CN111289648B - Method for establishing traditional Chinese medicine compound preparation fingerprint and fingerprint thereof - Google Patents

Method for establishing traditional Chinese medicine compound preparation fingerprint and fingerprint thereof Download PDF

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CN111289648B
CN111289648B CN202010157005.0A CN202010157005A CN111289648B CN 111289648 B CN111289648 B CN 111289648B CN 202010157005 A CN202010157005 A CN 202010157005A CN 111289648 B CN111289648 B CN 111289648B
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peak
retention time
relative retention
fingerprint
chinese medicine
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CN111289648A (en
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张爱军
李帅
唐玉
朱红梅
毛九州
陈东辉
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Sichuan Academy of Chinese Medicine Sciences SACMS
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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
    • G—PHYSICS
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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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N30/54—Temperature
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
    • G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
    • G—PHYSICS
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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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material
    • G—PHYSICS
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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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G01N2030/146—Preparation by elimination of some components using membranes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate

Abstract

The invention relates to the field of pharmaceutical analysis, in particular to a method for establishing a fingerprint of a traditional Chinese medicine compound preparation and a fingerprint thereof. The method for establishing the fingerprint of the traditional Chinese medicine compound preparation has the characteristics of high accuracy, high stability and good repeatability; the results of the detection of 11 Chinese herbal compound preparations in different batches show that the RSD of the relative retention time of chromatographic peaks at the same position is within 5 percent, which shows that the fingerprint provided by the invention has better reproducibility and the method for establishing the fingerprint of the Chinese herbal compound preparation provided by the invention has higher reliability; the fingerprint spectrum of the compound traditional Chinese medicine preparation established by the method has 29 common peaks achieving effective separation, can effectively represent the quality of the compound traditional Chinese medicine preparation, and is beneficial to comprehensively monitoring the quality of the medicine.

Description

Method for establishing traditional Chinese medicine compound preparation fingerprint and fingerprint thereof
Technical Field
The invention relates to the field of pharmaceutical analysis, in particular to a method for establishing a fingerprint of a traditional Chinese medicine compound preparation and a fingerprint thereof.
Background
The Chinese medicinal compound preparation for treating gastric cancer and other digestive tract tumors is a compound preparation of cutis Bufonis, Gekko Swinhonis, rhizoma Fagopyri Dibotryis, fructus Camptothecae Acuminatae, radix Panacis Quinquefolii, herba et Gemma Agrimoniae and Ganoderma, wherein cutis Bufonis and Gekko Swinhonis (Gekko Swinhonis) have effects of eliminating lump, removing blood stasis and relieving pain, and are monarch drugs; the wild buckwheat rhizome and the common camptotheca fruit are used as ministerial drugs for eliminating cancer, detoxifying, resolving masses, breaking blood and dissolving stasis; american ginseng, hairyvein agrimony and glossy ganoderma benefit qi and yin, calm the nerves, nourish the five internal organs, and regulate the toxicity of cutis Bufonis and gecko. Monarch drugs, ministerial drugs, adjuvant drugs and conductant drugs are combined to play the roles of eliminating mass, detoxifying, tonifying qi, activating blood, dissolving stasis and relieving pain, so that healthy qi can be reserved, and pathogenic factors can not invade, the tumor treatment principle of 'great dominance and slight supplement, blood circulation activation and qi regulation' is embodied, and the tumor treatment principle is met. The composition is mainly used for the clinical synergistic treatment of gastric cancer and other digestive tract tumors with blood stasis, and is used for the synergistic treatment after the chemotherapy of the gastric cancer in the advanced stage.
The traditional Chinese medicine fingerprint is an effective method for controlling the quality of a traditional Chinese medicine compound and a preparation thereof in recent years, and the traditional Chinese medicine fingerprint technology provides a wider visual field for the quality control research of the traditional Chinese medicine compound. The traditional Chinese medicine fingerprint has the characteristic of integrity and highlights the complete appearance of the traditional Chinese medicine. Meanwhile, the fingerprint spectrums of the same kind of medicinal materials have the characteristic of similarity, comprehensive evaluation of internal chemical components of the traditional Chinese medicine and comprehensive control of the whole quality are realized by means of the fingerprints spectrums, and the components of the traditional Chinese medicine preparation are more controllable.
HPLC fingerprint is the most widely used chromatographic analysis method at present. The HPLC method is a generally applicable analysis method for detecting various chemical components in the traditional Chinese medicine, and is widely applied to the detection of the chemical components of the traditional Chinese medicine with the advantages of high pressure, high sensitivity, high efficiency, automation and the like, so that the components of the traditional Chinese medicine preparation are more controllable.
Disclosure of Invention
In view of the above, the invention provides a method for establishing a fingerprint of a compound traditional Chinese medicine preparation and a fingerprint thereof, and the provided fingerprint can comprehensively reflect the components of the compound traditional Chinese medicine preparation.
The invention provides a method for establishing a fingerprint spectrum of a compound traditional Chinese medicine preparation, which is characterized by comprising the following steps of:
(1) performing ultrasonic extraction on the traditional Chinese medicine compound preparation by adopting methanol to obtain a test solution;
(2) testing the test solution by high performance liquid chromatography, and generating the fingerprint of the compound Chinese medicinal preparation from the obtained chromatographic data according to the similarity evaluation system 2012A edition of the chromatographic fingerprint of the Chinese medicinal material;
the chromatographic conditions of the high performance liquid chromatography are as follows:
a chromatographic column: c18A chromatographic column;
detection wavelength: 200-220 nm;
mobile phase: the mobile phase A is phosphoric acid aqueous solution with volume fraction of 0.05-0.3%, and the mobile phase B is acetonitrile;
the elution mode is gradient elution, and the elution procedure of the gradient elution is shown in table 1.
