CN117007711B - Method for detecting characteristic spectrum of matrine and related preparations thereof combined with one standard for multiple tests - Google Patents

Method for detecting characteristic spectrum of matrine and related preparations thereof combined with one standard for multiple tests Download PDF

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CN117007711B
CN117007711B CN202310970855.6A CN202310970855A CN117007711B CN 117007711 B CN117007711 B CN 117007711B CN 202310970855 A CN202310970855 A CN 202310970855A CN 117007711 B CN117007711 B CN 117007711B
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matrine
peak
solution
sample
phosphoric acid
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CN117007711A (en
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刘洪超
徐兴燕
汪冰
齐红
林永强
于雅萌
孙佩霖
牛艳
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Shandong Institute for Food and Drug Control
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Shandong Institute for Food and Drug Control
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • 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
    • 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
    • G01N30/34—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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
    • 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
    • G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention belongs to the field of quality analysis and evaluation of crude drugs and preparations thereof used in Chinese patent medicines, and particularly relates to a characteristic spectrum detection method for combination of matrine and related preparations thereof with one standard and multiple tests. According to the invention, the characteristic spectrum is detected by using a liquid chromatography, when 11 specified characteristic peaks can be effectively detected in the characteristic spectrum of the sample to be detected, the content analysis of index components is carried out by using correction factors, and the peak area ratio of the specified characteristic peaks is calculated. The detection method provided by the invention combines qualitative and quantitative analysis methods by using the characteristic spectrum and one-mark-multiple measurement, is more convenient, accurate and efficient, has good repeatability, accuracy and stability in verification and strong specificity, and can more effectively control the quality of the matrine and the preparation thereof.

Description

Method for detecting characteristic spectrum of matrine and related preparations thereof combined with one standard for multiple tests
Technical Field
The invention belongs to the field of quality analysis and evaluation of crude drugs and preparations thereof used in Chinese patent medicines, and particularly relates to a characteristic spectrum detection method for combination of matrine and related preparations thereof with one standard and multiple tests.
Background
Radix Sophorae Flavescentis is radix Sophorae Flavescentis of LeguminosaeSophora flavescensThe dry root of Ait has cold nature and bitter taste, has the effects of clearing heat, drying dampness, killing parasites and promoting urination, is a common variety of clinical traditional Chinese medicine, and is commonly used for treating dysentery, hematochezia, jaundice, uroschesis, leucorrhea with reddish discharge, yin swelling, pruritus vulvae, eczema, skin pruritus, scabies and leprosy; it is used for external treatment of trichomonas vaginitis. Research shows that the kuh-seng has definite antibacterial activity on staphylococcus aureus and pseudomonas aeruginosaThe bacteria and fungi such as the cytomycetes, the escherichia coli, the candida albicans and the like can play a role in bacteriostasis, wherein the alkaloid components have remarkable pharmacological effects in the process. The alkaloid contained in the kuh-seng is mainly quinolone-lixivium, and is approved to be prepared into kuh-seng total alkaloid as a raw material medicine of various traditional Chinese medicine preparations due to the exact pharmacological activity. The oxymatrine/sophocarpine can be regarded as a prodrug of matrine/sophocarpine when aiming at specific bacterial infection, and the conversion of the oxymatrine/sophocarpine in vivo can be understood as a slow release process to a certain extent, so that the physical action time is prolonged, and the drug effect is improved. In mass production, the matrine is usually extracted from radix sophorae flavescentis by using water, ethanol, acid water and the like as extraction media in the extraction modes of soaking, percolating, decocting, refluxing and the like, and the contained alkaloid components can effectively resist arrhythmia, can be clinically used as auxiliary medicines for arrhythmia, have the effects of clearing heat and removing dampness, moistening lung and relieving cough, activating blood and killing parasites, are commonly used for treating various infectious inflammations such as enteritis, fever, pneumonia, eczema and the like, can also prevent cardiovascular and cerebrovascular diseases and various cancers, increase the flow of cerebral vessels, regulate the immune function of the body, relieve the fatigue of the body, accelerate the metabolism of the body and inhibit the breeding of bacteria, and can be externally used for treating skin diseases in the clinical of traditional Chinese medicine. In particular, when the composition is used for treating novel coronavirus (2019-nCoV), pharmacological experimental data show that the antiviral efficacy is not weaker than that of arbidol and clitoral (lopinavir/ritonavir), and the composition has wide clinical medicinal prospect. In addition, the matrine has relatively low price and is widely applied to the production of biological pesticides.
As the extract of the specific part of the traditional Chinese medicine kuh-seng, the kuh-seng total alkali loses the original identification characteristics of medicinal materials, and if the commercial extract has hidden danger of safety and effectiveness due to low standard, the commercial extract brings great hidden danger to the administration of the Chinese medicine to people. The reason why the ginkgo leaf event in summer 2015 has a strong adverse effect at the moment is that enterprises do not use extraction solvents in a standard way to reduce the cost and improve the output, and the extraction process is not changed in a standard way, but the product still can meet the standard requirement, and the legal inspection method cannot be used for effective screening.
