at least one selected from water, methanol and ethanol, and is preferably water or methanol aqueous solution.
Illustratively, the preparation method of the test solution comprises the following steps: precisely adding solvent into the sample, sealing, weighing, ultrasonic treating, taking out, cooling, weighing again, adding solvent to make up the lost weight, shaking, filtering, and collecting the filtrate.
Further, the construction method comprises a step of preparing a reference substance solution by adding a solvent to at least one of caffeic acid, chlorogenic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, cryptochlorogenic acid, rutin, arnica Montanone D, mauredin C and kaempferol-3-O-rutinoside, and a step of detecting the reference substance solution to obtain a reference substance map by ultra-high performance liquid chromatography in the construction method according to any one of claims 1 to 4; preferably, the solvent is selected from methanol and/or ethanol.
Further, the fingerprint of the centipeda minima medicinal preparation has 26 common characteristic peaks, the peaks corresponding to chlorogenic acid, 3, 5-di-O-caffeoylquinic acid and brevifolin A are respectively an S1 peak, an S2 peak and an S3 peak, the relative retention time of the peaks 1 to 12 and the S1 peak is within the range of +/-10% of a specified value, and the specified value is as follows: 0.15 (peak 1), 0.25 (peak 2), 0.34 (peak 3), 0.43 (peak 4), 0.51 (peak 5), 0.61 (peak 6), 0.89 (peak 7), 0.96 (peak 8), 1.00 (peak 9), 1.22 (peak 10), 1.33 (peak 11), 1.53 (peak 12); the relative retention times of peaks 13 to 22 and the S2 peak are within ± 10% of the specified values: 0.77 (peak 13), 0.80 (peak 14), 0.84 (peak 15), 0.93 (peak 16), 0.94 (peak 17), 1.00 (peak 18), 1.07 (peak 19), 1.10 (peak 20), 1.22 (peak 21), 1.24 (peak 22); the relative retention times of peaks 23-26 and the S3 peak are within ± 10% of the specified values: 0.78 (Peak 23), 0.86 (Peak 24), 0.97 (Peak 25), 1.00 (Peak 26).
The invention also provides a content testing method of the centipeda minima medicinal preparation, which comprises the following steps:
preparing a test solution and a reference solution;
detecting the test solution and the reference solution by adopting ultra-high performance liquid chromatography, wherein the chromatographic conditions comprise that an Agilent InfinityLab Poroshell 120SB-Aq chromatographic column is adopted, acetonitrile is taken as a mobile phase A, an aqueous solution containing phosphoric acid is taken as a mobile phase B, and gradient elution is carried out according to the following procedures:
0-15min, the volume ratio of A to B is 3%:97% → 8%: the content of the active ingredients is 92%,
15-20min, wherein the volume ratio of A to B is 8%:92% → 12%:88 percent of the total weight of the mixture,
20-50min, wherein the volume ratio of A to B is 12%:92 → 20%:80 percent of the total weight of the mixture,
50-60min, wherein the volume ratio of A to B is 20%:88% → 20%:80 percent of the total weight of the mixture,
60-70min, the volume ratio of A to B is 20%:80% → 25%:75 percent of the total weight of the mixture,
70-90min, wherein the volume ratio of A to B is 25%:75% → 30%:70 percent of the total weight of the mixture,
90-100min, the volume ratio of A to B is 30%:70% → 35%:65 percent;
the elution time is less than 55-65 minutes, the detection wavelength is 320-330nm, the elution time is more than or equal to 55-65 minutes, the detection wavelength is 210-230nm, the column temperature is 23-27 ℃, and the flow rate is 0.19-0.25mL/min.
Further, an Agilent InfinityLab Poroshell 120SB-Aq column having a size of 2.1X 150mm,1.9 μm was used; and/or the sample amount of the test solution is 1-3 mu L; and/or the flow rate is 0.20-0.25mL/min; and/or the volume percentage of the phosphoric acid in the phosphoric acid-containing aqueous solution is 0.1-0.4%.
Further, the preparation method of the test solution comprises the following steps: weighing a sample, adding a solvent for extraction to obtain an extracting solution, carrying out solid-liquid separation, and taking a liquid to obtain a sample solution, wherein preferably, the preparation method of the sample solution further satisfies any one or more of the following A-E:
A. the ratio of the mass of the sample to the volume of the solvent is 0.3-0.5:10-50, preferably 0.4:10-50 parts of; the relation between the mass and the volume is g/mL;
B. the extraction mode is reflux extraction or ultrasonic extraction;
C. the extraction time is more than or equal to 10min, preferably 15-60min;
D. the solid-liquid separation is selected from centrifugation or filter membrane filtration;
E. the solvent is at least one selected from water, methanol and ethanol.
Further, the control solution is prepared by adding at least one of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, brevifolin A and caffeic acid with a solvent, preferably, the solvent is selected from at least one of methanol, ethanol and water.
The invention also provides a quality detection method of the centipeda minima medicinal preparation, which comprises the step of comparing the fingerprint of the centipeda minima product to be detected with the reference fingerprint of the centipeda minima medicinal preparation and/or the step of determining the content of the centipeda minima product to be detected by adopting any one of the methods for testing the content of the centipeda minima medicinal preparation; the fingerprint of the centipeda minima product to be detected is obtained by using the centipeda minima product to be detected and constructed according to any one of the construction methods, and the control fingerprint of the medicinal preparation of the centipeda minima is selected from any one of the following (1) to (3):
(1) The centipeda minima fingerprint spectrum of the medicinal preparation has 26 common characteristic peaks, the peaks corresponding to chlorogenic acid, 3, 5-di-O-caffeoylquinic acid and brevifolin A are respectively an S1 peak, an S2 peak and an S3 peak, the relative retention time of the peaks from 1 to 12 and the S1 peak is within the range of +/-10% of a specified value, and the specified value is as follows: 0.15 (peak 1), 0.25 (peak 2), 0.34 (peak 3), 0.43 (peak 4), 0.51 (peak 5), 0.61 (peak 6), 0.89 (peak 7), 0.96 (peak 8), 1.00 (peak 9), 1.22 (peak 10), 1.33 (peak 11), 1.53 (peak 12); the relative retention times of peaks 13-22 and the S2 peak are within ± 10% of the specified values: 0.77 (peak 13), 0.80 (peak 14), 0.84 (peak 15), 0.93 (peak 16), 0.94 (peak 17), 1.00 (peak 18), 1.07 (peak 19), 1.10 (peak 20), 1.22 (peak 21), 1.24 (peak 22); the relative retention times of peaks 23-26 and the S3 peak are within ± 10% of the specified values: 0.78 (peak 23), 0.86 (peak 24), 0.97 (peak 25), 1.00 (peak 26);
(2) The fingerprint of the centipeda minima medicinal preparation is obtained by using a single batch or multiple batches of centipeda minima medicinal preparation according to any one of the construction methods;
(3) And preparing a control fingerprint by using the fingerprints obtained by the multiple batches of the medicinal preparation of the centipeda minima according to any one of the construction methods through an average value method or a median method.
Wherein the centipeda minima product to be detected is a medicinal preparation of the centipeda minima.
The similarity of the centipeda minima formula particles to the fingerprint spectrum is more than 0.900.