Preferably, the Chinese medicinal compound preparation comprises cutis Bufonis, Gekko Swinhonis, rhizoma Fagopyri Dibotryis, fructus Camptothecae Acuminatae, radix Panacis Quinquefolii, herba et Gemma Agrimoniae and Ganoderma; the mass ratio of the cutis Bufonis, the gecko, the wild buckwheat rhizome, the fructus camptothecae acuminatae, the American ginseng, the hairyvein agrimony and the ganoderma lucidum is 1-9: 30-50: 20-40: 6-16: 10-30: 6-24.
Preferably, the chromatographic conditions further comprise:
flow rate: 0.5-1.5 mL/min;
column temperature: 15-35 ℃;
specification of chromatographic column: 250mm × 4.6mm, 5 μm;
sample introduction volume: 5-20 mu L;
sample introduction concentration: 0.01g/mL to 1.00 g/mL.
Preferably, the method for preparing the test solution comprises the following steps:
(a) mixing the Chinese medicinal compound preparation with methanol, performing ultrasonic treatment, filtering, and concentrating the filtrate under reduced pressure to obtain concentrated solution; the power of the ultrasonic wave is 200-400W, the frequency is 30-50 kHz, and the time is 20-60 min;
(b) and (3) adopting a methanol aqueous solution to perform constant volume on the concentrated solution, and then filtering a supernatant in the constant volume solution by using a microporous membrane to obtain a test solution.
Preferably, the volume concentration of the methanol aqueous solution in the step (b) is 10-100%.
Preferably, the pore diameter of the microporous filter membrane in the step (b) is 0.2-0.5 μm.
Preferably, the method also comprises the step of preparing a mixed pairThe preparation method of the mixed reference solution comprises the following steps: mixing ginsenoside Re and ginsenoside Rg1Camptothecin, ginsenoside Rb1Dissolving epicatechin and bufothionine in methanol to obtain mixed control solution;
testing the mixed reference substance solution by adopting the same high performance liquid chromatography condition as the test substance solution to obtain a chromatogram of the mixed reference substance solution; and comparing the chromatogram of the mixed reference solution with the obtained fingerprint of the compound Chinese medicinal preparation, and identifying the chromatographic peak in the fingerprint of the compound Chinese medicinal preparation.
The invention also provides a fingerprint spectrum of the traditional Chinese medicine compound preparation obtained by the method of the technical scheme, and ginsenoside Rb is used1The chromatographic peak is used as reference, and the fingerprint spectrum of the traditional Chinese medicine compound preparation comprises 29 common peaks, which are as follows:
peak 1, relative retention time 0.064; peak 2, relative retention time 0.079;
peak No. 3, relative retention time 0.088; peak No. 4, relative retention time 0.097;
peak 5, relative retention time 0.108; peak 6, relative retention time 0.119;
peak 7, relative retention time 0.134; peak 8, relative retention time 0.143;
peak 9, relative retention time 0.160; peak 10, relative retention time 0.282;
peak 11, relative retention time 0.294; peak No. 12, relative retention time 0.311;
peak 13, relative retention time 0.341; peak 14, relative retention time 0.352;
peak 15, relative retention time 0.411; peak 16, relative retention time 0.436;
peak 17, relative retention time 0.520; peak No. 18, relative retention time 0.547;
peak 19, relative retention time 0.570; peak No. 20, relative retention time 0.639;
peak 21, relative retention time 0.667; peak 22, relative retention time 0.687;
peak 23, relative retention time 0.898; peak 24, relative retention time 0.929;
peak 25, relative retention time 0.939; peak 26, relative retention time 0.990;
peak 27, relative retention time 1; peak 28, relative retention time 1.058;
peak 29, relative retention time 1.169.
Preferably, ginsenoside Rb is used1The chromatographic peak is used as reference, and the 13 th peak in the fingerprint is bufothionine; peak 17 is epicatechin; peak 23 is ginsenoside Re and Rg1(ii) a Peak 24 is camptothecin; 27 peak is ginsenoside Rb1。
Compared with the prior art, the method for establishing the fingerprint spectrum of the traditional Chinese medicine compound preparation has the characteristics of high accuracy, high stability and good repeatability; the results of the detection of 11 Chinese herbal compound preparations in different batches show that the RSD of the relative retention time of chromatographic peaks at the same position is within 5 percent, which shows that the fingerprint provided by the invention has better reproducibility and the method for establishing the fingerprint of the Chinese herbal compound preparation provided by the invention has higher reliability; the fingerprint spectrum of the compound traditional Chinese medicine preparation established by the method has 29 common peaks achieving effective separation, can effectively represent the quality of the compound traditional Chinese medicine preparation, and is beneficial to comprehensively monitoring the quality of the medicine.
Drawings
FIG. 1 is a high performance liquid chromatogram of a mixed control of example 4 of the present invention;
FIG. 2 is a stacked atlas of 11 batches of Chinese herbal compound preparation fingerprints in example 4 of the present invention;
FIG. 3 is a control spectrum generated based on the spectra of 11 test samples in example 4 of the present invention;
FIG. 4 is an attribution chromatogram of each common peak in example 4 of the present invention;
FIG. 5 is a different wavelength chromatogram of example 4 of the present invention and comparative example 1;
FIG. 6 shows different eluent chromatograms of example 4 of the present invention and comparative example 2;
FIG. 7 is a chromatogram of different extraction solvents of example 4 and comparative example 3 of the present invention.