The current legal standard of the matrine is WS-10001- (HD-1106) -2002, and the identification and the content measurement in the main qualitative and quantitative method are physicochemical reactions, wherein the identification can be positive reaction to various alkaloids, the content measurement by titration method has no selectivity to various alkaline components, and the specificity of the whole standard has great defects. The Chinese medicinal preparation kuh-seng suppository is kuh-seng total alkali single preparation, only the oleaginous matrix auxiliary material required by suppository is added, its detection method basically adopts the corresponding method of kuh-seng total alkali, and the standard regulation of total alkali content is calculated according to dry product, and the kuh-seng total alkali is contained in the form of oxymatrine (C 15 H 24 N 2 O 2 ) Meter … … ", description is also under-specified. The detection method has no specificity, the specification description mode is less strict, and the pharmacological activity/toxicity of alkaloid components such as matrine, oxymatrine and the like which are mainly contained in the detection method are slightly different, so that the conversion in the processing and storage processes affects the efficacy to a certain extent, and the toxic and side effects are enhanced, so that the quality control mode is required to be further optimized and improved.
In the prior art, when relevant components in the kuh-seng preparation are measured, an amino column is used for detection together with a normal phase chromatographic system, but the chromatographic column has high cost, short service life and serious target chromatographic peak with multiple front delays; using conventional C 18 The column is matched with a high pH (more than or equal to 8.0) buffer salt mobile phase, and gradient elution is carried out, so that the service life of the chromatographic column is seriously influenced. And all reports do not carry out qualitative and quantitative analysis on the total quality of the matrine and the preparation thereof at the same time.
The characteristic map is a detection method of traditional Chinese medicine/Chinese patent medicine which is widely used day by day. The sample is subjected to proper pretreatment, and chromatographic/spectral/mass spectrum data capable of showing the whole mass are obtained by using an instrument analysis means such as HPLC/GC/MS. Compared with the traditional single-component or multi-component qualitative/quantitative detection, the method can more comprehensively reflect the types and the amounts of chemical components contained in the traditional Chinese medicine, and further reflect the quality of the traditional Chinese medicine and the overall curative effect reflected by the traditional Chinese medicine. The effective components in the traditional Chinese medicine at the present stage are mostly not yet defined, the integrality and the ambiguity of the fingerprint/characteristic spectrum of the traditional Chinese medicine are more close to the requirements of quality control of the traditional Chinese medicine, and the traditional Chinese medicine has more scientificity and comprehensiveness. As a comprehensive and quantifiable identification means, the characteristic spectrum can be used for identifying the authenticity and the quality of the traditional Chinese medicine and the extracts and preparations thereof, and evaluating whether the quality of the traditional Chinese medicine is stable and uniform. In the prior art, no related report on the quality control of matrine, kuh-seng suppository and other related preparations is available.
Disclosure of Invention
Aiming at the problems of relatively simple quality standard, poor specificity and higher cost when using various reference substances for content measurement of the matrine and the matrine preparation in the prior art, the invention provides a more convenient and exclusive quality evaluation method, which combines qualitative and quantitative analysis methods by using a characteristic map and one-mark multi-measurement, is more convenient, accurate and efficient, has good verification repeatability, accuracy and stability and strong specificity, and can more effectively control the quality of the matrine and the matrine preparation.
The invention is realized by the following scheme:
the invention provides a matrine and related preparation combination one mark and multiple measurement characteristic spectrum detection method, the method uses matrine as a contrast to locate characteristic peaks, 11 characteristic peaks with relative retention time meeting the regulation should be detected in a sample, the regulation values are 0.38, 0.55, 0.66, 0.79, 0.92, 1.00, 1.26, 1.35, 1.79, 2.18 and 2.71 in sequence, and the regulation range is +/-10%; the maximum absorption wavelength of each characteristic peak in the range of 200-400 nm is in accordance with the following rule: peak 1 and peak 3 have two peak absorption at 231nm and 304nm, peak 4 has peak absorption at 210nm, peak 7 and peak 9 have peak absorption at 207nm, and the maximum absorption of the other characteristic peaks is at 200nm end, and no obvious peak absorption is in the range of 200-400 nm.
The invention provides a feature map detection method, which specifically comprises the following steps:
(1) Preparation of test solution: adding radix Sophorae Flavescentis total alkaloids into phosphoric acid water solution, shaking to dissolve uniformly, diluting with phosphoric acid water solution, and filtering to obtain radix Sophorae Flavescentis total alkaloids sample solution; adding phosphoric acid aqueous solution into kuh-seng suppository, shaking and extracting after water bath, refrigerating, taking out, and rapidly filtering to obtain a kuh-seng suppository test solution;
(2) Preparation of a control solution: weighing matrine reference substance, and adding phosphoric acid solution to dissolve the matrine reference substance to obtain reference substance solution;
(3) And (3) performing qualitative and quantitative analysis by adopting high performance liquid chromatography to obtain a characteristic spectrum and a fitting spectrum formed by common peaks.