The technical scheme of the invention has the following advantages:
1. according to the method for constructing the fingerprint of the centipeda minima medicinal preparation, the Agilent InfinityLab Poroshell 120SB-Aq chromatographic column, a specific elution program, and control of column temperature and flow rate are adopted, so that the separation effect of various effective components is obviously improved while the detection time is shortened, more characteristic peaks are contained in the fingerprint, and the information of the fingerprint is greatly enriched.
2. According to the content testing method of the centipeda minima medicinal preparation, provided by the invention, the content of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, brevifolin A, caffeic acid and other components can be simultaneously measured by adopting an Agilent InfiniyLab Poroshell 120SB-Aq chromatographic column and a specific elution program, and controlling the column temperature and the flow rate, and the separation degree of each component is good, and the construction method is high in precision, stability and repeatability, so that the effective components and the content thereof in the centipeda minima formula particles can be comprehensively and quickly detected.
3. According to the method for constructing the fingerprint of the centipeda minima pharmaceutical preparation, chlorogenic acid (S1 peak), 3, 5-di-O-caffeoylquinic acid (S2 peak) and brevifolin A (S3 peak) can be selected as internal reference peaks in the fingerprint, 26 common characteristic peaks of the centipeda minima formula particles can be determined, the relative retention time of each common characteristic peak can be calculated according to the chlorogenic acid (S1 peak), the 3, 5-di-O-caffeoylquinic acid (S2 peak) and the brevifolin A (S3 peak), the comprehensive quality detection and the overall quality control of the centipeda minima formula particles are facilitated, and the safety and the stability of the use of the medicine are improved.
4. The method for detecting the quality of the fingerprint of the centipeda minima medicinal preparation can simultaneously construct the fingerprint of the centipeda minima to be detected and carry out content determination, and is simple and convenient for comprehensively controlling the quality of the centipeda minima.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a finger print of 18 batches of centipeda minima formula granules obtained by high performance liquid chromatography in example 1 of the invention; FIG. 2 is a control fingerprint generated from 18 batches of centipeda minima formula granules according to example 1 of the present invention; FIG. 3 is one of the maps of the control fingerprint and the control with different characteristic peaks in example 1 of the present invention; FIG. 4 is a second location chart of the comparison fingerprint and the comparison product with different characteristic peaks in example 1 of the present invention; FIG. 5 is a chromatogram of a precision experiment of a centipeda minima formula of batch No. 2109001S; FIG. 6 is a fingerprint of the centipeda minima formula granules in the proprietary experiment of example 2 of the present invention; FIG. 7 is a fingerprint of a negative control solution in the specificity experiment of example 2 of the present invention; FIG. 8 is a fingerprint of the condition of 0.1% phosphoric acid solution in example 3 of the present invention; FIG. 9 is a fingerprint of the conditions of the 0.4% phosphoric acid solution in example 3 of the present invention; FIG. 10 is a fingerprint at a flow rate of 0.15mL/min in example 4 of the present invention; FIG. 11 is a fingerprint at a flow rate of 0.20mL/min in example 4 of the present invention; FIG. 12 is a fingerprint obtained by liquid phase detection of example 7 of the present invention, no. 2; FIG. 13 is a fingerprint obtained by liquid phase assay of example 7 of the present invention in (3); FIG. 14 is a fingerprint of a centipeda minima formula particle for a proprietary experiment according to example 9 of the invention; FIG. 15 is a fingerprint of a negative control solution in a specificity experiment of example 9 of the present invention; FIG. 16 is a fingerprint at a detection wavelength of 325nm in comparative example 1 of the present invention; FIG. 17 is a fingerprint at a detection wavelength of 225nm in comparative example 1 of the present invention; FIG. 18 is a fingerprint measured by the column (2) of comparative example 2 of the present invention; FIG. 19 is a fingerprint obtained by detecting the chromatographic column No. 3 in comparative example 2 of the present invention; FIG. 20 is a fingerprint at a column temperature of 20 ℃ in comparative example 3 of the present invention; FIG. 21 is a fingerprint at a column temperature of 30 ℃ in comparative example 3 of the present invention; FIG. 22 is a fingerprint obtained in comparative example 4 of the present invention.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The apparatus and reagents used in the present invention are as follows:
high performance liquid chromatograph 1: waters Arc UPLC, PDA detector, empower chromatography management system; high performance liquid chromatography 2: thermo Vanqish ultra high performance liquid phase, pump, colum component, autoSumpler, photometer; high performance liquid chromatograph 3: agilent 1290 ultra high performance liquid phase, pump, gum component, autosampler, photometer DAD; electronic analytical balance: METTLER TOLEDO (Mettler, switzerland) XS-205, XS-204, XSE-205, XPE56; an ultrasonic generator: SK5200H shanghai kogi ultrasonic instruments ltd; an ultrapure water system: millipore (Millipore) USA; acetonitrile is chromatographic purity (Merk company, germany), water is ultrapure water (resistivity 18.2m omega. Cm), and other reagents are analytical purity; and (3) chromatographic column: (1) Agilent InfinityLab Poroshell 120SB-Aq 2.1X 150mm 1.9 μm; (2) Waters HSS T3C 18.1X 150mm 1.8 μm; (3) Thermo Hypersil GOLD aQ 2.1X 150mm 1.9 μm; chlorogenic acid reference (batch No. 110753-202018, china institute for testing food and drug); 3, 5-O-dicaffeoylquinic acid reference substance (batch number: 111782-201807, china institute for food and drug testing); 4, 5-O-dicaffeoylquinic acid (batch No. 111894-202104, purity: 98%, shanghai Hongyong Biotech Co., ltd.); brevlin A reference (batch No. 112067-202001, china food and drug testing research institute); neochlorogenic acid (batch No. 19011731, purity 98%, shanghai Hotan Biotechnology Co., ltd.); caffeic acid (batch No. 110885-201703, purity: 100%, china institute for food and drug testing); rutin (batch number: 100080-202012, purity: 91.7%, china institute for food and drug testing); 3, 4-O-dicaffeoylquinic acid (batch No. 250035-201907, purity: 98%, shanghai hong Yongsheng, co., ltd.); arnica lactone D (batch No. 190141-201907, purity: 98%, shanghai hong Yongsheng Biotech limited); coreopsin C (batch No. 240095-201909, purity: 95%, shanghai hong Yongsheng Biotech limited); centipeda minima reference drug (batch No. 121053-201804, china institute for food and drug testing). The fingerprint spectrum method draft particle batch number: 2109001S
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1
A method for constructing a fingerprint of a centipeda minima medicinal preparation comprises the following steps:
(1) Preparation of a test solution: taking about 0.2g of centipeda minima medicinal preparation powder, precisely weighing, placing into a conical flask with a plug, adding 25ml of 50% methanol, carrying out ultrasonic treatment (power 200W, frequency 53 kHz) for 30 minutes, taking out, cooling, shaking up, filtering, and taking out a subsequent filtrate to obtain the centipeda minima medicinal preparation. The medicinal preparation of the centipeda minima is prepared by the following method:
taking centipeda minima decoction pieces, heating and refluxing for extraction for at least 1 time, adding 6-12 times of water by weight for extraction for at least 0.5h each time, filtering, combining filtrates, concentrating the filtrate to the relative density of 1.05-1.10g/mL at 60 ℃, adding conventional auxiliary materials, and preparing clinically acceptable tablets, capsules, pills, granules, honeyed pills, sustained release preparations, quick release preparations, controlled release preparations, oral liquid preparations or injection preparations according to the conventional process. The pharmaceutically acceptable auxiliary materials are as follows: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, bases, and the like. The filler comprises: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; the disintegrating agent comprises: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, cross-linked sodium carboxymethyl cellulose, etc.; the lubricant comprises: magnesium stearate, sodium lauryl sulfate, talc, silica, and the like; the suspending agent comprises: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, and the like; the adhesive comprises starch slurry, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; the sweetener comprises: saccharin sodium, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid and the like; the flavoring agent comprises: sweeteners and various essences; the preservative comprises: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroacetidine acetate, eucalyptus oil, etc.; the matrix comprises: PEG6000, PEG4000, insect wax, etc.