Detailed Description
The invention provides a method for establishing a fingerprint spectrum of a compound traditional Chinese medicine preparation, which comprises the following steps:
(1) performing ultrasonic extraction on the traditional Chinese medicine compound preparation by adopting methanol to obtain a test solution;
(2) testing the test solution by high performance liquid chromatography, and generating the fingerprint of the compound Chinese medicinal preparation from the obtained chromatographic data according to the similarity evaluation system 2012A edition of the chromatographic fingerprint of the Chinese medicinal material;
the chromatographic conditions of the high performance liquid chromatography are as follows:
a chromatographic column: c18A chromatographic column;
detection wavelength: 200-220 nm;
mobile phase: the mobile phase A is phosphoric acid aqueous solution with volume fraction of 0.05-0.3%, and the mobile phase B is acetonitrile;
the elution mode is gradient elution, and the elution program of the gradient elution is shown in table 1:
TABLE 1 gradient elution procedure
Figure GDA0003348948910000051
The invention adopts methanol to carry out ultrasonic extraction on the traditional Chinese medicine compound preparation to obtain a test solution.
In the invention, the traditional Chinese medicine compound preparation preferably comprises dried toad skin, gecko, wild buckwheat rhizome, common camptotheca fruit, American ginseng, hairyvein agrimony and lucid ganoderma; the mass ratio of the cutis Bufonis, the gecko, the wild buckwheat rhizome, the fructus camptothecae acuminatae, the American ginseng, the hairyvein agrimony and the ganoderma lucidum is preferably 1-9: 30-50: 20-40: 6-16: 10-30: 6-24.
In the present invention, the method for preparing the test solution preferably comprises the steps of:
(a) mixing the Chinese medicinal compound preparation with methanol, performing ultrasonic treatment, filtering, and concentrating the filtrate under reduced pressure to obtain concentrated solution; the power of the ultrasonic wave is 200-400W, the frequency is 30-50 kHz, and the time is 20-60 min;
(b) and (3) adopting a methanol aqueous solution to perform constant volume on the concentrated solution, and then filtering a supernatant in the constant volume solution by using a microporous membrane to obtain a test solution.
In the invention, the dosage ratio of the compound traditional Chinese medicine preparation in the step (a) to methanol is preferably 1g: 50-150 mL, more preferably 1g: 70-130 mL, and even more preferably 1g:100 mL; the power of the ultrasonic wave is preferably 200-400W, more preferably 220-380W, and even more preferably 240-360W; the frequency of the ultrasonic wave is preferably 30-50 kHz, and more preferably 35-45 kHz; the ultrasonic treatment time is preferably 20-60 min, and more preferably 30-50 min. In the present invention, the filtration in the step (a) is preferably a conventional filter paper filtration. The present invention is not particularly limited to the embodiment of concentration under reduced pressure, and may be carried out in a manner known to those skilled in the art.
After the concentrated solution is obtained, the concentrated solution is subjected to constant volume by adopting a methanol water solution, and then the supernatant in the constant volume solution is subjected to microfiltration membrane filtration to obtain a test solution. In the present invention, the volume concentration of the methanol aqueous solution is preferably 10% to 100%, more preferably 30% to 100%, and still more preferably 50% to 100%. In the embodiment of the present invention, the concentrate is preferably made to a volume of 5 mL. In the present invention, the pore diameter of the microfiltration membrane is preferably 0.20 to 0.50 μm, more preferably 0.25 to 0.45 μm, and still more preferably 0.3 to 0.45 μm.
After the test solution is obtained, the test solution is tested by adopting high performance liquid chromatography, and the fingerprint of the compound traditional Chinese medicine preparation is generated according to the obtained chromatographic data of the similarity evaluation system 2012A edition of the chromatographic fingerprint of traditional Chinese medicine.
In the present invention, the chromatographic conditions of the high performance liquid chromatography are as follows:
a chromatographic column: c18Chromatography column, preferably Diamonsil C18A chromatographic column;
detection wavelength: 200-220 nm, preferably 203 nm;
mobile phase: the mobile phase A is a phosphoric acid aqueous solution with the volume fraction of 0.05-0.3%, preferably a phosphoric acid aqueous solution with the volume fraction of 0.1%; the mobile phase B is acetonitrile;
the elution mode is gradient elution, and the elution procedure of the gradient elution is shown in the table 1;
the chromatographic conditions preferably further comprise:
flow rate: 0.5-1.5 mL/min, preferably 1.0 mL/min;
column temperature: 15-35 ℃, preferably 25-30 ℃;
specification of chromatographic column: 250mm × 4.6mm, 5 μm;
sample introduction volume: 5-20 muL, preferably 10-15 muL;
sample introduction concentration: 0.01 to 1.00g/mL, preferably 0.10 to 0.50g/mL, and more preferably 0.10 to 0.30 g/mL.
The method adopts the same high performance liquid chromatography condition as the test solution to test the mixed reference solution to obtain the chromatogram of the mixed reference solution; comparing the chromatogram of the mixed reference solution with the fingerprint of the compound Chinese medicinal preparation obtained from the test sample, and identifying the main chromatographic peak in the fingerprint of the compound Chinese medicinal preparation.
In the present invention, the preparation method of the mixed control solution preferably comprises the following steps: mixing ginsenoside Re and ginsenoside Rg1Camptothecin, ginsenoside Rb1Epicatechin and bufothionine controls were dissolved in methanol to give a mixed control solution.