Further, in the step (1), the specific preparation method of the sample solution is as follows: taking 0.2g of matrine, precisely adding 50mL of 0.1% phosphoric acid aqueous solution, shaking to dissolve uniformly, precisely diluting 10-15 times with 0.1% phosphoric acid aqueous solution, and filtering to obtain matrine sample solution; taking 0.2g of kuh-seng suppository, precisely adding 50mL of 0.1% phosphoric acid aqueous solution, melting in water bath at a temperature above 50 ℃, shaking and extracting for 5 minutes, refrigerating at a temperature of 4 ℃ for 30 minutes, taking out, and rapidly filtering to obtain a kuh-seng suppository sample solution.
Further, in the step (2), the concentration of the reference solution is 50. Mu.g.mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The concentration of the phosphoric acid solution was 0.1%.
Further, in the step (3), the conditions of the high performance liquid chromatography are as follows: chromatographic column, VAST Hadesil-Bio C 18 4.6mm.times.250mm, 5 μm; mobile phase, methanol-0.1% phosphoric acid solution isocratic elution; flow rate of 1.0 mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the Column temperature, 30 ℃; sample volume, 10 μl. The qualitative and quantitative analysis detection wavelength is 220nm.
Preferably, the mobile phase is adjusted to pH 2.8 with triethylamine; the ratio of methanol-0.1% phosphoric acid solution in the above isocratic elution was 5:95.
Further, in the quantitative detection, the concentration of the matrine reference substance and the peak area are combined with a correction factor to calculate peak 6 to peak 10, and the correction factor is calculatedfThe correction factors are calculated in the following modes of 1.00, 0.46, 1.35, 0.51 and 0.94 in sequence:
wherein A is S Peak area of matrine in sample, C S For the concentration of matrine in the test sample, A X C is the peak area of the component to be detected in the sample X In the test sampleThe concentration of the component to be measured; the total amount of 5 components corresponding to the peaks 6 to 10 is calculated, and the relative peak area ratio of the peak 6 to the peak 10 is calculated to be not less than 70.0% based on the dry product, and not more than 1.0.
Further, the peaks 6 to 10 correspond to matrine, sophocarpine, sophoridine, sophocarpine oxide and matrine oxide respectively.
According to the invention, the characteristic spectrum is detected by using a liquid chromatography, when 11 specified characteristic peaks can be effectively detected in the characteristic spectrum of the sample to be detected, the content analysis of index components is carried out by using correction factors, and the peak area ratio of the specified characteristic peaks is calculated.
In the method provided by the invention, the target component is quantitatively detected, and characteristic peaks 6-10 are detected in the sample and respectively correspond to matrine, sophocarpine, sophoridine, sophocarpine oxide and matrine oxide, and if the 5 characteristic peaks cannot be effectively identified, corresponding reference substances can be used for identification.
The beneficial effects of the invention are that
1. The research of the invention establishes a detection method of the matrine and the matrine preparation, the data obtained by comparing with the original titration method is verified by methods such as precision, repeatability, stability and the like, the method is accurate and reliable, the specified characteristic peak information is rich, the relative proportion can control the content of the effective components and the reasonable/compliance of sample storage, and the quality can be effectively reflected and evaluated by the listed qualitative/quantitative analysis;
2. in the aspect of using the contrast substance, only matrine which is low in price and easy to obtain is used as a reference, and the one-standard-multiple-measurement method is accurate and efficient, so that the cost of the contrast substance is saved, and the rigorous and reliable measurement result is ensured;
3. the polarity of the components contained in the sample to be detected is concentrated, and isocratic elution used in the method can avoid characteristic peak drift caused by instrument difference, gradient transformation and the like when the method is reproduced in a laboratory, so that the relative retention time is more stable;
4. the characteristic spectrum of 11 peaks is established, a limiting rule is carried out on a component with conversion/degradation, the relative peak area ratio of the matrine of peak 6 to the matrine of peak 10 is calculated, the influence of storage environment and aging on the stability of the components of the matrine is reflected when the matrine is used as a raw material and is prepared into a patent medicine, whether the storage conditions in storage and circulation links are reasonable can be evaluated, and the method and the obtained conclusion can provide scientific reference for the quality control of the matrine and the matrine suppository.