The test sample in the embodiment is selected from centipeda minima formula particles, and the specific preparation method of the centipeda minima formula particles comprises the following steps: taking centipeda minima decoction pieces, heating and refluxing for 2 times, adding 12 times of water by weight for the first time, soaking for 30min, heating and refluxing for 0.5h, filtering, adding 10 times of water by weight for the second time, extracting for 0.5h, filtering, combining filtrates, concentrating the filtrate to the relative density of 1.05g/mL at 60 ℃, carrying out spray drying, adding maltodextrin as an auxiliary material into dry powder, uniformly mixing, carrying out dry granulation, and preparing into granules.
(2) Chromatographic conditions of ultra-high performance liquid chromatography
A chromatographic column: agilent InfinityLab Poroshell 120SB-Aq column (2.1X 150mm 1.9 μm); acetonitrile-0.2% phosphoric acid solution was used as mobile phase, gradient elution is shown in table 1; flow rate: 0.2mL/min; the column temperature was 25 ℃; the detection wavelength was 325nm (first 60 minutes), and the conversion was 225nm. The number of theoretical plates is not less than 5000 calculated according to the peak of chlorogenic acid, and the sample amount is as follows: 1 μ L.
TABLE 1 gradient elution procedure
The common mode of the fingerprint spectrum of the centipeda minima formula particles is established according to the method.
Taking 15 batches of centipeda minima formula particle samples to obtain the fingerprint of the centipeda minima formula particle according to the method, as shown in figure 1. In fig. 1, the lot numbers of S1 to S18 are S1:1605001W, S2:1609001W, S3:1701001W, S4:1702001W, S5:1708001W, S6:1710001W, S7:1806001W, S8:1805001W, S9:1806001W, S10:1909001S, S11:1806001S, S12:1911001W, S13:2003001W, S14:2005001W, S15:2007001W, S16:2007001S, S17:2108001S, S18:2109001S. R (26) is the comparison fingerprint of the centipeda minima formula granules.
Preparation of reference solutions: precisely weighing appropriate amount of neochlorogenic acid, caffeic acid, chlorogenic acid, 3, 4-di-O-caffeoylquinic acid, 3, 5-di-O-caffeoylquinic acid, 4, 5-di-O-caffeoylquinic acid and brevifolin A reference substances, placing in a brown measuring flask, and adding methanol to obtain solutions containing 50 μ g of each reference substance per 1 mL; precisely weighing appropriate amount of cryptochlorogenic acid, rutin, arnica herb lactone D, coreopsin C, and kaempferol-3-O-rutinoside reference substances, and adding methanol to obtain solutions each containing 100 μ g of reference substance per 1 mL. Taking the reference substance solution, detecting according to the above HPLC chromatographic analysis conditions to obtain a control map, which is shown in FIG. 3.
The control fingerprint is generated by using fingerprint similarity evaluation software 'traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2012 edition' compiled by pharmacopoeia committee, as shown in figure 2. The detection results of the fingerprint of the formula granules can be analyzed and compared according to the reference fingerprint shown in figure 2, and the reference fingerprint is used for quality control of the formula granules. The specific method for controlling the quality by using the reference fingerprint spectrum comprises the following steps:
as can be seen from fig. 1, 2 and 3, the control fingerprint shows 26 characteristic peaks. Wherein the retention times of peak 9, peak 18 and peak 26 respectively correspond to the retention times of the reference peaks of chlorogenic acid, 3, 5-di-O-caffeoylquinic acid and agrimonine A reference substance; the peaks corresponding to chlorogenic acid, 3, 5-di-O-caffeoylquinic acid and brevifolin A are respectively an S1 peak, an S2 peak and an S3 peak, and the relative retention time of the peaks from 1 to 12 to the S1 peak is as follows: 0.15 (peak 1), 0.25 (peak 2), 0.34 (peak 3), 0.43 (peak 4), 0.51 (peak 5), 0.61 (peak 6), 0.89 (peak 7), 0.96 (peak 8), 1.00 (peak 9), 1.22 (peak 10), 1.33 (peak 11), 1.53 (peak 12); the relative retention times of peaks 13-22 and the S2 peak are: 0.77 (peak 13), 0.80 (peak 14), 0.84 (peak 15), 0.93 (peak 16), 0.94 (peak 17), 1.00 (peak 18), 1.07 (peak 19), 1.10 (peak 20), 1.22 (peak 21), 1.24 (peak 22); the relative retention times of peaks 23-26 and the S3 peak are: 0.78 (Peak 23), 0.86 (Peak 24), 0.97 (Peak 25), 1.00 (Peak 26), see Table 3.
18 batches of centipeda minima formula particle fingerprint spectrum calculates the relative retention time of the peak 1-8, the peak 10-12 and the S1 peak, the relative retention time is within the range of +/-10% of the specified value, and the specified value is as follows: 0.15 (peak 1), 0.25 (peak 2), 0.34 (peak 3), 0.43 (peak 4), 0.51 (peak 5, neochlorogenic acid), 0.61 (peak 6), 0.89 (peak 7, caffeic acid), 0.96 (peak 8, cryptochlorogenic acid), 1.22 (peak 10), 1.33 (peak 11), 1.53 (peak 12); taking the corresponding peak of the 3, 5-di-O-caffeoylquinic acid reference peak as the S2 peak, calculating the relative retention time of the peaks 13-17, 19-22 and the S2 peak, wherein the relative retention time is within +/-10% of the specified value, and the specified value is as follows: 0.77 (peak 13), 0.80 (peak 14, rutin), 0.84 (peak 15), 0.93 (peak 16, kaempferol-3-O-rutinoside), 0.94 (peak 17,3, 4-di-O-caffeoylquinic acid), 1.07 (peak 19), 1.10 (peak 20,4, 5-di-O-caffeoylquinic acid), 1.22 (peak 21), 1.24 (peak 22); taking the peak corresponding to the reference peak of the brevifolin A as an S3 peak, calculating the relative retention time of the peaks 23-25 and the S3 peak, wherein the relative retention time is within +/-10% of a specified value, and the specified value is as follows: 0.78 (Peak 23), 0.86 (Peak 24, arnica lactone D), 0.97 (Peak 25, calcesin C). The fingerprint similarity of 18 centipeda minima formula particles is larger than 0.90, the obtained results are shown in tables 2-5, and the reference product positioning of different characteristic peaks is shown in figure 3.
