CN115389654B

CN115389654B - Construction method and content testing method of fingerprint of centipeda minima medicinal preparation

Info

Publication number: CN115389654B
Application number: CN202210932265.XA
Authority: CN
Inventors: 江斌; 刘华明; 张辉; 赵伟志; 谭沛; 李璐
Original assignee: China Resources Sanjiu Modern Traditional Chinese Medicine Pharmaceutical Co ltd
Current assignee: China Resources Sanjiu Modern Traditional Chinese Medicine Pharmaceutical Co ltd
Priority date: 2022-08-04
Filing date: 2022-08-04
Publication date: 2023-10-27
Anticipated expiration: 2042-08-04
Also published as: CN115389654A

Abstract

The invention relates to the field of detection of traditional Chinese medicine preparations, and particularly provides a method for constructing a fingerprint spectrum of a pharmaceutical preparation of Centipeda minima and a content testing method, wherein a sample solution is adopted for super-performance liquid chromatography detection, chromatographic conditions comprise adopting a Agilent InfinityLabPoroshell SB-Aq chromatographic column, acetonitrile as a mobile phase A, and a 0.2% phosphoric acid solution as a mobile phase B, and eluting according to a program defined in the description. The invention can effectively and comprehensively detect the pharmaceutical preparation of the centipeda minima.

Description

Construction method and content testing method of fingerprint of centipeda minima medicinal preparation

Technical Field

The invention relates to the field of traditional Chinese medicine detection, in particular to a method for constructing a fingerprint spectrum of a pharmaceutical preparation of centipeda minima and a content testing method.

Background

The centipeda minima formula granule is prepared by extracting, concentrating and granulating traditional Chinese medicine centipeda minima. The herba Centipedae is dry whole herb of Centipeda minima (L.) A.Br.et ascers of Compositae, mainly contains volatile oil, sterols, flavonoids, triterpenes, guaiac esters and pseudo guaiac lactone, and is mainly used for treating wind-cold headache, cough, traumatic sprain, pertussis, malaria, acute and chronic rhinitis, allergic rhinitis, etc.

The quality control of the centipeda minima in the 2020 edition of Chinese pharmacopoeia comprises the items of original plant species, decoction piece processing, decoction piece characters, physical and chemical identification, extract content measurement and the like, and the literature also describes the chemical components in the centipeda minima, including compounds containing volatile oil, sterols, flavonoids, triterpenes, guaiac esters, pseudo guaiac lactones and the like. However, on the one hand, the Centipeda minima formula particles cannot be detected and controlled on the whole by the content measurement or identification of the above-mentioned single components; on the other hand, the identification of the centipeda minima formula particles by the content measurement of a single component and the combination of other components is time-consuming and labor-consuming, and is difficult to be widely applied to production practice.

In the prior art, the quality control of the centipeda minima is aimed at the centipeda medical material, but not the pharmaceutic preparation aimed at the centipeda minima, and the construction method aimed at the centipeda medical material in the prior art cannot be suitable for the pharmaceutic preparation of the centipeda minima. Therefore, a method for comprehensively and rapidly detecting the pharmaceutical preparation of the centipeda minima is established, and the method has important significance for the comprehensive quality detection and the overall quality control of the pharmaceutical preparation.

The laboratory discloses a construction method of a centipeda minima fingerprint in Chinese patent document CN112858526A, wherein the fingerprint only has 14 characteristic peaks, and the detection time is 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 method for constructing the fingerprint of the centipeda herb preparation disclosed in the prior art, thereby providing a method for constructing the fingerprint of the centipeda herb preparation and a method for testing the content of the centipeda herb preparation.

A method for constructing fingerprint of herba Centipedae pharmaceutical preparation comprises preparing sample solution;

the method comprises the steps of detecting a sample solution by adopting an ultra-high performance liquid chromatography, wherein chromatographic conditions are that an Agilent InfinityLab Poroshell 120 SB-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%: 92%,

15-20min, the volume ratio of A to B is 8%:92% -12%: 88 percent,

20-50min, wherein the volume ratio of A to B is 12%: 92-20%: 80 percent,

50-60min, the volume ratio of A to B is 20%:88% -20%: 80 percent,

60-70min, wherein the volume ratio of A to B is 20%:80% -25%: 75 percent,

70-90min, wherein the volume ratio of A to B is 25%:75% -30%: 70 percent,

90-100min, the volume ratio of A to B is 30%:70% -35%: 65%;

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, a Agilent InfinityLab Poroshell 120 SB-Aq column with a specification of 2.1X105 mm,1.9 μm was used; and/or the sample injection amount of the sample solution is 1-3 mu L; and/or a flow rate of 0.20-0.25mL/min; and/or the volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid is 0.1-0.4%.

Further, the preparation method of the sample solution comprises the following steps: weighing the sample, extracting with solvent to obtain extractive solution, separating solid from liquid, and collecting the liquid to obtain sample solution.

The preparation method of the sample solution also satisfies any one or more of the following A-E:

A. the ratio of the mass of the test 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 filtration by a filter membrane;

E. the solvent is at least one selected from water, methanol and ethanol, preferably water or methanol aqueous solution.

Illustratively, the method of preparing the test solution is: taking a sample, precisely adding a solvent, sealing, weighing, performing ultrasonic treatment, taking out, cooling, weighing again, supplementing the reduced weight with the solvent, shaking uniformly, filtering, and collecting the subsequent filtrate.

Further, the construction method further comprises the step of preparing a reference substance solution by using 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 lactone D, eleutheroside C and kaempferol-3-O-rutinoside as a solubilizer, and the step of detecting the reference substance solution according to the ultra-high performance liquid chromatography in any of the construction methods to obtain a reference substance map; preferably, the solvent is selected from methanol and/or ethanol.