Drawings
FIG. 1 is a liquid chromatography overlay of the total matrine of example 1 of the present invention;
FIG. 2 is a liquid chromatography overlay of 10 batches of kuh-seng suppositories in example 1 of the present invention;
FIG. 3 is a chromatogram of example 1 of the present invention fitted with 6 matrine batches and 10 matrine batches by software;
FIG. 4 is a chromatogram of 2 samples purchased randomly on the market in example 1 of the present invention;
FIG. 5 is a liquid chromatogram of 3 batches of samples with a super-period of the invention in example 1;
FIG. 6 is a chromatogram of a self-made kuh-seng suppository negative sample (matrix adjuvant) in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. It will be apparent that the following embodiments are part, but not all, of the embodiments of the present application and, therefore, the following detailed description of the embodiments does not limit the scope of the application claimed, and equivalents and modifications thereof by one of ordinary skill in the art without making any inventive effort are intended to be included in the scope of the present invention.
Example 1 construction of a characteristic map of Sophora flavescens Total alkaloid and Sophora flavescens suppository
Instrument (one): the Shimadzu LC-20A high performance liquid chromatograph, waters 2695-2998 high performance liquid chromatograph, equipped with diode array detector; a mertretolidox XSR 205DU one ten million electronic analytical balance; kunshan ultrasonic KQ-500DE type numerical control ultrasonic cleaner; the large constant-temperature oscillating water tank in the gold jar.
(II) chromatographic column: vast Hadesil-Bio C 18 ，Phenomenex Luna C 18 ，Waters XBridge C 18 All specifications were 4.6 mm. Times.250 mm, 5. Mu.m.
(III) reagent: methanol, acetonitrile, phosphoric acid and triethylamine are all chromatographic purity, water is purified water prepared by a MILLIPORE water purifier, and the rest reagents are analytical purity.
(IV) reference substance source:
matrine (lot number: 110805-202010, purity: 98.7%, national food and drug verification institute); sophocarpine (lot number: 112052-202001, purity: 91.8%, national institute of food and drug testing); sophoridine (lot number: 110784-202207, purity: 98.2%, national institute of food and drug testing); sophocarpine oxide (lot number: 111652-202202, purity: 93.1%, national institute of food and drug testing); matrine oxide (lot number: 110780-202210, purity: 92.8%, national food and drug verification institute). And (V) the research matrine and kushen suppository are provided by Shandong step Shenzhou pharmaceutical Co., ltd (BCP). Wherein, the matrine is supplied by a third party Ningxia cercis flower pharmaceutical Co., ltd, and the approval document is national medicine standard H64020285; the kuh-seng suppository in the 10 batches of expiration date and the kuh-seng suppository in the 3 batches of expiration date are produced and stored by BCP; samples 2 batches of the same specification of the A, B company are randomly purchased in the market. Details are shown in Table 1.
TABLE 1 sample information
Method (six) and results
1. Chromatographic conditions and system suitability test: high performance liquid chromatography measurement, using octadecylsilane chemically bonded silica as filler; isocratic elution with methanol-0.1% aqueous phosphoric acid (triethylamine to pH 2.8) (5:95); the flow rate is 1 mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the The column temperature is 30 ℃; the detection wavelength is 220nm, and the full wavelength scanning is carried out within the range of 200-400 nm; the number of theoretical plates is not less than 5000 according to the peak of matrine oxide.
2. Preparation of reference substance solution, matrine and kuh-seng suppository sample solution
(1) Preparation of a control solution: taking matrine reference substance, adding 0.1% phosphoric acid solution to prepare 50 μg solution containing matrine per 1mL, and taking the solution as matrine reference substance solution; and adding 0.1% phosphoric acid solution into matrine, sophocarpine, sophoridine, oxidized sophocarpine and oxidized matrine reference substance to obtain mixed solution containing matrine, sophocarpine, sophoridine and oxidized sophocarpine 50 μg each and 70 μg each 1mL, and using as mixed reference substance solution.
(2) Preparation of sample solution: taking about 0.2g of total matrine, precisely adding 50mL of 0.1% phosphoric acid aqueous solution, shaking to dissolve and disperse uniformly, precisely diluting about 15 times with 0.1% phosphoric acid aqueous solution, and filtering to obtain total matrine sample solution; taking 0.2g of kuh-seng suppository, precisely adding 50mL of 0.1% phosphoric acid aqueous solution, melting in water bath at a temperature above 50 ℃, shaking and extracting for 5 minutes, refrigerating at a temperature of 4 ℃ for 30 minutes, taking out, and rapidly filtering to obtain a kuh-seng suppository sample solution.
3. Assay: precisely sucking 10 μl of reference solution and 10 μl of sample solution, and injecting into high performance liquid chromatograph for measurement.
4. Determining detection wavelength
And sucking the reference substance and the sample solution, recording a chromatogram under 220nm according to the chromatographic conditions, synchronously recording spectrum information under 200-400 nm wave bands by using a diode array detector, and monitoring the ultraviolet absorption condition of the detected peak. Analysis shows that the maximum ultraviolet absorption of a plurality of components such as matrine, oxymatrine and the like is below 200nm at the ultraviolet end, exceeds the cut-off use wavelength of methanol, and according to related research data, the characteristic peaks still have stronger absorption at 220nm, the interference of target peaks is less, and the characteristic peak information is more abundant, so that detection at 220nm is selected.