TABLE 2 common mode relative retention times for centipeda minima formula granules
TABLE 3 common pattern matching data for centipeda minima formula granules
TABLE 4 fingerprint measurement results of centipeda minima formula granules of 18 batches
TABLE 5 fingerprint similarity results for centipeda minima formula granules of batch 18
Through the construction method in the embodiment, the fingerprint spectrum with good separation degree of each characteristic peak can be effectively obtained, subsequent tests prove that the construction method can simultaneously measure the contents of the components such as caffeic acid, chlorogenic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid and the like, the chlorogenic acid (S1 peak), the 3, 5-di-O-caffeoylquinic acid (S2 peak) and the brevifolin A (S3 peak) are used as reference peaks in the fingerprint spectrum, 26 common characteristic peaks of the centipeda minima formula particles can be determined, and the relative retention time of each common characteristic peak can be calculated according to the chlorogenic acid (S1 peak), the 3, 5-di-O-caffeoylquinic acid (S2 peak) and the brevifolin A (S3 peak), so that the comprehensive quality detection and the overall quality control of the centipeda minima formula particles are facilitated, and the safety and the stability of the medicine in use are improved.
Example 2
The present embodiment provides the process and the result of the detection on precision, stability, repeatability, and the like by the construction method in the above embodiment 1, and the specific steps are as follows:
1 precision
1.1 precision test
The same sample solution (Centipeda minima formula of batch No. 2109001S) prepared in example 1 was sampled repeatedly 6 times according to the chromatographic conditions of example 1 (2), and the relative retention times of 26 common peaks were determined, wherein the RSD values of the relative retention times of the peaks are: 0.25%, 0.22%, 0.27%, 0.26%, 0.19%, 0.20%, 0.14%, 0.04%, 0%, 0.05%, 0.03%, 0.06%, 0.10%, 0.04%, 0.01%, 0%, 0.04%, 0.02%, 0.07%, 0.08%, 0.03%, 0.02%, 0.01%, 0%, and as a result, as shown in table 6, the chromatogram of the precision experiment is shown in fig. 5.
TABLE 6 results of similarity of precision experiments
As can be seen from table 6 and fig. 5, RSD of the relative retention time of each characteristic peak and the reference S peak is less than 2.0%, and the similarity with the fingerprint of the centipeda minima formula particle is greater than 0.90, which indicates that the precision is better.
1.2 repeatability tests
Taking 6 portions of the same batch of test sample (batch number is 2109001S), respectively measuring the relative retention time of 26 common peaks according to the method of example 1, wherein the RSD values of the relative retention time of each peak are respectively as follows: 0.25%, 0.40%, 0.51%, 0.43%, 0.29%, 0.31%, 0.17%, 0.03%, 0%, 0.03%, 0.04%, 0.09%, 0.04%, 0.08%, 0.05%, 0.02%, 0%, 0.04%, 0.03%, 0.07%, 0.03%, 0.02%, 0.01%, 0%, the results are shown in Table 7.
TABLE 7 similarity results of the repeatability tests (n = 6)
As can be seen from Table 7, the RSD of the relative retention time of each characteristic peak and the reference substance S peak of the result is less than 2.0%, and the similarity of each characteristic peak and the fingerprint of the control of the centipeda minima formula particles is greater than 0.90, which indicates that the method has better repeatability.
2 Special Property experiment
The test sample adopts centipeda minima formula particles with the batch number of 2109001S, the test sample solution is prepared according to the method of the embodiment 1, 0.4g of maltodextrin is weighed to replace the test sample, the negative control solution is prepared according to the same method of the test sample solution, the detection is carried out according to the method of the embodiment 1, the detection result is shown in figures 6 and 7, and the result shows that the negative control solution is not interfered.
3 stability test
The same batch of test samples (batch number 2109001S) are taken, the construction method operation of the embodiment 1 is carried out, the samples are injected at 0 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours respectively, the relative retention time of 26 common peaks is determined, and the RSD value of each peak is respectively: 0.18%, 0.38%, 0.54%, 0.52%, 0.35%, 0.39%, 0.21%, 0.04%, 0%, 0.05%, 0.07%, 0.13%, 0.07%, 0.05%, 0.07%, 0.01%, 0%, 0.04%, 0.03%, 0.06%, 0.07%, 0.03%, 0.01%, 0%, and the results are shown in Table 8.
Table 8 similarity results of stability experiments
As shown in table 8, RSD of the relative retention time of each characteristic peak and the reference S peak was less than 2.0%, and the similarity to the control fingerprint of the centipeda minima formula granules was greater than 0.90, respectively; this indicates that the test solution is stable within 24 hours and meets the measurement requirements.
Example 3
This example is different from example 1 in that the concentrations of the mobile phases B in this example are different, namely, a 0.1% phosphoric acid solution and a 0.4% phosphoric acid solution. The test was carried out using the centipeda minima formula granules with the batch number of 2109001S, and the test results are shown in fig. 8 and 9. As can be seen from fig. 8 and 9, the 0.1-0.4% phosphoric acid solution is effectively suitable for separating all components of the centipeda minima formula granules.
TABLE 9 examination results of phosphoric acid-acetonitrile mobile phase systems of different concentrations
Example 4
The difference between this example and example 1 is that the flow rates in this example are 0.15mL/min and 0.25mL/min, respectively. The test was carried out using the centipeda minima formula granules with the batch number of 2109001S, and the test results are shown in fig. 10 and 11. As can be seen from fig. 10 and 11, the effect of analyzing the characteristic spectrum of the centipeda minima formula particles under the flow rate condition of 0.15mL/min to 0.25mL/min is large, and when the flow rate is 0.15mL/min, the separation effect of all components of the centipeda minima formula particles is poor, and the number of characteristic peaks is small. When the flow rate is 0.20 and 0.25mL/min, the separation effect of all the components of the centipeda minima formula particles is good, and the peak information content is large.
TABLE 10 investigation results of different flow rates
Example 5
The present example differs from example 1 in that the sample volumes in the present example are different, and the detection results when the sample volumes were 1. Mu.L, 2. Mu.L, and 3. Mu.L were examined. That is, the swan chow formula granules with a lot number of 2109001S were used for testing, and the system adaptability parameters for different sample amounts are shown in table 11.
TABLE 11 System Adaptation parameters for chromatographic peaks of different sample size
As can be seen from table 11: the number of the detected chromatographic peaks and the system adaptability parameters of each chromatographic peak are used as evaluation indexes, and 1-3 muL can meet the detection requirement.
Example 6
This example differs from example 1 in that the test was carried out using a centipeda minima formula with a lot number of 2109001S, the test solutions of this example having different treatment parameters. The method comprises the following specific steps:
(1) Investigation of different extraction modes
In this example, water bath reflux and ultrasound were examined, and the remaining processes and conditions were as in example 1, and the results showed that the chromatogram separation effects of the extraction methods were substantially consistent, wherein the chromatogram separation effects and system adaptability parameters exhibited by the water bath reflux and ultrasound solvents were relatively good, and the system adaptability parameters of the main characteristic peaks obtained after the extraction treatment with the above-mentioned different solvents were as shown in table 12.
TABLE 12 System Adaptation parameters for chromatograms for different extraction regimes
From the chromatogram and the results shown in table 12, it was found that there was no significant difference in the dissolution effect and the chromatogram between the different extraction methods.
(2) Investigation of different extraction solvents
In the embodiment, water, ethanol, methanol, 50% methanol and 50% ethanol are considered, the rest processes and conditions are as in example 1, the results show that the chromatogram separation effects of the extraction solvents are basically consistent, the extraction effects of the water, ethanol, methanol, 50% methanol and 50% ethanol on the centipeda minima formula particles are considered, the extraction solvents can enable the sample to be well extracted, the separation degree of each peak is good, the number of the mixed peaks is small, the chromatogram base line is flat, the system adaptability parameters are excellent, and the system adaptability parameters of the main characteristic peaks obtained after the extraction treatment of the different solvents are shown in table 13.