In the invention, in the mixed reference solution, the concentration of the ginsenoside Re is preferably 90-110 mu g/mL, and the ginsenoside Rg1The concentration of (A) is preferably 60-80 mug/mL, the concentration of the camptothecin is preferably 30-40 mug/mL, and the ginsenoside Rb is1The concentration of (a) is preferably 280-320 mu g/mL, the concentration of epicatechin is preferably 35-45 mu g/mL, and the concentration of bufothionine is preferably 15-25 mu g/mL. The concentration of each control in the mixed control solution is preferably controlled within the above range, which is advantageous for the mixed control solutionThe concentration of each substance in the test solution is close to the concentration of the corresponding substance in the test solution.
The invention also provides a fingerprint spectrum of the traditional Chinese medicine compound preparation obtained by the method of the technical scheme, and ginsenoside Rb is used1The chromatographic peak is used as reference, and the fingerprint spectrum of the traditional Chinese medicine compound preparation comprises 29 common peaks, which are as follows:
peak 1, relative retention time 0.064;
peak 2, relative retention time 0.079;
peak No. 3, relative retention time 0.088;
peak No. 4, relative retention time 0.097;
peak 5, relative retention time 0.108;
peak 6, relative retention time 0.119;
peak 7, relative retention time 0.134;
peak 8, relative retention time 0.143;
peak 9, relative retention time 0.160;
peak 10, relative retention time 0.282;
peak 11, relative retention time 0.294;
peak No. 12, relative retention time 0.311;
peak 13, relative retention time 0.341;
peak 14, relative retention time 0.352;
peak 15, relative retention time 0.411;
peak 16, relative retention time 0.436;
peak 17, relative retention time 0.520;
peak No. 18, relative retention time 0.547;
peak 19, relative retention time 0.570;
peak No. 20, relative retention time 0.639;
peak 21, relative retention time 0.667;
peak 22, relative retention time 0.687;
peak 23, relative retention time 0.898;
peak 24, relative retention time 0.929;
peak 25, relative retention time 0.939;
peak 26, relative retention time 0.990;
peak 27, relative retention time 1;
peak 28, relative retention time 1.058;
peak 29, relative retention time 1.169.
In the invention, ginsenoside Rb is used1The chromatographic peak is used as a reference, and the attribution of the common peak in the fingerprint spectrum of the traditional Chinese medicine compound preparation comprises the following steps:
the 13 th peak in the fingerprint is bufothionine;
peak 17 is epicatechin;
peak 23 is ginsenoside Re and Rg1；
Peak 24 is camptothecin;
27 peak is ginsenoside Rb1。
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
In the examples of the present invention, the following instruments and reagents were used:
the instrument equipment comprises: agilent 1260 high performance liquid chromatograph equipped with quaternary pump and DAD detector (Agilent Corp.); JY5002 electronic balance (Shanghai Liangping Instrument Co., Ltd.); XS205 electronic balance (Mettler-Toledo, Switzerland); RE-52AA rotary evaporator (Shanghai Yangrong Biochemical apparatus factory); SHB-IIIG circulating water type multipurpose vacuum pump (Zhengzhou great wall science, industrial and trade Co., Ltd.); model KQ-300B ultrasonic cleaner (ultrasonic instruments ltd, kunshan).
Reference, test and reagent:
comparison products: ginsenoside Re (batch number: 110754-1(batch: 110703-201731 with mass fraction of 93.6%), camptothecin (batch: 100532-201702 with mass fraction of 99.8%), ginsenoside Rb1(batch: 110704-201827 with mass fraction of 91.2%)Epicatechin (batch number 110878-201703, mass fraction 99.7%), institute of food and drug assay in China; bufothionine, home-made.
And (3) testing the sample: the batch numbers of the traditional Chinese medicine compound preparation used by the invention are respectively as follows: 20180516, 20180517, 20181205, 20181218, 20181224, 20190118, 20190121, 20190122, 20190123, 20190124 and 20190125 are all made by self, and the preparation methods of the batch number test products are the same and are all as follows:
mixing cutis Bufonis, Gekko Swinhonis, rhizoma Fagopyri Dibotryis, fructus Camptothecae Acuminatae, radix Panacis Quinquefolii, herba et Gemma Agrimoniae and Ganoderma with methanol, ultrasonic filtering, and concentrating the filtrate under reduced pressure to obtain concentrated solution; the mass ratio of the cutis Bufonis to the Gekko Swinhonis to the rhizoma Fagopyri Dibotryis to fructus Camptothecae Acuminatae, radix Panacis Quinquefolii to herba et Gemma Agrimoniae to Ganoderma is 1:1:8:6:2:4: 3; the power of the ultrasonic wave is 300W, the frequency is 40kHz, and the time is 40 min; and (3) adopting a methanol aqueous solution to perform constant volume on the concentrated solution, and then filtering a supernatant in the constant volume solution by using a microporous membrane to obtain a test solution.
Reagent: acetonitrile, chromatographically pure, Tedia corporation, usa; the water is pure water; the other reagents are analytically pure.
Example 1
Precision experiment
Taking a compound Chinese medicinal preparation (20190122 batches), precisely weighing 0.5g of compound Chinese medicinal preparation powder, adding 50mL of methanol as an extraction solvent, carrying out ultrasonic treatment for 40min at the power of 300W and the frequency of 40kHz, cooling, shaking up, filtering, concentrating the filtrate under reduced pressure, dissolving the filtrate by using a methanol aqueous solution with the volume concentration of 50%, fixing the volume to 5mL, sucking the supernatant, and enabling the supernatant to pass through a 0.45-micron microporous filter membrane to obtain a test solution.