Further, the fingerprint of the pharmaceutical preparation of the centipeda minima has 26 common characteristic peaks, the peaks corresponding to chlorogenic acid, 3, 5-di-O-caffeoylquinic acid and Brevibacterium sarcinum A are respectively S1 peak, S2 peak and 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: 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 time of the peaks 13 to 22 and the S2 peak is within + -10% of the prescribed value, the prescribed value is: 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 time of peaks 23 to 26 and S3 is within + -10% of the prescribed value, which is: 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 pharmaceutical preparation of the centipeda minima, which comprises the following steps:

preparing a sample solution and a reference substance solution;

detecting the sample solution and the reference substance solution by adopting an ultra-high performance liquid chromatography, wherein chromatographic conditions comprise adopting a Agilent InfinityLab Poroshell SB-Aq chromatographic column, taking acetonitrile as a mobile phase A, taking an aqueous solution containing phosphoric acid as a mobile phase B, and performing gradient elution according to the following procedures:

0-15min, the volume ratio of A to B is 3%:97% -8%: 92%,

15-20min, the volume ratio of A to B is 8%:92% -12%: 88 percent,

20-50min, wherein the volume ratio of A to B is 12%: 92-20%: 80 percent,

50-60min, the volume ratio of A to B is 20%:88% -20%: 80 percent,

60-70min, wherein the volume ratio of A to B is 20%:80% -25%: 75 percent,

70-90min, wherein the volume ratio of A to B is 25%:75% -30%: 70 percent,

90-100min, the volume ratio of A to B is 30%:70% -35%: 65%;

Further, a Agilent InfinityLab Poroshell 120SB-Aq column with a specification of 2.1X105 mm,1.9 μm was used; and/or the sample injection amount of the sample solution is 1-3 mu L; and/or a flow rate of 0.20-0.25mL/min; and/or the volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid is 0.1-0.4%.

Further, the preparation method of the sample solution comprises the following steps: weighing a test sample, adding a solvent for extraction to obtain an extracting solution, carrying out solid-liquid separation, and taking the liquid as a test sample solution, wherein the preparation method of the test sample solution preferably further satisfies any one or more of the following A-E:

A. the ratio of the mass of the test sample to the volume of the solvent is 0.3-0.5:10-50, preferably 0.4:10-50; 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;

E. the solvent is at least one selected from water, methanol and ethanol.

Further, 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, geraniin A and caffeic acid is used for preparing a reference substance solution, and preferably, the solvent is at least one selected from methanol, ethanol and water.

The invention also provides a quality detection method of the pharmaceutical preparation of the centipeda minima, which comprises the step of comparing the fingerprint of the product of the centipeda minima to be detected with the reference fingerprint of the pharmaceutical preparation of the centipeda minima and/or the step of measuring the content of the product of the centipeda minima by adopting the content test method of any one of the pharmaceutical preparations of the centipeda minima; the fingerprint of the centipeda minima product to be detected is obtained by constructing the centipeda minima product to be detected according to any one of the construction methods, and the reference fingerprint of the centipeda minima pharmaceutical preparation is selected from any one of the following (1) - (3):

(1) 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 Brevibacterium officinalis A are respectively S1 peak, S2 peak and S3 peak, the relative retention time of the peaks 1-12 and the S1 peak is within +/-10% of a specified value, and the specified value is: 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 time of the peaks 13 to 22 and the S2 peak is within + -10% of the prescribed value, the prescribed value is: 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 time of peaks 23 to 26 and S3 is within + -10% of the prescribed value, which is: 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 obtained by using single-batch or multi-batch centipeda minima medicinal preparation according to any one of the above construction methods;

(3) The fingerprint obtained by using a plurality of batches of the pharmaceutical preparation of the centipeda minima according to any one of the construction methods is prepared into a control fingerprint by an average value or a median method.

Wherein the centipeda minima product to be detected is a pharmaceutical preparation of the centipeda minima.

The similarity of the centipeda minima formula particles in comparison with the fingerprint is greater 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 120-SB-Aq chromatographic column, the specific elution program and the control of the column temperature and the flow rate are adopted, so that the detection time is shortened, the separation effect of various active ingredients is obviously improved, more characteristic peaks are contained in the fingerprint, and the spectrum information is greatly enriched.

2. According to the content testing method of the pharmaceutical preparation of the centipeda minima, 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, geraniin A, caffeic acid and other components can be measured simultaneously by adopting a Agilent InfinityLab Poroshell 120-SB-Aq chromatographic column, a specific elution program and control of column temperature and flow rate, the separation degree of each component is good, and the construction method has high precision, stability and repeatability, so that the effective components and the content of the effective components in the centipeda minima prescription granule can be comprehensively and rapidly detected.

3. According to the method for constructing the fingerprint of the centipeda minima medicinal preparation, chlorogenic acid (S1 peak), 3, 5-di-O-caffeoyl quinic acid (S2 peak) and geraniin 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, 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-caffeoyl quinic acid (S2 peak) and the geraniin A (S3 peak), so that the comprehensive quality detection and the overall quality control of the centipeda minima formula particles are facilitated, and the use safety and the use stability of the medicine are improved.

4. The quality detection method of the finger print of the pharmaceutical preparation of the centipeda minima can simultaneously construct the finger print of the centipeda minima to be detected, measure the content, 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 that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a fingerprint of 18 batches of Centipeda minima formula particles obtained by high performance liquid chromatography in example 1 of the present invention; FIG. 2 is a control fingerprint generated by 18 batches of Centipeda minima formula particles in example 1 of the present invention; FIG. 3 is one of the localization maps of the comparison fingerprint in example 1 of the present invention and the comparison products of different characteristic peaks; FIG. 4 is a second positioning chart of the comparison fingerprint in the embodiment 1 of the present invention and the comparison products with different characteristic peaks; FIG. 5 is a precision experimental chromatogram of the Centipeda minima formula particle of lot number 2109001S; FIG. 6 is a fingerprint of Centipeda minima formula particles in the specificity 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 condition of 0.4% phosphoric acid solution in example 3 of the present invention; FIG. 10 is a fingerprint spectrum at a flow rate of 0.15mL/min in example 4 of the present invention; FIG. 11 is a fingerprint spectrum 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 (2) in example 7 of the present invention; FIG. 13 is a fingerprint obtained by liquid phase detection of (3) in example 7 of the present invention; FIG. 14 is a fingerprint of Centipeda minima formula particles in the specificity experiment of example 9 of the present invention; FIG. 15 is a fingerprint of a negative control solution in the specificity experiment of example 9 of the present invention; FIG. 16 is a fingerprint spectrum under the condition of a detection wavelength of 325nm in comparative example 1 of the present invention; FIG. 17 is a fingerprint spectrum under the condition of a detection wavelength of 225nm in comparative example 1 of the present invention; FIG. 18 is a fingerprint obtained by detection on the column (2) in comparative example 2 of the present invention; FIG. 19 is a fingerprint obtained by detection on the column (3) in comparative example 2 of the present invention; FIG. 20 is a fingerprint of comparative example 3 of the present invention at a column temperature of 20deg.C; FIG. 21 is a fingerprint of comparative example 3 of the present invention at a column temperature of 30 ℃; fig. 22 is a fingerprint obtained in comparative example 4 of the present invention.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The instruments and reagents used in the invention are as follows:

high performance liquid chromatograph 1: waters Arc UPLC, PDA detector, empower chromatography management system; high performance liquid chromatography 2: thermo Vanquish ultra high performance liquid phase, pump, colum Compartment, autosumpler, photometer; high performance liquid chromatograph 3: agilent 1290 ultra high performance liquid phase, pump, colum Compartment, autosimpler, photometer DAD; electronic analytical balance: METTER TOLEDO (Metrehler, switzerland) XS-205, XS-204, XSE-205, XPE56; ultrasonic generator: SK5200H Shanghai Kogyo ultrasonic instruments Co., ltd; ultrapure water system: millipore, inc., milribo, USA; acetonitrile was chromatographic pure (Merk, germany), water was ultrapure water (resistivity 18.2mΩ.cm), and the other reagents were analytical pure; chromatographic column: (1) Agilent InfinityLab Poroshell 120 SB-Aq 2.1X105 mm 1.9 μm; (2) Waters HSS T3C 18.1X105 mm 1.8 μm; (3) Thermo Hypersil GOLD aQ 2.1.1X105 mm 1.9 μm; chlorogenic acid reference (lot number: 110753-202018, national food and drug verification institute); 3, 5-O-dicaffeoylquinic acid control (lot number: 111782-201807, china food and drug inspection institute); 4, 5-O-dicaffeoylquinic acid (lot number: 111894-202104, purity: 98%, shanghai Yongsheng Biotechnology Co., ltd.); geraniin A reference (lot number: 112067-202001, national institute of food and drug testing); chlorogenic acid (lot number: 19011731, purity: 98%, shanghai Hotan Biotechnology Co., ltd.); caffeic acid (lot number: 110885-201603, purity: 100%, national food and drug verification institute); rutin (lot number 100080-202012, purity 91.7%, national food and drug verification institute); 3, 4-O-dicaffeoylquinic acid (lot number: 250035-201907, purity: 98%, shanghai Yongsheng Biotechnology Co., ltd.); arnica lactone D (lot number: 190141-201907, purity: 98%, shanghai Yongsheng Biotechnology Co., ltd.); carpesium chrysanthemic acid C (lot number 240095-201609, purity 95%, shanghai HongYongsheng Biotech Co., ltd.); centipeda minima control drug (lot number: 121053-201634, china food and drug inspection institute). Fingerprint method grassing granule lot number: 2109001S

The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

Example 1

A method for constructing a fingerprint of a pharmaceutical preparation of Centipeda minima comprises the following steps:

(1) Preparation of test solution: taking about 0.2g of the herba Centipedae medicinal preparation powder, precisely weighing, placing into a conical flask with a plug, adding 25ml of 50% methanol, performing ultrasonic treatment (power 200W, frequency 53 kHz) for 30 minutes, taking out, cooling, shaking, filtering, and collecting the subsequent filtrate. The pharmaceutical preparation of the centipeda minima is prepared by the following steps:

heating and reflux-extracting herba Centipedae decoction pieces for at least 1 time, adding 6-12 times of water by weight for extraction for at least 0.5h each time, filtering, mixing filtrates, concentrating the filtrate to relative density of 1.05-1.10g/mL at 60deg.C, adding conventional adjuvants, and making into clinically acceptable tablet, capsule, pill, granule, honeyed pill, delayed release preparation, quick release preparation, controlled release preparation, oral liquid preparation or injection according to conventional process. The pharmaceutically acceptable auxiliary materials are as follows: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, matrices, and the like. The filler comprises: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, and the like; the disintegrating agent comprises: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, crosslinked sodium carboxymethyl cellulose, and the like; 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 binder includes starch slurry, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; the sweetener comprises: saccharin sodium, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid, etc.; the flavoring agent comprises: sweetener and various flavors; the preservative comprises: nipagin, benzoic acid, sodium benzoate, sorbic acid and salts thereof, benzalkonium bromide, chlorhexidine acetate, eucalyptus oil and the like; the matrix comprises: PEG6000, PEG4000, insect wax, and the like.

The sample in this embodiment is a Centipeda minima formula particle, and the specific preparation method of the Centipeda minima formula particle comprises the following steps: taking herba Centipedae decoction pieces, heating and reflux-extracting for 2 times, firstly adding 12 times of water by weight for soaking for 30min, heating and reflux-extracting for 0.5h, filtering, secondly adding 10 times of water by weight for extracting for 0.5h, filtering, combining filtrates, concentrating the filtrate to a relative density of 1.05g/mL at 60 ℃, spray-drying, adding auxiliary material maltodextrin into dry powder, uniformly mixing, granulating by a dry method, and making into granules.

(2) Chromatographic conditions of ultra-high performance liquid chromatography

Chromatographic column: agilent InfinityLab Poroshell 120 SB-Aq chromatography column (2.1X105 mm 1.9 μm); gradient elution was performed using acetonitrile-0.2% phosphoric acid solution as mobile phase as shown in table 1; flow rate: 0.2mL/min; the column temperature is 25 ℃; the detection wavelength was 325nm (first 60 minutes) and post-conversion was 225nm. The theoretical plate number is not less than 5000 according to chlorogenic acid peak calculation, and the sample injection amount is: 1 mul.