TABLE 13 systematic Adaptation parameters of chromatograms of different extraction solvents
As can be seen from table 13 in combination with the detection spectra, the extraction effect of the centipeda minima formula particles by adding different extraction solvents is similar, wherein the extraction solvent is preferably water or methanol aqueous solution, and more preferably 50% methanol, the obtained chromatogram has a better peak shape and relatively best system adaptability parameters.
(2) Investigation of extraction time under the condition that the extraction solvent is methanol
According to the determined extraction solvent, different sonication times were investigated: the extraction effect on the centipeda minima formula granules is 15min, 30min and 45min, and the rest processes and conditions are as in example 1. The system adaptation parameters for different extraction times are shown in table 14.
TABLE 14 chromatogram parameters of centipeda minima formula granules at different extraction times
From the results shown in table 14, it was found that there was no significant difference in the dissolution effect and the chromatogram at different ultrasonic times.
(3) Selection of the amount of extraction solvent added
Different addition amounts of methanol were investigated according to the defined extraction solvent (50% methanol): extraction efficiency of the centipeda minima formula granules of 10mL, 25mL and 50mL, the rest of the process and conditions are as in example 1, and the systematic adaptability parameters of the addition amounts of different extraction solvents are shown in Table 15.
TABLE 15 systematic Adaptation parameters for chromatographic peaks for different amounts of extraction solvent
From the results shown in table 15, it can be seen that the extraction effect of the centipeda minima formula particles by adding the extraction solvent with different volumes is similar, and the chromatographic separation effect has no obvious difference.
Example 7
The difference between this example and example 1 is that the brand of the apparatus in this example is different, and this example discloses that the apparatus of different brands detects the centipeda minima formula granules with the batch number of 2109001S, and the chromatographic columns of different models are as follows:
(1)Waters Arc UPLC；
(2) Thermo Vanqish ultra high performance liquid phase;
(3) Agilent 1290 ultra-high performance liquid phase; the remaining processes and conditions were as in example 1.
The chromatogram obtained by the detection of the apparatus (1) is shown in FIG. 6, and the chromatograms obtained by the detection of the apparatuses (2) to (3) are shown in FIGS. 12 and 13. The detection results show that no obvious difference exists in chromatographic separation effect.
TABLE 16 results of different chromatograph examinations
Example 8 a method for determining the content of a centipeda minima pharmaceutical preparation, comprising the following steps:
(1) Octadecylsilane chemically bonded silica is used as a filler (the column length is 150mm, the inner diameter is 2.1mm, and the particle size is 1.9 mu m); acetonitrile is taken as a mobile phase A,0.2 percent phosphoric acid is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the flow rate was 0.2ml per minute; the column temperature was 25 ℃; the detection wavelength was 325nm (first 60 minutes), and the conversion was 225nm. The number of theoretical plates is not less than 5000 calculated according to chlorogenic acid peak.
(2) Preparation of reference solution chlorogenic acid reference, caffeic acid reference, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-di-O-caffeoylquinic acid reference, 3, 5-di-O-caffeoylquinic acid reference, and appropriate amount of 4, 5-di-O-caffeoylquinic acid, and brevifolin A reference are precisely weighed, and 50% methanol is added to prepare mixed solution containing 0.05mg per 1 ml.
(3) Preparing a test solution, grinding an appropriate amount of the test solution, taking about 0.4g of the test solution, precisely weighing, placing in a conical flask with a plug, adding 25ml of 50% methanol, carrying out ultrasonic treatment (power 200W and frequency 53 kHz) for 30 minutes, taking out, cooling, shaking up, filtering, and taking out a subsequent filtrate.
(4) The determination method comprises precisely sucking reference solution and sample solution 1 μ l each, injecting into liquid chromatograph, and determining.
Example 9
This example differs from example 8 in that the test was carried out using a centipeda minima formula with a lot number of 2109001S, the test solutions of this example having different treatment parameters. The method comprises the following specific steps:
(1) Investigation of different extraction modes
In this example, water bath reflux and sonication were considered, and the remaining processes and conditions were as in example 8, specifically: respectively taking a proper amount of the product, grinding, precisely weighing about 0.4g, placing into a conical flask with a plug, precisely adding 10ml of 50% methanol, weighing, refluxing in water bath and performing ultrasonic treatment (power 200W and frequency 53 kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss by 50% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the product.
TABLE 17 measurement results of contents of index components of different extraction methods
RSD values are less than 2.0%, the content of the sample is low in a water bath reflux mode, and ultrasound is recommended to be selected as a sample extraction mode.
(2) Investigation of different extraction solvents
This example considers water, ethanol, methanol, 50% ethanol, and the remaining processes and conditions are as in example 8, as follows: taking a proper amount of the product, grinding, precisely weighing about 0.4g, placing in a conical flask with a plug, precisely adding 10ml of water, methanol, ethanol, 50% methanol and 50% ethanol, weighing, ultrasonically treating (with the power of 200W and the frequency of 53 kHz) for 30 minutes, cooling, weighing again, complementing the lost weight with 50% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product.
TABLE 18 measurement results of contents of index components of different extraction solvents
According to results, the content of components with different extraction volume indexes is close to that of components with different extraction volume indexes, and the content is consistent with that of the components in the construction method, and 50% methanol is recommended to be selected as an extraction solvent of a test sample.
(3) Investigation of different extraction times
Investigation of different ultrasound times: the extraction effect on the centipeda minima formula granules is 15min, 30min and 60min, and the rest processes and conditions are as in example 8. The method specifically comprises the following steps: respectively taking a proper amount of the product, grinding, precisely weighing about 0.4g, placing into a conical flask with a plug, precisely adding 10ml of 50% methanol, weighing, respectively carrying out ultrasonic treatment (power 200W and frequency 53 kHz) for 15 minutes, 30 minutes and 60 minutes, cooling, weighing again, complementing the lost weight with 50% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product.
TABLE 19 measurement results of contents of different extraction volume index components
The results show that the content of index components of different extraction volumes is close, and the RSD values are all less than 2.0%, which indicates that the extraction effect of the extraction time is close.
(3) Selection of the amount of extraction solvent added
On the basis of the determined extraction solvent (50% methanol), different additions of methanol were examined: 10mL, 25mL and 50mL of the effect of extracting the centipeda minima formula particles, and the rest of the process and conditions are as in example 8, concretely, taking a proper amount of the product, grinding, taking about 0.4g, precisely weighing, placing into a conical flask with a plug, precisely adding 10mL of 50% methanol, weighing, carrying out ultrasonic treatment (power 200W and frequency 53 kHz) for 30 minutes, cooling, weighing again, complementing the lost weight with 50% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the centipeda minima formula particles.
TABLE 20 measurement results of contents of different extraction volume index components
As can be seen from the results, the contents of the different extraction volume index components are close, indicating that 10-50ml of extraction solvent can be used.
(3) Selection of sample size
Examining the influence of the sampling amount of the centipeda minima formula particles on the extraction effect, and carrying out other processes and conditions as in example 8, specifically, taking a proper amount of the product, grinding, precisely weighing about 0.3g, 0.4g or 0.5g, placing into a conical flask with a plug, precisely adding 25ml of 50% methanol, weighing, carrying out ultrasonic treatment (power 200W and frequency 53 kHz) for 30 minutes, cooling, weighing again, complementing the weight loss with 50% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the centipeda minima formula particles.