The following high performance liquid chromatography conditions were used for the tests:
a chromatographic column: diamonsil C18A chromatographic column;
detection wavelength: 203 nm;
mobile phase: the mobile phase A is phosphoric acid aqueous solution with the volume fraction of 0.1 percent; the mobile phase B is acetonitrile;
the elution mode is gradient elution, and the elution procedure of the gradient elution is shown in the table 1;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
specification of chromatographic column: 250mm × 4.6mm, 5 μm;
sample introduction volume: 10 mu L of the solution;
sample introduction concentration: 0.10 g/mL.
Continuously injecting sample for 6 needles according to the above chromatographic conditions, and adding ginsenoside Rb1The peak is a reference peak, the relative peak area and relative retention time of each common peak and the reference peak are calculated, and the RSD value is calculated, and the results are shown in tables 2 and 3:
TABLE 2 precision experiments (relative peak area of common peaks)
Figure GDA0003348948910000101
Figure GDA0003348948910000111
Note: (S) is a reference peak
TABLE 3 precision experiments (relative retention time of the respective common peaks)
Figure GDA0003348948910000112
Figure GDA0003348948910000121
As is clear from tables 2 and 3, the relative peak area RSD% of each common peak is less than 5%, and the relative retention time RSD% is less than 5%, indicating that the detection method provided by the present invention has good precision.
Example 2
Repeatability test
Taking compound Chinese medicinal preparation (20190122 batches), weighing 6 parts in parallel, preparing 6 parts of test solution according to the preparation method of the test solution in example 1, injecting sample according to the chromatographic condition in example 1, and using ginsenoside Rb1The peak is a reference peak, and each common peak is calculatedThe relative peak areas and relative retention times of the peaked and reference peaks were calculated and the RSD values were calculated as shown in tables 4 and 5:
TABLE 4 repeatability tests (relative peak area of each common peak)
Figure GDA0003348948910000131
Figure GDA0003348948910000141
TABLE 5 repeatability tests (relative retention time of the common peaks)
Figure GDA0003348948910000142
Figure GDA0003348948910000151
As can be seen from tables 4 and 5, the relative peak area RSD% of each common peak is less than 5%, and the relative retention time RSD% is less than 5%, indicating that the method has good reproducibility.
Example 3
Stability test
Taking compound Chinese medicinal preparation (20190122 batches), preparing the test solution according to the preparation method of the test solution in example 1, injecting samples under the conditions of 0h, 3h, 6h, 9h, 12h and 24h respectively according to the chromatographic condition in example 1, and taking ginsenoside Rb as the reference1The peak was a reference peak, the relative peak area and relative retention time of each common peak to the reference peak were calculated, and RSD values were calculated, and the results are shown in tables 6 and 7:
TABLE 6 stability test (relative peak area of common peaks)
Figure GDA0003348948910000152
Figure GDA0003348948910000161
TABLE 7 stability test (relative retention time of the respective common peaks)
Figure GDA0003348948910000162
Figure GDA0003348948910000171
Figure GDA0003348948910000181
As is clear from tables 6 and 7, the relative peak area RSD% < 5% and the relative retention time RSD% < 5% of each common peak indicate that the test solution is substantially stable within 24 hours.
Example 4
Establishment of traditional Chinese medicine compound preparation fingerprint
(1) Preparation of test solution
Taking the 11 batches of the Chinese medicinal compound preparation, sequentially carrying out batches of 20180516, 20180517, 20181205, 20181218, 20181224, 20190118, 20190121, 20190122, 20190123, 20190124 and 20190125, respectively numbering S1, S2, S3, S4, S5, S6, S7, S8, S9, S10 and S11, respectively and precisely weighing 0.5g of powder, adding 50mL of methanol as an extraction solvent, carrying out ultrasonic treatment for 40min at the power of 300W and the frequency of 40kHz, cooling, shaking uniformly, filtering, concentrating the filtrate under reduced pressure, dissolving with 50% methanol in volume concentration and fixing the volume to 5mL, sucking supernatant, and enabling the supernatant to pass through a 0.45-micrometer microporous filter membrane to obtain 11 batches of Chinese medicinal compound preparation sample solution.
(2) Preparation of Mixed control solutions
Collecting ginsenoside Re and ginsenoside Rg1Camptothecin, ginsenoside Rb1Epicatechin, bufothionine control, precisely weighed, placed in 10mL volumetric flask, usedDissolving the methanol, fixing the volume to a scale, shaking up to obtain a mixed reference substance solution. Wherein the ginsenoside Re and ginsenoside Rg in the mixed reference solution1Camptothecin, ginsenoside Rb1The mass concentrations of epicatechin and bufothionine were 99.54. mu.g/mL, 71.88. mu.g/mL, 33.88. mu.g/mL, 306.38. mu.g/mL, 41.83. mu.g/mL and 19.60. mu.g/mL, respectively.
(3) High performance liquid chromatography conditions
A chromatographic column: diamonsil C18Column (specification 250mm × 4.6mm, 5 μm);
mobile phase: the mobile phase A is a phosphoric acid aqueous solution with the volume fraction of 0.1 percent, and the mobile phase B is acetonitrile;
the elution mode is gradient elution, and the gradient elution mode is shown in table 1;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: 203 nm.
(4) Respectively and precisely sucking 10 μ L of each of the test solution and the reference solution, and testing by adopting the high performance liquid chromatography conditions to respectively obtain the liquid chromatogram of the test solution and the liquid chromatogram of the reference solution.