TABLE 1 gradient elution procedure

The common mode of the centipeda minima formula particle fingerprint is established according to the method.

15 batches of samples of the centipeda minima formula particles are taken, and the fingerprint of the centipeda formula particles is obtained 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, S: 2007001W, S: 2007001S, S: 2108001S, S18:2109001S. R (26) is a control fingerprint of the centipeda minima formula particle.

Preparation of reference solution: taking a proper amount of reference substances 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 geraniin A, precisely weighing, placing into a brown measuring flask, and adding methanol to prepare 50 mug of solution containing each reference substance per 1 mL; taking appropriate amounts of reference substances of cryptochlorogenic acid, rutin, arnica lactone D, elemene C and kaempferol-3-O-rutinoside, precisely weighing, and adding methanol to obtain 100 μg of each reference substance per 1 mL. And detecting the reference substance solution according to the high performance liquid HPLC chromatographic analysis conditions to obtain a control map, wherein the control map is shown in figure 3.

The comparison fingerprint is generated by adopting fingerprint similarity evaluation software compiled by the pharmacopoeia committee, namely a 2012 edition of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, as shown in figure 2. The detection results of the fingerprint of the formula particles can be analyzed and compared according to the control fingerprint shown in fig. 2, and the detection results are used for quality control of the formula particles. The specific method for quality control by adopting the reference fingerprint spectrum is as follows:

as can be seen from fig. 1, 2 and 3, 26 characteristic peaks are shown in the control fingerprint. Wherein the retention times of peak 9, peak 18, peak 26 correspond to the retention times of chlorogenic acid, 3, 5-di-O-caffeoylquinic acid, and short She Laohe oxacin a control reference peaks, respectively; the peaks corresponding to chlorogenic acid, 3, 5-di-O-caffeoylquinic acid and geraniin A are respectively S1 peak, S2 peak and S3 peak, and the relative retention time of the peaks 1 to 12 and 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 to 22 and S2 were: 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 S3 were: 0.78 (peak 23), 0.86 (peak 24), 0.97 (peak 25), 1.00 (peak 26), see Table 3.

Calculating the relative retention time of peaks 1-8, peaks 10-12 and S1 of the fingerprint of the 18 batches of centipeda minima formula particles, wherein the relative retention time is within +/-10% of a specified value, and the specified value is: 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); calculating the relative retention time of the peaks 13 to 17, 19 to 22 and the S2 peak by taking the corresponding peak of the 3, 5-di-O-caffeoylquinic acid reference substance peak as the S2 peak, wherein the relative retention time is within the range of +/-10% of a specified value, and the specified value is: 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); calculating the relative retention time of the peaks 23 to 25 and the S3 peak by taking the peak corresponding to the peak of the geraniin A reference substance as the S3 peak, wherein the relative retention time is within +/-10% of a specified value, and the specified value is: 0.78 (peak 23), 0.86 (peak 24, arnitine D), 0.97 (peak 25, core print C). The fingerprint similarity of the 18 batches of centipeda minima formula particles is greater than 0.90, the obtained results are shown in tables 2-5, and the reference substance positioning of different characteristic peaks is shown in figure 3.

TABLE 2 common mode relative Retention time of Centipeda minima formula particles

Table 3 common Pattern matching data for Centipeda minima formula particles

TABLE 4 fingerprint determination of 18 batch of Centipeda minima formula particles

Table 5 results of similarity of fingerprints of 18 batches of Centipeda minima formula particles

The fingerprint with good separation degree of each characteristic peak can be effectively obtained through the construction method in the embodiment, the subsequent experiments prove that the method can also simultaneously measure the contents of 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, chlorogenic acid (S1 peak), 3, 5-di-O-caffeoylquinic acid (S2 peak) and brevifolin A (S3 peak) are taken as internal reference peaks in the fingerprint, 26 common characteristic peaks of the centipeda minima formula particles can be determined, and the relative retention time of each common characteristic peak is calculated according to chlorogenic acid (S1 peak), 3, 5-di-O-caffeoylquinic acid (S2 peak) and brevifolin A (S3 peak), so that the method is beneficial to the quality detection and the overall quality control of the centipeda minima formula particles, thereby being beneficial to improving the safety and the stability of the drug in use.

Example 2

The present example provides the process and results of the above construction method of example 1 for detecting precision, stability, repeatability, etc., and is specifically as follows:

1 precision degree

1.1 precision experiments

Taking the same sample solution (the centipeda minima formula granule with batch number 2109001S) prepared in the method of example 1, repeating sample injection for 6 times according to the chromatographic condition in the item (2) of example 1, and determining the relative retention time of 26 common peaks, wherein the relative retention time RSD value of each peak is respectively as follows: 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%, and 0% are shown in Table 6, and the chromatogram of the precision test is shown in FIG. 5.

TABLE 6 similarity results 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 similarity with the reference fingerprint of the centipeda minima formula particle is greater than 0.90, which indicates that the precision is better.

1.2 repeatability experiments

6 parts of the same sample (sample number 2109001S) were taken, and the relative retention times of 26 common peaks were measured in the same manner as in example 1, and the relative retention times RSD values of the peaks were: the results of 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%, and 0% are shown in table 7.

Table 7 similarity results of repeatability experiments (n=6)

As shown in Table 7, 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 respectively greater than 0.90, which indicates that the method has better repeatability.

2 specificity experiments

The sample is prepared from herba Centipedae formula particles with lot number of 2109001S, a sample solution is prepared according to the method of the embodiment 1, 0.4g of maltodextrin is weighed to replace the sample, a negative control solution is prepared according to the same method of the sample solution, the detection is performed according to the method of the embodiment 1, the detection result is shown in fig. 6 and 7, and the result shows that the negative control solution has no interference.

3 stability test

The same sample (lot 2109001S) was sampled as in example 1, and samples were taken at 0, 2, 4, 8, 12 and 24 hours, respectively, to determine the relative retention times of 26 common peaks, and the relative retention time RSD values of the peaks were: the results of 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%, and 0% are shown in table 8.