TABLE 21 measurement results of contents of different extraction volume index components
From the results, it is known that the content of different pressure-removing index components is close, which indicates that the extraction effect of different sampling amounts is close, but in the operation process, it is found that when the sampling amount is 0.3g, the response of chromatographic peak is low due to the relatively low concentration of the sample, and the system adaptability is relatively poor, and when the sampling amount is 0.5g, part of the sample is not completely dissolved, and the extraction effect is reduced, so that it is recommended to select the sampling amount to be 0.4g.
(4) Determination of preparation method of test solution
Taking a proper amount of the product, grinding, precisely weighing about 0.4g, placing into a conical flask with a plug, precisely adding 25ml of 50% methanol, weighing, carrying out ultrasonic treatment (power 200W and frequency 53 kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by 50% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the product.
Example 9 methodological validation
1. Specificity
The sample solution (lot number: 2109001S) and the negative control solution (0.4 g of maltodextrin was weighed instead of the sample solution to prepare the negative control solution according to the same method as the sample solution in example 8) prepared in example 8 were taken, HPLC analysis was performed under the chromatographic conditions described in example 8, and a chromatogram was recorded, as shown in FIG. 14 and FIG. 15, which shows that the method was good in specificity without interference in the negative result.
TABLE 22 System Adaptation Table
2. Investigation of linear relationships
Taking appropriate amount of chlorogenic acid, caffeic acid peak, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, and brevifolin A reference, precisely weighing, adding 50% methanol to respectively prepare current reference solutions, precisely absorbing 1 μ l of the above 6 reference solutions with different concentrations, injecting into a liquid chromatograph, measuring peak area under the chromatographic conditions described in example 8, drawing a standard curve with the source sample amount as abscissa and the peak area integral value as ordinate, to obtain a linear regression equation
TABLE 23 results of linear relationship of ingredients
3. Precision test
(1) Precision test of instrument
The same centipeda minima formula particle sample solution (batch number: 2109001S) is taken, sample injection is repeated for 6 times according to the chromatographic conditions described in the embodiment 8, and the content and RSD value of chlorogenic acid, caffeic acid, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid and brevifolin A are measured, the RSD value of 9 components is 0.12-1.90%, and the precision of the instrument is good.
(2) Intermediate precision investigation by different personnel
The same batch of centipeda minima formula particles (batch number: 2109001S) are separately and independently operated by A, B and C experimenters, treated by the preparation method of the test sample under the embodiment 8, the contents of chlorogenic acid, caffeic acid, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid and brevifolin A are measured, the RSD values of all the components are calculated, the RSD values of 9 components are between 0.15 and 1.35 percent, and the result shows that the precision of the method is good among different operators.
(3) Repeatability inspection
6 parts of the same batch of centipeda minima formula granules (batch No. 2109001S) are taken, prepared according to the preparation method of the test sample solution, measured according to the chromatographic conditions described in the example 8 and calculated to obtain the content result RSD%. The RSD of the chlorogenic acid content is 1.1 percent, the RSD of the caffeic acid content result is 1.0 percent, and the RSD of the chlorogenic acid, the 3, 5-di-O-caffeoylquinic acid, the 4, 5-di-O-caffeoylquinic acid and the 3, 4-di-O-caffeoylquinic acid is 0.5-1.6 percent, which indicates that the method has good repeatability.
(4) Accuracy survey
Namely, in the sample-adding recovery test, 9 samples (lot number 2109001S) of the same batch of centipeda minima formula particles are precisely weighed, about 0.20g of each sample is placed in a conical flask with a plug, 25ml of control solutions with different concentrations are precisely added, 3 parts of each concentration are respectively added, 2 accompanying samples are prepared simultaneously according to the preparation method of the test sample in example 8, the contents of chlorogenic acid, caffeic acid, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid and brevifolin A are measured, and the RSD value of each component is calculated. The recovery rate is calculated by using the content of the accompanying sample, and the recovery rate of 9 components is 99.60-101.10%. The experimental result shows that the method accuracy meets the regulations.
4. Durability examination
(1) Stability of the solution
The same test sample solution (batch number: 2109001S) of the centipeda minima formula particles is taken, sample injection is carried out at 0, 2, 4, 8, 10, 12 and 24 hours after preparation, peak areas of chlorogenic acid, caffeic acid, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid and brevifolin A are measured according to the chromatographic conditions described in the embodiment 8, the RSD value of each component is calculated, and the RSD value of 9 components is 0.32-1.02%. The experimental result shows that the test solution is basically stable within 24 hours and can meet the measurement requirement.
(2) Durability investigation of different flow rates
The centipeda minima formula particles (batch number: 2109001S) are precisely weighed, are processed according to the preparation method of the test sample in the embodiment 8, and are respectively tested at different flow rates of 0.19ml/min, 0.20ml/min and 0.21ml/min, the contents of chlorogenic acid, caffeic acid, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid and brevifolin A are measured under the chromatographic conditions in the embodiment 8, the RSD value is calculated, the RSD values of 9 components are between 0.2% and 1.3%, and the results show that the different flow rates have small influence on the contents of index components and good stability.
(3) Durability investigation of different column temperatures
The centipeda minima formula particles (batch number: 2109001S) are precisely weighed, are processed according to the preparation method of the test sample in example 8, are respectively tested at different flow rates of 23 ℃, 25 ℃ and 27 ℃, the contents of chlorogenic acid, caffeic acid, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid and brevifolin A are measured according to the chromatographic conditions in example 8 for the rest, and the RSD value is calculated, wherein the RSD values of 9 components are between 0.2% and 1.4%, and the results show that the contents of index components are not greatly influenced by different injection temperatures and the stability is good.
(4) Investigation of different chromatographic column durability
The centipeda minima formula particles (batch number: 2109001S) are precisely weighed, are processed according to the preparation method of the test sample of example 8, are respectively tested under chromatographic columns of the same brand and different models, the contents of chlorogenic acid, caffeic acid, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid and brevifolin A are measured according to the chromatographic conditions described in example 8 for the rest, the RSD value is calculated, the RSD value of 9 components is between 0.3% and 1.4%, and the result shows that the different chromatographic columns have little influence on the contents of index components.
(5) Investigation of durability of different instruments
The centipeda minima formula particles (batch number: 2109001S) are precisely weighed, are processed according to the preparation method of the test sample in the embodiment 8, are respectively tested by different instruments, the contents of chlorogenic acid, caffeic acid, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid and brevifolin A are measured according to the chromatographic conditions in the embodiment 8, the RSD value is calculated, the RSD value of 9 components is between 0.4 and 1.8 percent, and the result shows that different instruments have little influence on the contents of index components and have good stability.
5. Content determination of multiple batches of centipeda minima formula particles
18 batches of the centipeda minima formula granules were treated according to the preparation method of the test sample of example 8, and the contents of chlorogenic acid, caffeic acid and phenolic acids (the total content of chlorogenic acid, 3, 4-di-O-caffeoylquinic acid, 3, 5-di-O-caffeoylquinic acid and 4, 5-di-O-caffeoylquinic acid) were measured according to the chromatographic conditions described in example 8, and the results are shown in the following table.