5. Determination of characteristic peaks
And (3) establishing a contrast characteristic map: according to the preparation method of the sample solution, 6 batches of matrine and 10 batches of matrine suppository sample solutions are prepared, detection is carried out according to the detection conditions, and chromatograms are recorded, wherein the chromatograms are shown in figures 1-2; and respectively analyzing 11 common peak information of the total matrine and the kuh-seng suppository, synthesizing liquid phase patterns of all 16 batches of standard samples by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), and establishing a comparison characteristic pattern, wherein the comparison characteristic pattern is shown in figure 3. The relative retention times were calculated using the corresponding data on 3 columns, see table 2:
TABLE 2 sample information
Through matching, 11 common peaks are determined, and the characteristic spectrum result is judged as follows: 11 characteristic peaks with the relative retention time meeting the regulation should be detected in the test sample, matrine is taken as an S peak, the relative retention time of each characteristic peak is sequentially regulated to be 0.38, 0.55, 0.66, 0.79, 0.92, 1.00, 1.26, 1.35, 1.79, 2.18 and 2.71, the maximum absorption wavelength of each characteristic peak in the range of 200-400 nm meets the regulation, wherein peak 2 and peak 6 have peak absorption at 231nm and 304nm, peak 7 has peak absorption at 210nm, peak 10 and peak 13 have peak absorption at 207nm, and the maximum absorption of other characteristic peaks are all at the tail end of 200nm without obvious peak absorption in the range of 200-400 nm.
The average value of the relative retention time of each characteristic peak of 3 chromatographic columns is a specified value, and the relative retention time of all characteristic peaks is within +/-10% of the specified value, so that the specified value is accurate and reasonable; the chromatographic peak confirmation condition is reported by literature, 5 alkaloid reference substances which are clearly contained in the kuh-seng are used for characteristic peak assignment, and the peak 6 to the peak 10 are sequentially matrine, sophocarpine, sophoridine, sophocarpine oxide and matrine oxide.
Characteristic peak-to-peak area definition: according to the chromatographic conditions and the preparation method of the sample solution, 1 batch of matrine (batch number 221217) and 1 batch of matrine plug (batch number 221101) are used for acceleration test and comparison of different storage conditions, the samples are sealed and then stored under different conditions of T37 ℃ and RH 75% (acceleration), below 20 ℃ (shady) and 10-30 ℃ (Room temperature), 3 samples are measured AT the time point of 2 months and compared with the time point of 0, and as a result, the shady storage and the normal temperature (RT) storage are basically indiscriminate, the absolute content of the sophocarpine oxide and the matrine oxide in the accelerated test (Accelerated tests, AT) samples is reduced by about 5%, and the content of the rest 3 alkaloids is relatively stable;
according to the chromatographic conditions and the preparation method of the sample solution, the kuh-seng suppository samples which are stored in the shade after 3 batches (batch numbers 160701, 160801 and 160802 and the validity period is up to 2019) exceed the validity period and are not regulated after the expiration period are detected, and partial characteristic peaks are found to be missing, so that the degradation/conversion phenomenon of oxidized alkaloids is basically and clearly found to exist, as shown in figure 5. The amounts of matrine and oxymatrine were determined to be relatively higher, and the relative peak area ratios were specified based on the data set forth in table 3:
table 3 relative peak area measurement data
Total matrine and kuh-seng suppository sample in effective periodA Matrine /A Oxymatrine The actual measurement value range is 0.17-0.94, the average value is 0.39, and the kuh-seng suppository sample which is stored in the shade without regulation is far higher than the sample in the period of validity. The storage conditions in the specifications of the BCP company are shady and cool (below 20 ℃), the temperature of the A company is 10-30 ℃, the temperature of the B company is below 30 ℃, and the storage time and the storage mode have certain influence on the conversion of specific components by combining the results of the 2-month acceleration test. Wherein the peak area ratio of the sample batch B-210102 reaches 0.94 during the measurement of the near-term. So it is provided withA Matrine /A Oxymatrine The limit of 1.0 limits the reasonable and compliant storage of the matrine and kuh-seng suppository. Commercial 2 batches all met the above specifications, see fig. 4; the self-made kuh-seng suppository negative samples were undisturbed, see figure 6.
EXAMPLE 2 establishment of a method for measuring a plurality of amounts of Sophora flavescens suppository and Sophora flavescens total alkaloids
The method is characterized in that the instrument, the reagent, the chromatographic conditions, the reference substance and the sample preparation are all established by the same characteristic spectrum method.