The liquid chromatogram of the control solution is shown in figure 1, wherein peak 1 is bufothionine control, peak 2 is epicatechin control, and peak 3 is ginsenoside Re and ginsenoside Rg1The reference substance, camptothecin reference substance at peak No. 4, and ginsenoside Rb at peak No. 51And (5) a reference substance.
Introducing the liquid chromatogram of the test solution into fingerprint similarity evaluation software, namely, traditional Chinese medicine chromatogram fingerprint similarity evaluation system 2012A edition, for analysis, taking S8 test solution as a comparison chromatogram, selecting 'time window width' to be 0.5min, calculating by adopting an average method, performing multi-point correction and data matching, and generating a superposition chromatogram, namely, a traditional Chinese medicine compound preparation fingerprint, as shown in figure 2; the control profile generated from the "consensus pattern" based on the profiles of 11 test samples is shown in FIG. 3. The fingerprints of 11 batches of the preparation and the generated control map are subjected to similarity analysis, the relative retention time of each common peak and a reference peak is calculated, and an RSD value is calculated. The results are shown in tables 8 and 9:
table 811 fingerprint similarity of batch preparations of Chinese herbs
Figure GDA0003348948910000191
Fingerprint spectrum relative retention time determination result of batch of traditional Chinese medicine compound preparation in Table 911
Figure GDA0003348948910000192
Figure GDA0003348948910000201
As can be seen from tables 8 and 9, the similarity between the fingerprint and the reference spectrum of the compound Chinese medicinal preparation of different batches is above 0.9, which indicates that the similarity between different batches is high; the relative retention time RSD of each common peak of the traditional Chinese medicine compound preparations in different batches is less than 5 percent, which shows that the test method provided by the invention has better stability.
Example 5
The positive control chromatogram and the negative control chromatogram of the gecko, the common camptotheca fruit, the American ginseng, the cutis Bufonis, the lucid ganoderma, the hairyvein agrimonia herb and the wild buckwheat rhizome in the traditional Chinese medicine compound preparation are respectively determined according to the method of example 1, the ultraviolet absorption of chromatographic peaks in the fingerprint of the traditional Chinese medicine compound preparation and the positive control chromatogram and the negative control chromatogram of each medicinal material is analyzed by a DAD detector, the retention time of the chromatographic peaks is compared, and finally the attribution peak of the common peak in the fingerprint of the traditional Chinese medicine compound preparation is determined. The results are shown in Table 10:
TABLE 10 common peaks on the herb spectra
Figure GDA0003348948910000211
Figure GDA0003348948910000221
Common peak assignment analysis in Gekko Swinhonis
The positive control chromatogram of Gekko Swinhonis and the chromatogram comparison chart of the positive control chromatogram and the fingerprint are shown in FIG. 4; the negative control chromatogram of Gekko Swinhonis and the chromatogram comparison graph of the negative control chromatogram and the fingerprint are shown in FIG. 4. In fig. 4, i is a negative test sample of gecko, j is a test sample of gecko, and o is the fingerprint of the compound Chinese medicinal preparation obtained in example 4. And comparing chromatograms to show that the No. 7 peak in the common peaks of the fingerprint of the compound Chinese medicinal preparation is derived from the gecko medicinal material.
Common peak assignment analysis in (II) camptotheca acuminata fruits
The positive control chromatogram of fructus Camptothecae Acuminatae and the chromatogram comparison graph of the positive control chromatogram and the fingerprint are shown in FIG. 4; the negative control chromatogram of fructus Camptothecae Acuminatae and the chromatogram comparison graph of the negative control chromatogram and the fingerprint are shown in FIG. 4. In FIG. 4, c is a negative test sample of fructus Camptothecae Acuminatae, and d is a test sample of fructus Camptothecae Acuminatae. By comparison of chromatograms, the common peaks of fingerprint chromatogram of the compound Chinese medicinal preparation, such as 11, 15, 24, and 26, are derived from fructus Camptothecae Acuminatae.
Common peak attribution analysis in (III) American ginseng
The positive control chromatogram of the American ginseng medicinal material and the chromatogram comparison graph of the positive control chromatogram and the fingerprint are shown in figure 4; the negative control chromatogram of radix Panacis Quinquefolii and the chromatogram comparison of the negative control chromatogram and the fingerprint are shown in FIG. 4. In fig. 4, a is a test sample of American ginseng negative, and b is a test sample of American ginseng. By comparison of chromatograms, the 23 # and 27 # peaks in the common peaks of the fingerprint of the compound Chinese medicinal preparation are from the American ginseng medicinal material.
Common peak assignment analysis In (IV) cymose buckwheat
The positive control chromatogram of rhizoma Fagopyri Dibotryis and the chromatogram comparison chart of the positive control chromatogram and the fingerprint are shown in FIG. 4; the negative control chromatogram of rhizoma Fagopyri Dibotryis and the chromatogram comparison of the negative control chromatogram and the fingerprint are shown in FIG. 4. In FIG. 4, e is a sample of wild buckwheat rhizome negative test sample, and f is a sample of wild buckwheat rhizome drug. And comparing the chromatograms to obtain 14 th peak of the common peaks of the fingerprint spectrum from wild buckwheat rhizome.
(V) analysis of consensus peaks in Agrimonia pilosa
The positive control chromatogram of herba et Gemma Agrimoniae and the chromatogram comparison graph of the positive control chromatogram and the fingerprint are shown in FIG. 4; the negative control chromatogram of herba et Gemma Agrimoniae and the chromatogram comparison graph of the negative control chromatogram and the fingerprint are shown in FIG. 4. In FIG. 4, m is a negative sample of Agrimonia pilosa Ledeb, and n is a sample of Agrimonia pilosa Ledeb. By comparing the chromatogram, the 18, 19, 20, 22 and 25 peaks in the common peaks of the fingerprint spectrum can be derived from herba et Gemma Agrimoniae.