Table 8 similarity results of stability experiments

As shown in Table 8, 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 respectively greater than 0.90; this indicates that the test solution is stable within 24 hours and meets the assay requirements.

Example 3

The difference between this example and example 1 is that the concentration of each mobile phase B in this example is different, and is 0.1% phosphoric acid solution and 0.4% phosphoric acid solution. The detection is carried out by adopting the centipeda minima formula particles with the batch number of 2109001S, and the detection results are shown in fig. 8 and 9. As can be seen from FIGS. 8 and 9, the 0.1-0.4% phosphoric acid solution is effectively applicable to the separation of all components of the Centipeda minima formula granule.

TABLE 9 examination results of different concentrations of phosphoric acid-acetonitrile mobile phase system

Example 4

The difference between this example and example 1 is that the flow rates in this example are different, 0.15mL/min and 0.25mL/min, respectively. The detection is carried out by adopting the centipeda minima formula particles with the batch number of 2109001S, and the detection results are shown in fig. 10 and 11. As can be seen from the graphs 10 and 11, the characteristic spectrum analysis method of the Centipeda minima formula particles is greatly influenced under the condition of the flow rate of 0.15-0.25 mL/min, and 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 0.15 mL/min. And when the flow rate is 0.20 and 0.25mL/min, the separation effect of all components of the centipeda minima formula particle is good, and the peak information amount is more.

Table 10 results of different flow rate investigation

Example 5

The difference between this example and example 1 is that the sample amounts in this example are different, and the detection results were examined when the sample amounts were 1. Mu.L, 2. Mu.L, and 3. Mu.L, respectively. Namely, the centipeda minima formula particles with the batch number of 2109001S are adopted for detection, and the system adaptability parameters of different sample injection amounts are shown in table 11.

TABLE 11 System Adaptation parameters of chromatographic peaks for different sample injection volumes

As can be seen from table 11: the detection requirement can be met by taking the number of detected chromatographic peaks and the system adaptability parameters of each chromatographic peak as the evaluation indexes and 1 mu L-3 mu L.

Example 6

The difference between this example and example 1 is that the batch number 2109001S of the Centipeda minima formula particles was used for detection, and the sample solution treatment parameters in this example were different. The method comprises the following steps:

(1) Investigation of different extraction modes

In this example, water bath reflux and ultrasound were examined, the rest of the process and conditions were as in example 1, and the results showed that the chromatogram separation effect of each extraction method was substantially consistent, wherein the chromatogram separation effect 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 above different solvent extraction treatments were as shown in table 12.

TABLE 12 System Adaptation parameters of chromatograms of different extraction modes

From the chromatogram and the results shown in Table 12, there was no obvious difference in the dissolution effect and the chromatogram of the different extraction modes.

(2) Investigation of different extraction solvents

In the embodiment, water, ethanol, methanol, 50% methanol and 50% ethanol are examined, the rest processes and conditions are as in the embodiment 1, the result shows that the chromatogram separation effect of each extraction solvent is basically consistent, and the extraction effects of water, ethanol, methanol, 50% methanol and 50% ethanol on the centipeda minima formula particles are examined, so that the extraction solvents can better extract the tested substances, the separation degree of each peak is good, the number of mixed peaks is small, the chromatogram baseline 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 the table 13.

TABLE 13 System Adaptation parameters of chromatograms of different extraction solvents

As can be seen from the combination of the detection patterns in Table 13, the extraction effect of the addition of different extraction solvents on the centipeda minima formula particles is similar, wherein the preferred extraction solvents are water or methanol aqueous solution, and the peak type of the obtained chromatograms is better and the system adaptability parameters are relatively best when the preferred extraction solvents are 50% methanol.

(2) Investigation of extraction time with methanol as the extraction solvent

According to the determined extraction solvent, different ultrasonic times are examined: the extraction effect of the centipeda minima formula particles is 15min, 30min and 45min, and the rest processes and conditions are as in example 1. The system adaptation parameters for the different extraction times are shown in table 14.

TABLE 14 parameters of the chromatograms of Centipeda minima formula particles at different extraction times

From the results shown in Table 14, there was no significant difference in the dissolution effect and the chromatogram at different ultrasonic times.

(3) Selection of the amount of extraction solvent to be added

Based on the determined extraction solvent (50% methanol), different addition amounts of methanol were examined: the extraction effect of 10mL, 25mL and 50mL on the centipeda minima formula particles, the rest processes and conditions are as in example 1, and the system adaptability parameters of the addition amounts of different extraction solvents are shown in Table 15.

TABLE 15 systematic adaptive parameters of chromatographic peaks for different amounts of extraction solvent

As shown in Table 15, the extraction effect of the addition of the extraction solvents with different volumes on the Centipeda minima formula particles was similar, and the chromatographic separation effect was not significantly different.

Example 7

The difference between this embodiment and embodiment 1 is that the brands of the instruments in this embodiment are different, and in this embodiment, it is disclosed that the instruments of different brands detect the Centipeda minima formula particles with a lot number of 2109001S, and the chromatographic columns of different models are as follows:

(1)Waters Arc UPLC；

(2) A Thermo Vanquish ultra high performance liquid phase;

(3) Agilent 1290 ultra-high performance liquid phase; the remaining process and conditions were as in example 1.

The chromatograms obtained by the detection of the (1) th instrument are shown in fig. 6, and the chromatograms obtained by the detection of the (2) to (3) th instruments are shown in fig. 12 and 13. As can be seen from the detection results, there was no significant difference in chromatographic separation effect.

Table 16 results of investigation by different chromatographs

Example 8 a method for determining the content of a pharmaceutical formulation of Centipeda minima, comprising:

(1) Octadecylsilane chemically bonded silica is used as filler (column length 150mm, inner diameter 2.1mm, particle diameter 1.9 μm); acetonitrile is taken as a mobile phase A,0.2% phosphoric acid is taken as a mobile phase B, and gradient elution is carried out according to the specifications in the following table; the flow rate is 0.2ml per minute; the column temperature is 25 ℃; the detection wavelength was 325nm (first 60 minutes) and post-conversion was 225nm. The theoretical plate number should be not less than 5000 calculated as chlorogenic acid peak.