(II) selection of quantitative index Components
(III) scanning the wavelength range of 200-400 nm, wherein the absorption of matrine, sophocarpine, sophoridine, sophocarpine oxide and matrine oxide is far higher than that of other characteristic peaks, and the 5 components are selected for measurement because the reference substances can be used for quantitative detection.
(IV) HPLC Multi-index constituent determination methodology test
(1) Precision investigation test
Taking a reference substance solution, preparing a sample solution according to the method specified in the embodiment 1, continuously injecting the sample solution for 6 times, 10 mu L each time, detecting according to the chromatographic conditions of the step (1) of the embodiment 1, recording the chromatograms, finding out corresponding peaks, calculating the relative retention time and the relative peak area of 5 specified components, and calculating the RSD value of the relative retention time and the relative peak area to be less than 1.0 percent, wherein the accuracy of the instrument is good.
(2) Repeatability test
Taking 6 parts of matrine and kuh-seng suppository samples, preparing a test solution according to the method specified in the example, detecting according to the chromatographic conditions in the example 1, recording a chromatogram, and calculating the relative retention time and the relative peak area of each target peak, wherein the RSD value is less than 1.0%, which indicates that the method has good repeatability.
(3) Stability investigation test
Taking matrine and kuh-seng suppository samples, preparing a sample solution according to the method of the step (2) of the example 1, detecting at a specified time point within 48 hours according to the chromatographic conditions of the step (1) of the example 1, recording a chromatogram, calculating the relative peak areas of all target peaks, wherein the RSD values of the target peaks are less than 1.0%, and the stability of the components to be detected in the sample solution under the extraction and measurement conditions of the method is good within 48 hours.
(V) calculation of transfer Rate for quantitative index transfer
10 batches of kuh-seng bolts and the corresponding raw materials of kuh-seng total alkaloids are taken, the contents of 5 index components are measured by using the method according to the combination of the feeding amount and the preparation amount of the enterprise related production process, the transfer rate from the raw materials to the finished product is calculated, and the result is shown in Table 4:
TABLE 4 transfer Rate data
The transfer rate of 5 alkaloid components is respectively 94.89% -102.58%, 99.03% -106.91%, 95.42% -102.00%, 94.19% -103.29%, 94.24% -97.36%, and the total alkali content transfer rate is 95.22% -99.47%. The 5 components in the matrine are relatively stable under the corresponding production conditions of the preparation, and are basically transferred completely, so that the quantitative detection and the research of the matrine and the preparation thereof can be combined.
(six) one-standard multi-measurement method for calculating 5 alkaloid contents
Because the components to be detected use the reference substances to raise the detection cost, the method is unified with the characteristic spectrum item method, and the sample is detected by using only one reference substance of matrine and a relative correction factor.
Linear test of control solution and peak area/concentration ratiofCalculation of values
Taking the control solution in the step (1), injecting 1 mu L, 3 mu L,5 mu L, 10 mu L, 15 mu L and 20 mu L according to the method specified in the example 1, and calculating a linear regression equation and a peak area/concentration ratio, wherein the result is shown in Table 5.
TABLE 5 Linear and Peak area/concentration ratio measurement data
With reference to the regulations of the relative correction factors reported in the related literature of the Chinese pharmacopoeia, the correction factor calculation is performed according to the following formula,A S to obtain the peak area of matrine in the sample,C s for the amount of matrine in the test sample,A x to specify the peak area of the component to be measured in the sample,C x the amounts of the components to be measured are specified for the test sample:
the 5 component correction factors were calculatedfSequentially 1.00, 0.46, 1.35, 0.51, 0.94.
Correction factorfIs used for testing the durability of chromatographic column
The correction factors of the two high performance liquid chromatographs of Waters 2695 and LC-20A and the 3 chromatographic column pairs under the item 1 of the example are examinedfIs carried out on 5 components on different chromatographs and different chromatographic columnsfThe RSD of the values is less than 1%, and the RSD is calculated by different chromatographs and different chromatographic columnsfThe values have no obvious difference, and the method has good durability. The data are shown in Table 6.
TABLE 6fValue measurement durability test data
The total amount of 5 components measured by liquid chromatography is compared with the results of acid-base titration in the original standard, and the data are shown in Table 7.
Table 7 data comparison of different determination methods (unit: total alkali mg/g; suppository mg/granule)
The relative average deviation between the total amount of 5 target components measured by the HPLC one-standard-multiple measurement method and the measurement result of the orthoalkaline titration method is less than 3.0%, and the method can effectively replace the original method.