(VI) analysis of common peak assignment in Ganoderma
The positive control chromatogram of Ganoderma and the chromatogram comparison graph of the positive control chromatogram and the fingerprint are shown in FIG. 4; the negative control chromatogram of Ganoderma and the chromatogram comparison graph of the negative control chromatogram and the fingerprint are shown in FIG. 4. In FIG. 4, k is the negative test sample of Ganoderma, and l is the test sample of Ganoderma. And comparing the chromatograms to determine that the 28 th peak in the common peaks of the fingerprint is from the ganoderma lucidum medicinal material.
Analysis of common peak assignment in (seventy) dried toad skin
The positive control chromatogram of the cutis Bufonis medicinal material and the chromatogram comparison graph of the positive control chromatogram and the fingerprint chromatogram are shown in FIG. 4; the negative control chromatogram of cutis Bufonis and the chromatogram comparison between the negative control chromatogram and fingerprint chromatogram are shown in FIG. 4. In fig. 4, g is a dried toad skin negative sample, and h is a dried toad skin medicinal material sample. And comparing chromatograms, wherein the 13 th peak in the common peaks of the fingerprint is from a dried toad skin medicinal material.
The peaks in FIG. 4 are assigned: the peak 13 corresponds to bufothionine control, the peak 17 corresponds to epicatechin control, and the peak 23 corresponds to ginsenoside Re and ginsenoside Rg1Reference substance, camptothecin reference substance corresponding to peak 24, and ginsenoside Rb corresponding to peak 271And (5) a reference substance.
Comparative example 1
The experiment was conducted in accordance with the preparation method and the test method of the test solution of batch S1 in example 4, except that comparative example 1 was conducted under the conditions of detection wavelengths of 225nm, 254nm, 280nm and 296nm, respectively. The chromatogram of the test solution of batch S1 in example 4 was compared with the chromatogram of the test solution of comparative example 1 at different detection wavelengths, and the results are shown in fig. 5. In FIG. 5, a is a chromatogram of example 4 at a detection wavelength of 203nm, b is a chromatogram of comparative example 1 at a detection wavelength of 225nm, c is a chromatogram of comparative example 1 at a detection wavelength of 254nm, d is a chromatogram of comparative example 1 at a detection wavelength of 280nm, and e is a chromatogram of comparative example 1 at a detection wavelength of 296 nm. As can be seen from FIG. 5, the detection wavelength is set to 203nm, the number of detectable chromatographic peaks is the largest, the response value of the chromatographic peak is higher, and the baseline is smoother.
Comparative example 2
The experiment was conducted according to the preparation method of the test solution of batch S1 in example 4 and the test method in which the gradient elution procedure is different from that of example 4, and the gradient elution procedure of comparative example 2 is shown in table 11; comparative example 2 experiments were carried out under the conditions that the mobile phase a was water, the mobile phase a was an aqueous phosphoric acid solution with a volume fraction of 0.2%, and the mobile phase a was an aqueous phosphoric acid solution with a volume fraction of 0.3%, respectively. The chromatogram of the test sample solution of batch S1 in example 4 was compared with the chromatogram of the test sample of comparative example 2 in different mobile phase a, and the results are shown in fig. 6. In FIG. 6, a is the chromatogram of comparative example 2 in which the mobile phase A is water, b is the chromatogram of example 4 in which the mobile phase A is a 0.1% volume fraction aqueous solution of phosphoric acid, c is the chromatogram of comparative example 2 in which the mobile phase A is a 0.2% volume fraction aqueous solution of phosphoric acid, and d is the chromatogram of comparative example 2 in which the mobile phase A is a 0.3% volume fraction aqueous solution of phosphoric acid. As can be seen from fig. 6, the present invention adopts the gradient elution procedure shown in table 1, and sets the mobile phase a to be a phosphoric acid aqueous solution with a volume fraction of 0.1%, so that the number of the chromatographic peaks that can be detected is the largest, the chromatographic peak response value is higher, and the baseline is smoother.
TABLE 11 gradient elution procedure
Figure GDA0003348948910000241
Comparative example 3
The experiment was conducted according to the preparation method of the test solution of batch S1 in example 4 and the test method in which the gradient elution procedure is different from that of example 4, and the gradient elution procedure of comparative example 3 is shown in table 12; comparative example 3 an experiment was performed under the conditions of water as an extraction solvent and methanol having a volume fraction of 50% as an extraction solvent, respectively. The chromatogram of the test sample solution of batch S1 in example 4 was compared with the chromatogram of the test sample in the different extraction solvents in comparative example 3, and the results are shown in fig. 7. In FIG. 7, a is the chromatogram of comparative example 3 in which the extraction solvent is water, b is the chromatogram of comparative example 3 in which the extraction solvent is a 50% methanol aqueous solution by volume fraction, and c is the chromatogram of example 4 in which the extraction solvent is methanol. As can be seen from FIG. 7, the gradient elution procedure shown in Table 1 and methanol as the extraction solvent are adopted in the present invention, which enables the maximum number of detectable chromatographic peaks, higher chromatographic peak response values and a more stable baseline to be detected.