TABLE 24 determination results of granule amounts of multiple batches of centipeda minima formulas
Comparative example 1
The comparative example differs from example 1 in that the detection wavelength is different in the comparative example, the comparative example discloses that the detection is carried out on the centipeda minima formula granules with the batch number of 2109001S under the wavelength conditions of 325nm and 225nm, the rest of the process and the conditions are as in example 1, and the obtained chromatograms are shown in fig. 16 and 17. The detection result under the single wavelength condition of 325nm and 225nm is obviously worse than that of the embodiment 1, wherein the chromatographic peak under 325nm is mainly distributed before 60min, the number of the characteristic peaks is obviously less than that of the chromatogram constructed in the embodiment 1, the characteristic peaks before 25min under 225nm are distributed in a centralized manner, the separation degree of a plurality of characteristic peaks is lower than 1.5, and the separation effect is worse.
Comparative example 2
The difference between the comparative example and the example 1 is that the types of the chromatographic columns in the comparative example are different, the comparative example discloses that the chromatographic columns of different types are used for detecting the centipeda minima formula granules with the batch number of 2109001S, and the chromatographic columns of different types are as follows:
(1)Waters HSS T3 C18 2.1×150mm 1.8μm；
(2) Thermo Hypersil GOLD aQ 2.1X 150mm 1.9 μm; the rest of the processes and conditions are as in example 1, and chromatograms obtained by detection in chromatographic columns (1) to (2) are shown in fig. 18 and fig. 19. As is clear from comparison with fig. 6, the chromatographic columns of nos. (1) to (2) have unsatisfactory separation effects.
Comparative example 3
The present comparative example is different from example 1 in that the column temperature of the column in the present comparative example is different, and the results of the detection of the centipeda minima formula granules with the lot number of 2109001S at 20 ℃ and 30 ℃ are disclosed in the present comparative example, as shown in fig. 20 and 21, fig. 20 is a chromatogram at 20 ℃ and fig. 21 is a chromatogram at 30 ℃. As can be seen from comparison with fig. 6, the detection result is significantly inferior to that of example 1 in the absence of peaks at 20 ℃ and 30 ℃ compared to 25 ℃.
TABLE 25 examination results of various column temperatures
Comparative example 4
This example differs from example 1 in the procedure of gradient elution, which is shown in table 26:
TABLE 26 gradient elution procedure at various ratios
The sample is detected by the centipeda minima formula particles with the batch number of 2109001S, the fingerprint obtained by detection is shown in figure 22, the baseline is unstable, the stability is poor, the number of characteristic peaks is small, and the constructed fingerprint cannot be used for quality detection.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A method for constructing a fingerprint of a centipeda minima medicinal preparation is characterized in that,
preparing a test solution;
detecting the test solution by adopting ultra-high performance liquid chromatography, wherein the chromatographic conditions comprise that an Agilent InfinityLab poroschel 120SB-Aq chromatographic column is adopted, acetonitrile is taken as a mobile phase A, a water solution containing phosphoric acid is taken as a mobile phase B, and gradient elution is carried out according to the following procedures:
0-15min, the volume ratio of the mobile phase A to the mobile phase B is 3%:97% → 8%: the content of the active ingredients is 92%,
15-20min, the volume ratio of the mobile phase A to the mobile phase B is 8%:92% → 12%:88 percent of the total weight of the mixture,
20-50min, the volume ratio of the mobile phase A to the mobile phase B is 12%:92 → 20%:80 percent of the total weight of the mixture,
50-60min, the volume ratio of the mobile phase A to the mobile phase B is 20%:88% → 20%:80 percent of the total weight of the mixture,
60-70min, the volume ratio of the mobile phase A to the mobile phase B is 20%:80% → 25%:75 percent of the total weight of the mixture,
70-90min, wherein the volume ratio of the mobile phase A to the mobile phase B is 25%:75% → 30%:70 percent of the total weight of the mixture,
90-100min, the volume ratio of the mobile phase A to the mobile phase B is 30%:70% → 35%:65 percent;
the elution time is less than 55-65 minutes, the detection wavelength is 320-330nm, the elution time is more than or equal to 55-65 minutes, the detection wavelength is 210-230nm, the column temperature is 23-27 ℃, and the flow rate is 0.19-0.25mL/min.
2. The method of claim 1, wherein an Agilent InfinityLab Poroshell 120SB-Aq column having a size of 2.1X 150mm,1.9 μm is used; and/or the sample amount of the test solution is 1-3 mu L; and/or the flow rate is 0.20-0.25mL/min; and/or the volume percentage of the phosphoric acid in the phosphoric acid-containing aqueous solution is 0.1-0.4%.
3. The construction method according to claim 1 or 2, wherein the test solution is prepared by: weighing a sample, adding a solvent for extraction to obtain an extracting solution, carrying out solid-liquid separation, and taking a liquid to obtain a sample solution, wherein preferably, the preparation method of the sample solution further satisfies any one or more of the following A-E:
A. the ratio of the mass of the sample to the volume of the solvent is 0.3-0.5:10-50; the relation between the mass and the volume is g/mL;
B. the extraction mode is reflux extraction or ultrasonic extraction;
C. the extraction time is more than or equal to 10min, preferably 15-60min;
D. the solid-liquid separation is selected from centrifugation or filter membrane filtration;
E. the solvent is at least one selected from water, methanol and ethanol, and is preferably water or methanol aqueous solution.
4. The method of construction according to any one of claims 1 to 3, further comprising a step of preparing a control solution by using a solvent for at least one of caffeic acid, chlorogenic acid, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, cryptochlorogenic acid, rutin, arninolide D, cejcin C, and kaempferol-3-O-rutinoside, and a step of detecting the control solution by ultra high performance liquid chromatography in the method of construction according to any one of claims 1 to 4 to obtain a control map; preferably, the solvent is selected from at least one of methanol, ethanol and water.
5. The construction method according to any one of claims 1 to 4, wherein the fingerprint of the centipeda minima medicinal preparation has 26 common characteristic peaks, the peaks corresponding to chlorogenic acid, 3, 5-di-O-caffeoylquinic acid and brevifolin A are respectively an S1 peak, an S2 peak and an S3 peak, the relative retention time of the peaks 1 to 12 and the S1 peak is within +/-10% of a specified value, and the specified value is as follows: 0.15 (peak 1), 0.25 (peak 2), 0.34 (peak 3), 0.43 (peak 4), 0.51 (peak 5), 0.61 (peak 6), 0.89 (peak 7), 0.96 (peak 8), 1.00 (peak 9), 1.22 (peak 10), 1.33 (peak 11), 1.53 (peak 12); the relative retention times of peaks 13-22 and the S2 peak are within ± 10% of the specified values: 0.77 (peak 13), 0.80 (peak 14), 0.84 (peak 15), 0.93 (peak 16), 0.94 (peak 17), 1.00 (peak 18), 1.07 (peak 19), 1.10 (peak 20), 1.22 (peak 21), 1.24 (peak 22); the relative retention times of peaks 23 to 26 and the S3 peak are within ± 10% of the specified values: 0.78 (Peak 23), 0.86 (Peak 24), 0.97 (Peak 25), 1.00 (Peak 26).