Claims (6)

1. A characteristic spectrum detection method combining matrine and related preparations thereof with one standard and multiple tests is characterized in that the characteristic peak is positioned by taking matrine as a reference, 11 characteristic peaks with the relative retention time meeting the regulation are detected in a sample, and the regulation values are sequentially 0.38, 0.55, 0.66, 0.79, 0.92, 1.00, 1.26, 1.35, 1.79, 2.18 and 2.71, and the regulation range is +/-10%; the maximum absorption wavelength of each characteristic peak in the range of 200-400 nm is in accordance with the following rule: peak 1 and peak 3 have two peak absorption at 231nm and 304nm, peak 4 has peak absorption at 210nm, peak 7 and peak 9 have peak absorption at 207nm, and the maximum absorption of the other characteristic peaks is at 200nm end, and no obvious peak absorption is generated in the range of 200-400 nm;
the method specifically comprises the following steps:
(1) Preparation of test solution: adding radix Sophorae Flavescentis total alkaloids into phosphoric acid water solution, shaking to dissolve uniformly, diluting with phosphoric acid water solution, and filtering to obtain radix Sophorae Flavescentis total alkaloids sample solution; adding phosphoric acid aqueous solution into kuh-seng suppository, shaking and extracting after water bath, refrigerating, taking out, and rapidly filtering to obtain a kuh-seng suppository test solution;
(2) Preparation of a control solution: weighing matrine reference substance, and adding phosphoric acid solution to dissolve the matrine reference substance to obtain reference substance solution;
(3) And (3) performing high performance liquid chromatography to obtain a characteristic spectrum and a fitting spectrum formed by common peaks, performing qualitative and quantitative analysis, wherein the chromatographic column uses octadecylsilane chemically bonded silica as a filler, the detection wavelength is 220nm, methanol-0.1% phosphoric acid aqueous solution is eluted at equal degree, the ratio of the methanol-0.1% phosphoric acid solution is 5:95, and the mobile phase is adjusted to pH 2.8 by triethylamine.
2. The method for detecting a characteristic spectrum according to claim 1, wherein in the step (1), the specific preparation method of the sample solution is as follows: taking 0.2g of matrine, precisely adding 50mL of 0.1% phosphoric acid aqueous solution, shaking to dissolve uniformly, precisely diluting 10-15 times with 0.1% phosphoric acid aqueous solution, and filtering to obtain matrine sample solution; taking 0.2g of kuh-seng suppository, precisely adding 50mL of 0.1% phosphoric acid aqueous solution, melting in water bath at a temperature above 50 ℃, shaking and extracting for 5 minutes, refrigerating at a temperature of 4 ℃ for 30 minutes, taking out, and rapidly filtering to obtain a kuh-seng suppository sample solution.
3. The method according to claim 1, wherein in the step (2), the concentration of the reference solution is 50. Mu.g/mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The concentration of the phosphoric acid solution was 0.1%.
4. The feature map detection method according to claim 1, wherein in step (3), the following is performedThe conditions for high performance liquid chromatography were: flow rate of 1.0 mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the Column temperature, 30 ℃; sample volume, 10 μl.
5. The method for detecting a feature map according to any one of claims 1 to 4, wherein, in the quantitative detection, peak 6 to peak 10 are calculated by using the concentration and peak area of matrine reference substance in combination with a correction factorfThe correction factors are calculated in the following modes of 1.00, 0.46, 1.35, 0.51 and 0.94 in sequence:
wherein A is S Peak area of matrine in sample, C S For the concentration of matrine in the test sample, A X C is the peak area of the component to be detected in the sample X Is the concentration of the component to be detected in the sample; the total amount of 5 components corresponding to the peaks 6 to 10 is calculated, and the relative peak area ratio of the peak 6 to the peak 10 is calculated to be not less than 70.0% based on the dry product, and not more than 1.0.