TABLE 12 gradient elution procedure
Figure GDA0003348948910000251
In conclusion, 29 common peaks in the fingerprint spectrum of the traditional Chinese medicine compound preparation provided by the invention are basically found out with definite affiliation in 7 medicinal materials of dried toad skin, gecko, common camptotheca fruit, American ginseng, lucid ganoderma, hairyvein agrimonia herb and bud and wild buckwheat rhizome positive control chromatograms. Therefore, the fingerprint spectrum of the preparation can basically represent the material basis of the preparation.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (6)

1. A method for establishing a fingerprint spectrum of a traditional Chinese medicine compound preparation is characterized by comprising the following steps:
(1) performing ultrasonic extraction on the traditional Chinese medicine compound preparation by adopting methanol to obtain a test solution; the Chinese medicinal compound preparation comprises cutis Bufonis, Gekko Swinhonis, rhizoma Fagopyri Dibotryis, fructus Camptothecae Acuminatae, radix Panacis Quinquefolii, herba et Gemma Agrimoniae and Ganoderma; the mass ratio of the cutis Bufonis, the gecko, the wild buckwheat rhizome, the fructus camptothecae acuminatae, the American ginseng, the hairyvein agrimony and the ganoderma lucidum is 1-9: 30-50: 20-40: 6-16: 10-30: 6-24;
(2) testing the test solution by high performance liquid chromatography, and generating the fingerprint of the compound Chinese medicinal preparation from the obtained chromatographic data according to the similarity evaluation system 2012A edition of the chromatographic fingerprint of the Chinese medicinal material;
the chromatographic conditions of the high performance liquid chromatography are as follows:
a chromatographic column: c18A chromatographic column;
detection wavelength: 200-220 nm;
mobile phase: the mobile phase A is phosphoric acid aqueous solution with volume fraction of 0.05-0.3%, and the mobile phase B is acetonitrile;
the chromatographic conditions further comprise:
flow rate: 0.5-1.5 mL/min;
column temperature: 15-35 ℃;
specification of chromatographic column: 250mm × 4.6mm, 5 μm;
sample introduction volume: 5-20 mu L;
sample introduction concentration: 0.01 g/mL-1.00 g/mL;
the elution mode is gradient elution, and the elution program of the gradient elution is shown in table 1:
TABLE 1 gradient elution procedure
Figure FDA0003348948900000011
The preparation method of the mixed reference substance solution comprises the following steps: mixing ginsenoside Re and ginsenoside Rg1Camptothecin, ginsenoside Rb1Dissolving epicatechin and bufothionine in methanol to obtain mixed control solution;
testing the mixed reference substance solution by adopting the same high performance liquid chromatography condition as the test substance solution to obtain a chromatogram of the mixed reference substance solution; and comparing the chromatogram of the mixed reference solution with the obtained fingerprint of the compound Chinese medicinal preparation, and identifying the chromatographic peak in the fingerprint of the compound Chinese medicinal preparation.
2. The method for establishing the fingerprint spectrum of the compound traditional Chinese medicine preparation according to claim 1, wherein the preparation method of the test solution comprises the following steps:
(a) mixing the Chinese medicinal compound preparation with methanol, performing ultrasonic treatment, filtering, and concentrating the filtrate under reduced pressure to obtain concentrated solution; the power of the ultrasonic wave is 200-400W, the frequency is 30-50 kHz, and the time is 20-60 min;
(b) and (3) adopting a methanol aqueous solution to perform constant volume on the concentrated solution, and then filtering a supernatant in the constant volume solution by using a microporous membrane to obtain a test solution.
3. The method for establishing the fingerprint spectrum of the compound traditional Chinese medicine preparation according to claim 2, wherein the volume concentration of the methanol water solution in the step (b) is 10-100%.
4. The method for establishing the fingerprint of the compound traditional Chinese medicine preparation according to claim 2, wherein the pore diameter of the microporous filter membrane in the step (b) is 0.2-0.5 μm.
5. The method for establishing the fingerprint of the compound Chinese medicine preparation according to any one of claims 1 to 4, wherein ginsenoside Rb is used1The chromatographic peak is used as reference, and the fingerprint spectrum of the traditional Chinese medicine compound preparation comprises 29 common peaks, which are as follows:
peak 1, relative retention time 0.064;
peak 2, relative retention time 0.079;
peak No. 3, relative retention time 0.088;
peak No. 4, relative retention time 0.097;
peak 5, relative retention time 0.108;
peak 6, relative retention time 0.119;
peak 7, relative retention time 0.134;
peak 8, relative retention time 0.143;
peak 9, relative retention time 0.160;
peak 10, relative retention time 0.282;
peak 11, relative retention time 0.294;
peak No. 12, relative retention time 0.311;
peak 13, relative retention time 0.341;
peak 14, relative retention time 0.352;
peak 15, relative retention time 0.411;
peak 16, relative retention time 0.436;
peak 17, relative retention time 0.520;
peak No. 18, relative retention time 0.547;
peak 19, relative retention time 0.570;
peak No. 20, relative retention time 0.639;
peak 21, relative retention time 0.667;
peak 22, relative retention time 0.687;
peak 23, relative retention time 0.898;
peak 24, relative retention time 0.929;
peak 25, relative retention time 0.939;
peak 26, relative retention time 0.990;
peak 27, relative retention time 1;
peak 28, relative retention time 1.058;
peak 29, relative retention time 1.169.
6. The method for establishing the fingerprint of the compound traditional Chinese medicine preparation according to claim 5, wherein ginsenoside Rb is used1The chromatographic peak is taken as a reference, and the chromatographic peak is taken as a reference,
the 13 th peak in the fingerprint is bufothionine;
peak 17 is epicatechin;
peak 23 is ginsenoside Re and Rg1；
Peak 24 is camptothecin;
27 peak is ginsenoside Rb1。
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