6. A content testing method of a centipeda minima medicinal preparation is characterized by comprising the following steps:
preparing a test solution and a reference solution;
detecting the test solution and the reference solution by adopting ultra-high performance liquid chromatography, wherein the chromatographic conditions comprise that an Agilent InfinityLab Poroshell 120SB-Aq chromatographic column is adopted, acetonitrile is taken as a mobile phase A, an aqueous solution containing phosphoric acid is taken as a mobile phase B, and gradient elution is carried out according to the following procedures:
0-15min, the volume ratio of the mobile phase A to the mobile phase B is 3%:97% → 8%: the content of the waste water is 92%,
15-20min, the volume ratio of the mobile phase A to the mobile phase B is 8%:92% → 12%:88 percent of the total weight of the mixture,
20-50min, the volume ratio of the mobile phase A to the mobile phase B is 12%:92 → 20%:80 percent of the total weight of the mixture,
50-60min, wherein the volume ratio of the mobile phase A to the mobile phase B is 20%:88% → 20%:80 percent of the total weight of the mixture,
60-70min, the volume ratio of the mobile phase A to the mobile phase B is 20%:80% → 25%:75 percent of the total weight of the mixture,
70-90min, wherein the volume ratio of the mobile phase A to the mobile phase B is 25%:75% → 30%:70 percent of the total weight of the mixture,
90-100min, the volume ratio of the mobile phase A to the mobile phase B is 30%:70% → 35%:65 percent;
the elution time is less than 55-65 minutes, the detection wavelength is 320-330nm, the elution time is more than or equal to 55-65 minutes, the detection wavelength is 210-230nm, the column temperature is 23-27 ℃, and the flow rate is 0.19-0.25mL/min.
7. The method according to claim 6, wherein an Agilent InfinityLab Poroshell 120SB-Aq column having a size of 2.1X 150mm,1.9 μm is used; and/or the sample amount of the test solution and/or the reference solution is 1-3 mu L; and/or the flow rate is 0.20-0.25mL/min; and/or the volume percentage of the phosphoric acid in the phosphoric acid-containing aqueous solution is 0.1-0.4%.
8. The content determination method according to claim 6 or 7, wherein the preparation method of the test solution is: weighing a sample, adding a solvent for extraction to obtain an extracting solution, carrying out solid-liquid separation, and taking a liquid to obtain a sample solution, wherein preferably, the preparation method of the sample solution further satisfies any one or more of the following A-E:
A. the ratio of the mass of the sample to the volume of the solvent is 0.3-0.5:10-50, preferably 0.4:10-50 parts of; the relation between the mass and the volume is g/mL;
B. the extraction mode is reflux extraction or ultrasonic extraction;
C. the extraction time is more than or equal to 10min, preferably 15-60min;
D. the solid-liquid separation is selected from centrifugation or filter membrane filtration;
E. the solvent is at least one selected from water, methanol and ethanol.
9. The content determination method according to any one of claims 6 to 8, wherein a control solution is prepared by adding a solvent to at least one of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, brevifolin A, and caffeic acid, preferably, the solvent is at least one selected from methanol, ethanol, and water.
10. A quality detection method of a centipeda minima medicinal preparation is characterized by comprising the step of comparing the fingerprint of a centipeda minima product to be detected with the reference fingerprint of the centipeda minima medicinal preparation and/or the step of determining the content of the centipeda minima product to be detected by adopting the content test method of the centipeda minima medicinal preparation of any one of claims 6 to 9; the fingerprint of the centipeda minima product to be detected is obtained by constructing the centipeda minima product to be detected according to the construction method of any one of claims 1 to 5, and the control fingerprint of the medicinal preparation of the centipeda minima is selected from any one of the following (1) to (3):
(1) The centipeda minima fingerprint spectrum has 26 common characteristic peaks, the peaks corresponding to chlorogenic acid, 3, 5-di-O-caffeoylquinic acid and brevifolin A are respectively an S1 peak, an S2 peak and an S3 peak, the relative retention time of the peaks from 1 to 12 and the S1 peak is within +/-10% of a specified value, and the specified value is as follows: 0.15 (peak 1), 0.25 (peak 2), 0.34 (peak 3), 0.43 (peak 4), 0.51 (peak 5), 0.61 (peak 6), 0.89 (peak 7), 0.96 (peak 8), 1.00 (peak 9), 1.22 (peak 10), 1.33 (peak 11), 1.53 (peak 12); the relative retention times of peaks 13 to 22 and the S2 peak are within ± 10% of the specified values: 0.77 (peak 13), 0.80 (peak 14), 0.84 (peak 15), 0.93 (peak 16), 0.94 (peak 17), 1.00 (peak 18), 1.07 (peak 19), 1.10 (peak 20), 1.22 (peak 21), 1.24 (peak 22); the relative retention times of peaks 23-26 and the S3 peak are within ± 10% of the specified values: 0.78 (peak 23), 0.86 (peak 24), 0.97 (peak 25), 1.00 (peak 26);
(2) Fingerprint of the centipeda minima pharmaceutical preparation obtained by the construction method according to any one of claims 1-5 by using a single batch or multiple batches of the centipeda minima pharmaceutical preparation;
(3) Preparing a control fingerprint by using fingerprints obtained by a plurality of batches of the pharmaceutical preparation of the centipeda minima according to the construction method of any one of claims 1 to 5 through a mean value method or a median method.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103235064A (en) * | 2013-05-16 | 2013-08-07 | 韩燕全 | Construction method of UPLC (Ultra Performance Liquid Chromatography) finger print and standard finger print of cocklebur fruit medicinal material |
| WO2017148426A1 (en) * | 2016-03-03 | 2017-09-08 | 石家庄以岭药业股份有限公司 | Method for determining fingerprint of chinese medicine composition |
| WO2017148418A1 (en) * | 2016-03-03 | 2017-09-08 | 石家庄以岭药业股份有限公司 | Method for determining component contents of chinese medicine composition |
| CN112858526A (en) * | 2021-03-05 | 2021-05-28 | 华润三九医药股份有限公司 | Centipeda minima fingerprint spectrum and construction method thereof |
| CN114689775A (en) * | 2020-12-31 | 2022-07-01 | 北京同仁堂科技发展股份有限公司 | Peony and licorice root decoction fingerprint spectrum, construction method thereof and detection method of peony and licorice root decoction product |
| CN114814042A (en) * | 2022-06-10 | 2022-07-29 | 广东一方制药有限公司 | Method for constructing and detecting characteristic spectrum of honeysuckle-forsythia medicine pair preparation |
-
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103235064A (en) * | 2013-05-16 | 2013-08-07 | 韩燕全 | Construction method of UPLC (Ultra Performance Liquid Chromatography) finger print and standard finger print of cocklebur fruit medicinal material |
| WO2017148426A1 (en) * | 2016-03-03 | 2017-09-08 | 石家庄以岭药业股份有限公司 | Method for determining fingerprint of chinese medicine composition |
| WO2017148418A1 (en) * | 2016-03-03 | 2017-09-08 | 石家庄以岭药业股份有限公司 | Method for determining component contents of chinese medicine composition |
| CN114689775A (en) * | 2020-12-31 | 2022-07-01 | 北京同仁堂科技发展股份有限公司 | Peony and licorice root decoction fingerprint spectrum, construction method thereof and detection method of peony and licorice root decoction product |
| CN112858526A (en) * | 2021-03-05 | 2021-05-28 | 华润三九医药股份有限公司 | Centipeda minima fingerprint spectrum and construction method thereof |
| CN114814042A (en) * | 2022-06-10 | 2022-07-29 | 广东一方制药有限公司 | Method for constructing and detecting characteristic spectrum of honeysuckle-forsythia medicine pair preparation |
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