(2) Preparing reference solution, namely accurately weighing 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, 4, 5-di-O-caffeoylquinic acid and a proper amount of geraniin A reference, and adding 50% methanol to prepare mixed solution containing 0.05mg each 1 ml.

(3) The sample solution is prepared into a proper amount of the sample, the sample is ground, about 0.4g is taken, precisely weighed, placed into a conical flask with a plug, added with 25ml of 50% methanol, subjected to ultrasonic treatment (power 200W and frequency 53 kHz) for 30 minutes, taken out, cooled, shaken uniformly, filtered, and the subsequent filtrate is taken, thus obtaining the product.

(4) The measurement method comprises precisely sucking 1 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.

Example 9

The difference between this example and example 8 is that the batch number 2109001S of the Centipeda minima formula particles was used for detection, and the sample solution treatment parameters in this example were different. The method comprises the following steps:

(1) Investigation of different extraction modes

This example examined water bath reflux and ultrasound, the rest of the process and conditions were as in example 8, specifically: respectively 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, respectively refluxing in water bath and performing ultrasonic treatment (power 200W, frequency 53 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking uniformly, filtering, and taking subsequent filtrate.

TABLE 17 results of measurement of the content of index Components of different extraction modes

RSD values are less than 2.0%, the extraction content is low in a water bath reflux mode, and ultrasound is recommended to be used as a sample extraction mode.

(2) Investigation of different extraction solvents

This example examined water, ethanol, methanol, 50% ethanol, the remainder of the process and conditions were as in example 8, and were as follows: 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 water, methanol, ethanol, 50% methanol and 50% ethanol, weighing, performing ultrasonic treatment (power 200W, frequency 53 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking, filtering, and taking the subsequent filtrate.

TABLE 18 determination of the content of index Components of different extraction solvents

From the results, the content of index components of different extraction volumes is close, and is consistent with the construction method, and 50% methanol is recommended to be selected as the extraction solvent of the sample.

(3) Investigation of different extraction times

Examine different ultrasound times: the extraction effect of the centipeda minima formula particles is 15min, 30min and 60min, and the rest processes and conditions are as in example 8. The method comprises the following steps: respectively 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, respectively performing ultrasonic treatment (power 200W and frequency 53 kHz) for 15 minutes, 30 minutes and 60 minutes, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking uniformly, filtering, and taking subsequent filtrate.

TABLE 19 determination of the content of the index Components of different extraction volumes

The results show that the contents of index components of different extraction volumes are close, and the RSD values are smaller than 2.0%, which indicates that the extraction effect of the extraction time is close.

(3) Selection of the amount of extraction solvent to be added

Based on the determined extraction solvent (50% methanol), different addition amounts of methanol were examined: the extraction effect of 10mL, 25mL and 50mL on the centipeda minima formula particles is as in example 8, and the other processes and conditions are as follows, specifically, a proper amount of the product is taken, ground, about 0.4g is taken, precisely weighed, placed in a conical flask with a plug, precisely added with 10mL of 50% methanol, weighed, subjected to ultrasonic treatment (power 200W, frequency 53 kHz) for 30 minutes, cooled, weighed again, and the reduced weight is complemented by 50% methanol, shaken uniformly, filtered, and the subsequent filtrate is taken, thus obtaining the centipeda minima.

TABLE 20 determination of the content of different extraction volume index Components

As can be seen from the results, the contents of the index components of different extraction volumes are close, which indicates that 10-50ml of extraction solvent can be used.

(3) Selection of the sampling quantity

Considering the influence of the sampling amount of the centipeda minima formula particles on the extraction effect, taking a proper amount of the sample, grinding, taking about 0.3g, 0.4g or 0.5g, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of 50% methanol, weighing, performing ultrasonic treatment (power 200W and frequency 53 kHz) for 30 minutes, cooling, weighing again, supplementing the reduced weight with 50% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the centipeda minima.

TABLE 21 determination of the content of different extraction volume index Components

As a result, the contents of the different pressure-removing index components were close, indicating that the extraction effect was close, but it was found that, in the course of the operation, the response of the chromatographic peak was low due to the relatively low concentration of the sample at 0.3g, the system adaptability was relatively poor, and at 0.5g, the extraction effect was reduced because part of the sample was not completely dissolved, and therefore, it was recommended to select the sampling amount to be 0.4g.

(4) Determination of the method for preparing a sample solution

Taking a proper amount of the product, grinding, taking about 0.4g, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of 50% methanol, weighing, performing ultrasonic treatment (power 200W, frequency 53 kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking uniformly, filtering, and taking the subsequent filtrate.

Example 9 methodological verification

1. Specialization of

The sample solution (lot number: 2109001S) and the negative control solution (0.4 g of maltodextrin was weighed to replace the sample and prepared in the same manner as the sample solution of example 8) obtained in example 8 were analyzed by HPLC under the chromatographic conditions described in example 8, and chromatograms were recorded, see FIG. 14 and FIG. 15, and the result was negative without interference, indicating that the method was excellent in specificity.

Table 22 System Adaptation Table

2. Linear relationship investigation

Providing 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 Geraniin A reference substances, precisely weighing, adding 50% methanol to obtain current reference substance solutions, precisely sucking 1 μl of above 6 reference substance solutions with different concentrations, and injecting into liquid chromatograph, measuring peak area under chromatographic conditions described in example 8, and drawing standard curve with source sample injection amount as abscissa and peak area integral value as ordinate to obtain linear regression equation

TABLE 23 results of Linear relationship of the Components

3. Precision test

(1) Precision test of instrument

Taking the same part of sample solution (batch number: 2109001S) of the centipeda minima formula particles, repeatedly sampling for 6 times according to the chromatographic conditions described in example 8, and measuring chlorogenic acid, caffeic acid, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, content of geraniin A and RSD value of 9 components, wherein the RSD value is 0.12-1.90%, which indicates that the instrument precision is good.