6. The method according to claim 5, wherein the peaks 6 to 10 correspond to matrine, sophocarpine, sophoridine, sophocarpine oxide, matrine oxide, respectively.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101458235A (en) * 2008-12-30 2009-06-17 上海纳米技术及应用国家工程研究中心有限公司 Matrine liquid chromatography measuring method
KR20100122222A (en) * 2009-05-12 2010-11-22 인하대학교 산학협력단 Method for extracting and separating active constituents from sophora flavescens ait. using liquid-liquid extraction and rp-hplc
CN102000140A (en) * 2010-11-08 2011-04-06 承德燕峰药业有限责任公司 Novel method for synchronously quantifying matrine and oxymatrine in kuh-seng and preparations of kuh-seng
CN113125595A (en) * 2021-03-29 2021-07-16 黄玉英 HPLC detection method of sophora flavescens component
CN114577917A (en) * 2020-12-02 2022-06-03 山西振东制药股份有限公司 Method for detecting content of active ingredients of compound sophora flavescens injection and fingerprint spectrum
CN114577918A (en) * 2020-12-02 2022-06-03 山西振东制药股份有限公司 Ultra-high performance liquid chromatography detection method for active ingredient content and fingerprint spectrum of compound sophora flavescens injection

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101458235A (en) * 2008-12-30 2009-06-17 上海纳米技术及应用国家工程研究中心有限公司 Matrine liquid chromatography measuring method
KR20100122222A (en) * 2009-05-12 2010-11-22 인하대학교 산학협력단 Method for extracting and separating active constituents from sophora flavescens ait. using liquid-liquid extraction and rp-hplc
CN102000140A (en) * 2010-11-08 2011-04-06 承德燕峰药业有限责任公司 Novel method for synchronously quantifying matrine and oxymatrine in kuh-seng and preparations of kuh-seng
CN114577917A (en) * 2020-12-02 2022-06-03 山西振东制药股份有限公司 Method for detecting content of active ingredients of compound sophora flavescens injection and fingerprint spectrum
CN114577918A (en) * 2020-12-02 2022-06-03 山西振东制药股份有限公司 Ultra-high performance liquid chromatography detection method for active ingredient content and fingerprint spectrum of compound sophora flavescens injection
CN113125595A (en) * 2021-03-29 2021-07-16 黄玉英 HPLC detection method of sophora flavescens component

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
Title
Characterization of alkaloids in Sophora flavescens Ait. by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry;Liu, GQ等;Journal of Pharmaceutical And Biomedical Analysis;第54卷(第05期);第1065-1072页 *
Liu, GQ等.Characterization of alkaloids in Sophora flavescens Ait. by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.Journal of Pharmaceutical And Biomedical Analysis.2011,第54卷(第05期),第1065-1072页. *
一测多评法测定苦参中5种生物碱的含量;陈静;王淑美;孟江;孙飞;梁生旺;;中国中药杂志(第09期);第1406-1410页 *
不同提取方法对苦参中生物碱提取率的影响;陈远谷;李春燕;王若焱;杨娟;;山地农业生物学报(第06期);第41-45页 *
吕昭云 ; 张艳红 ; 乔培香 ; 潘显道 ; .高效液相色谱法测定苦参素注射液中氧化槐果碱杂质含量.中国现代应用药学.2007,(第05期),第401-402页. *
周刚 ; 梁生旺 ; 荆鲁 ; 陈静 ; .苦参药材的HPLC特征图谱.中国新药杂志.2013,(第10期),第1218-1221页. *
周莹 ; 孙秀梅 ; 张兆旺 ; 王京龙 ; 陈丽静 ; .苦参3种方法提取液HPLC指纹图谱比较.辽宁中医药大学学报.2012,第34-36页. *
基于指纹图谱结合化学计量学及多成分含量测定的壁钱质量研究;石丽;焦阳;周广涛;汪冰;崔伟亮;刘丽;徐丽丽;林永强;;中国药学杂志;第57卷(第22期);第1935-1941页 *
王一奇 ; 马源源 ; 吴雁 ; 张跃飞 ; 李发美 ; .苦参配方颗粒的高效液相色谱指纹图谱研究及定量分析.分析化学.2006,(第12期),第1791-1793页. *
王盼盼 ; 王小敏 ; 薛艳许 ; 贾严征 ; 魏彩彩 ; .苦参配方颗粒高效液相色谱指纹图谱及定量分析研究.亚太传统医药.2016,(第06期),第27-28页. *
石丽 ; 焦阳 ; 周广涛 ; 汪冰 ; 崔伟亮 ; 刘丽 ; 徐丽丽 ; 林永强 ; .基于指纹图谱结合化学计量学及多成分含量测定的壁钱质量研究.中国药学杂志.2022,第57卷(第22期),第1935-1941页. *
苦参药材的HPLC特征图谱;周刚;梁生旺;荆鲁;陈静;;中国新药杂志(第10期);第1218-1221页 *
苦参药材质量标准的研究;韩颂;于喜水;赵敏;;中医药学报(第02期);第40-42+83页 *
苦参配方颗粒的高效液相色谱指纹图谱研究及定量分析;王一奇;马源源;吴雁;张跃飞;李发美;;分析化学(第12期);第1791-1793页 *
苦参配方颗粒高效液相色谱指纹图谱及定量分析研究;王盼盼;王小敏;薛艳许;贾严征;魏彩彩;;亚太传统医药(第06期);第27-28页 *
陈远谷 ; 李春燕 ; 王若焱 ; 杨娟 ; .不同提取方法对苦参中生物碱提取率的影响.山地农业生物学报.2015,(第06期),第41-45页. *
陈静 ; 王淑美 ; 孟江 ; 孙飞 ; 梁生旺 ; .一测多评法测定苦参中5种生物碱的含量.中国中药杂志.2013,(第09期),第1406-1410页. *
韩颂 ; 于喜水 ; 赵敏 ; .苦参药材质量标准的研究.中医药学报.2009,(第02期),第40-42+83页. *
高效液相色谱法测定苦参素注射液中氧化槐果碱杂质含量;吕昭云;张艳红;乔培香;潘显道;;中国现代应用药学(第05期);第401-402页 *

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