(2) Investigation of intermediate precision of different personnel

The same batch of centipeda minima formula particles (batch number: 2109001S) are separately and independently operated by experiment personnel A, B and C, and are treated according to the preparation method of the 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 geraniin A are measured, the RSD value of each component is calculated, the RSD value of 9 components is between 0.15 and 1.35%, and the result shows that the method has good intermediate precision among different operation personnel.

(3) Repeatability investigation

6 parts of the same batch of centipeda minima formula particles (batch number: 2109001S) are taken and prepared according to the preparation method of the test solution, and the content result RSD% is measured and calculated according to the chromatographic conditions described in example 8. The RSD of chlorogenic acid content is 1.1%, the RSD of caffeic acid content is 1.0%, and the RSD of chlorogenic acid, 3, 5-di-O-caffeoylquinic acid, 4, 5-di-O-caffeoylquinic acid and 3, 4-di-O-caffeoylquinic acid are 0.5-1.6%, which shows that the method has good repeatability.

(4) Accuracy investigation

Namely, in a sample collection test, 9 parts of samples (batch number is 2109001S) of the same batch of centipeda minima formula particles are precisely weighed and respectively put into conical flasks with plugs, 25ml of control solutions with different concentrations are precisely added, each concentration is 3 parts, the samples are processed according to the preparation method of the sample of the example 8, 2 parts of follow-up samples are prepared at the same time, and the content 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 geraniin A is measured, so that the RSD value of each component is calculated. The recovery rate was calculated using the following sample content, and the recovery rate of 9 components was 99.60 to 101.10%. Experimental results show that the accuracy of the method meets the regulations.

4. Durability inspection

(1) Stability of the solution

Taking the same part of sample solution (batch number: 2109001S) of the centipeda minima formula particles, injecting samples at 0, 2, 4, 8, 10, 12 and 24 hours after preparing, measuring 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 geraniin A according to the chromatographic conditions described in example 8, calculating RSD values of all components, and calculating RSD values of 9 components to be 0.32% -1.02%. The experimental result shows that the test sample solution is basically stable within 24 hours, and can meet the measurement requirement.

(2) Investigation of durability at different flow rates

The preparation method of the sample of the example 8 is used for testing at different flow rates of 0.19ml/min, 0.20ml/min and 0.21ml/min, and 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 geraniin A are measured according to the chromatographic conditions described in the example 8, RSD values are 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 content of index components and good stability.

(3) Investigation of durability at different column temperatures

The preparation method of the sample of the example 8 is used for testing at the temperature of 25 ℃ and the temperature of 27 ℃ at different flow rates, and 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 the example 8, so that RSD values are calculated, the RSD values of 9 components are between 0.2% and 1.4%, and the results show that different injection temperatures have little influence on the content of index components and good stability.

(4) Durability inspection of different chromatographic columns

The preparation method of the sample of the example 8 is used for testing the sample under different types of chromatographic columns of the same brand, and 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 all measured according to the chromatographic conditions described in the example 8, so that the RSD value is calculated, the RSD value of 9 components is between 0.3 and 1.4 percent, and the result shows that the influence of different chromatographic columns on the content of index components is not great.

(5) Durability investigation of different instruments

The preparation method of the sample of the example 8 is used for testing the components of the centipeda minima prescription granule (batch number: 2109001S), the components are precisely weighed, the components are treated according to the preparation method of the sample of the example 8, 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 geraniin A are measured according to the chromatographic conditions described in the example 8, the RSD value is calculated, the RSD value of 9 components is between 0.4% and 1.8%, and the results show that the different instruments have little influence on the content of index components and good stability.

5. Content determination of multiple batches of centipeda minima formula particles

18 batches of Centipeda minima formula particles were taken, treated according to the sample preparation method of example 8, and the chlorogenic acid, caffeic acid and phenolic acid contents (total amount 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 results of determination of the amount of particles of the multiple batches of Centipeda minima formulation

Comparative example 1

The difference between this comparative example and example 1 is that the detection wavelength is different in this comparative example, detection of the Centipeda minima formula particles with lot number 2109001S is disclosed under the conditions of 325nm and 225nm wavelength, and the other processes and conditions are as in example 1, and the obtained chromatograms are shown in FIG. 16 and FIG. 17. The detection result is obviously worse than that of the embodiment 1 under the conditions of single wavelength of 325nm and 225nm, wherein the number of the chromatographic peaks at 325nm is obviously less than that of the chromatographic chart constructed in the embodiment 1 before 60min, the characteristic peaks at 225nm are intensively distributed before 25min, the separation degree of a plurality of characteristic peaks is lower than 1.5, and the separation effect is poor.

Comparative example 2

The difference between this comparative example and example 1 is that the types of chromatographic columns in this comparative example are different, and in this comparative example, the detection of the Centipeda minima formula granule with lot number 2109001S by the different types of chromatographic columns is disclosed as follows:

(1)Waters HSS T3 C18 2.1×150mm 1.8μm；

(2) Thermo Hypersil GOLD aQ 2.1.1X105 mm 1.9 μm; the other processes and conditions were as in example 1, and chromatograms obtained by column detection of (1) - (2) are shown in fig. 18 and 19. As can be seen from comparison with FIG. 6, the chromatographic columns (1) to (2) have unsatisfactory separation effects.

Comparative example 3

The comparative example differs from example 1 in that the column temperature of the chromatographic column in the comparative example is different, and the detection results of 20 ℃ and 30 ℃ for the detection of the centipeda minima formula particles with a lot number of 2109001S are disclosed in the 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 that the peak absence occurs at both the temperatures of 20℃and 30℃as compared with 25 ℃.

Table 25 results of different column temperature investigation

Comparative example 4

The difference between this example and example 1 is the gradient elution procedure, which is shown in Table 26:

TABLE 26 gradient elution procedure for different ratios

The sample is detected by using the centipeda minima formula particles with the lot 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 is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims

1. A method for constructing a fingerprint of a pharmaceutical preparation of centipeda minima is characterized by comprising the steps of,

preparing a sample solution and a reference substance solution; the extraction solvent of the sample solution is at least one selected from water, methanol and ethanol; the reference substance solution is prepared by adding solvents into chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, rutin, 3, 4-O-dicaffeoylquinic acid, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, geraniin A and caffeic acid;

0-15min, the volume ratio of mobile phase A to mobile phase B is 3%:97% -8%: 92%,

15-20min, the volume ratio of the mobile phase A to the mobile phase B is 8%:92% -12%: 88 percent,

20-50min, the volume ratio of the mobile phase A to the mobile phase B is 12%: 92-20%: 80 percent,

50-60min, the volume ratio of the mobile phase A to the mobile phase B is 20%:88% -20%: 80 percent,

60-70min, the volume ratio of the mobile phase A to the mobile phase B is 20%:80% -25%: 75 percent,

70-90min, the volume ratio of mobile phase A to mobile phase B is 25%:75% -30%: 70 percent,

90-100min, the volume ratio of the mobile phase A to the mobile phase B is 30%:70% -35%: 65%;

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;

the fingerprint of the pharmaceutical preparation of the centipeda minima 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 of the peaks 1 to 12 is: 0.15, 0.25, 0.34, 0.43, 0.51, 0.61, 0.89, 0.96, 1.00, 1.22, 1.33, 1.53; the relative retention time of the peaks 13 to 22 and the S2 peak is within + -10% of the prescribed value, and the prescribed value of the peaks 13 to 22 is: 0.77, 0.80, 0.84, 0.93, 0.94, 1.00, 1.07, 1.10, 1.22, 1.24; the relative retention time of the peaks 23 to 26 and the S3 peak is within + -10% of the prescribed value, and the prescribed values of the peaks 23 to 26 are: 0.78, 0.86, 0.97, 1.00.

2. The method according to claim 1, wherein a Agilent InfinityLab Poroshell SB-Aq chromatographic column with a specification of 2.1X105 mm and 1.9 μm is used; and/or the sample injection amount of the sample solution is 1-3 mu L; and/or a flow rate of 0.20-0.25mL/min; and/or the volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid is 0.1-0.4%.

3. The construction method according to claim 1 or 2, wherein the preparation method of the test solution is: weighing the sample, extracting with solvent to obtain extractive solution, separating solid from liquid, and collecting the liquid to obtain sample solution.

4. A method of constructing as claimed in claim 3 wherein the method of preparing the test solution further satisfies any one or more of the following a-D:

B. the extraction mode is reflux extraction or ultrasonic extraction;

C. the extraction time is more than or equal to 10min;

D. the solid-liquid separation is selected from centrifugation or filtration with a membrane.

5. The method according to claim 4, wherein the sample solution is prepared for 15-60min.

6. The method according to claim 1, wherein the solvent is at least one selected from the group consisting of methanol, ethanol and water.

7. The method for testing the content of the pharmaceutical preparation of the centipeda minima is characterized by comprising the following steps of:

8. The method according to claim 7, wherein a Agilent InfinityLab Poroshell 120 SB-Aq column having a specification of 2.1×150mm and 1.9 μm is used; and/or the sample injection amount of the sample solution and/or the reference substance solution is 1-3 mu L; and/or a flow rate of 0.20-0.25mL/min; and/or the volume percentage of phosphoric acid in the aqueous solution containing phosphoric acid is 0.1-0.4%.

9. The content testing method according to claim 7 or 8, wherein the preparation method of the test sample solution comprises: weighing the sample, extracting with solvent to obtain extractive solution, separating solid from liquid, and collecting the liquid to obtain sample solution.

10. The content testing method according to claim 9, wherein the method for preparing the test solution further satisfies any one or more of the following a-D:

B. the extraction mode is reflux extraction or ultrasonic extraction;

C. the extraction time is more than or equal to 10min;

11. The method according to claim 9, wherein the extraction time is 15-60min.

12. The content testing method according to claim 9, wherein in the preparation method of the test sample solution, the ratio of the mass of the test sample to the volume of the solvent is 0.4:10-50; the mass versus volume relationship is g/mL.

13. The method according to claim 7, wherein the solvent is at least one selected from the group consisting of methanol, ethanol and water.

14. A quality detection method of a pharmaceutical preparation of centipeda minima, which is characterized by comprising the step of comparing a fingerprint of a centipeda minima product to be detected with a reference fingerprint of the pharmaceutical preparation of centipeda minima and/or the step of measuring the content of the centipeda minima product to be detected by adopting the content test method of the pharmaceutical preparation of centipeda minima according to any one of claims 7-13; 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 6, and the reference fingerprint of the pharmaceutical preparation of the centipeda minima is selected from any one of the following (1) to (3):

(1) The method has 26 common characteristic peaks, wherein the peaks corresponding to chlorogenic acid, 3, 5-di-O-caffeoylquinic acid and Brevibacterium geraniin A are respectively S1 peak, S2 peak and 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 of the peaks 1 to 12 is as follows: 0.15, 0.25, 0.34, 0.43, 0.51, 0.61, 0.89, 0.96, 1.00, 1.22, 1.33, 1.53; the relative retention time of the peaks 13 to 22 and the S2 peak is within + -10% of the prescribed value, and the prescribed value of the peaks 13 to 22 is: 0.77, 0.80, 0.84, 0.93, 0.94, 1.00, 1.07, 1.10, 1.22, 1.24; the relative retention time of the peaks 23 to 26 and the S3 peak is within + -10% of the prescribed value, and the prescribed values of the peaks 23 to 26 are: 0.78, 0.86, 0.97, 1.00;

(2) Fingerprint of a pharmaceutical preparation of Centipeda minima obtained by using a single batch or multiple batches of pharmaceutical preparations of Centipeda minima according to the construction method of any one of claims 1-6;

(3) The fingerprints obtained by using a plurality of batches of the pharmaceutical preparation of centipeda minima according to the construction method of any one of claims 1 to 6 are prepared into a control fingerprint by an average value or a median method.