Fingerprint detection method for quality control of Huanglong cough and asthma capsules / Huanglong cough granules

Abstract

The invention discloses a fingerprint detection method for quality control of Huanglong cough and asthma capsules / Huanglong cough granules, and belongs to the technical field of quality control of traditional Chinese medicine preparations. According to the method, a high performance liquid chromatography-mass spectrometry technology is adopted, 5-hydroxymethylfurfural, vitexin, hyperoside, irisflorentin, epimedin B, icariin, irisflorentin, sub-irisflorentin and baohuoside I are taken as reference substances, and different batches of test samples are detected by optimizing extraction conditions and chromatographic parameters, so that the fingerprint spectrum is successfully constructed. The method is simple and easy to implement, high in precision and good in reproducibility, multi-component synchronous identification can be achieved at a time, the detection efficiency is greatly improved, and time and cost are saved; the quality of a plurality of characteristic components of the Huanglong cough and asthma capsule / Huanglong cough granules can be accurately, quickly and simultaneously monitored, and a powerful guarantee is provided for controlling the quality and ensuring the clinical curative effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of quality control of traditional Chinese medicine preparation, in particular to a fingerprint detection method for quality control of Huanglong Cough and Asthma Capsules / Huanglong Cough Granules. BACKGROUND

[0002] Huanglong Cough and Asthma Capsules (State Food and Drug Administration approval number: Z20025060) and Huanglong Cough Granules (State Food and Drug Administration approval number: B20020710) are commonly used traditional Chinese medicine in clinical practice. Both of them are composed of nine medicinal materials, i.e. Radix Astragali, Earthworm, Herba Epimedii, Pulp of Crataegus Pinnatifida Bunge, Platycodon Grandiflorum, Houttuynia Cordata, Belamcanda Chinensis, Honey Ephedra and Semen Lepidii Descurainii, and have the effects of tonifying qi and kidney, clearing lung and reducing phlegm, relieving cough and asthma. They are used for treating cough and asthma caused by deficiency of lung and kidney and phlegm-heat obstructing lung, and diseases such as acute and chronic bronchitis and bronchial asthma. In the prescription composition, each medicinal material plays a unique and key role. Radix Astragali is good at tonifying qi and ascending yang and consolidating the exterior to stop sweating, Earthworm can clear heat and calm convulsion and dredge collaterals to relieve asthma, Herba Epimedii can tonify kidney yang, strengthen sinews and bones and dispel wind-damp, Pulp of Crataegus Pinnatifida Bunge can digest food and invigorate the stomach and remove blood stasis, Platycodon Grandiflorum can ventilate the lung and relieve the throat and expel phlegm and pus, Houttuynia Cordata can clear heat and resolve toxicity and resolve abscess and pus, Belamcanda Chinensis can clear heat and resolve toxicity and reduce phlegm and relieve the throat, Honey Ephedra can sweat and dispel cold and ventilate the lung to relieve asthma, and Semen Lepidii Descurainii can purge the lung to relieve asthma and drain water and reduce swelling. The combination of the above medicinal materials complements each other and has the synergistic effects of tonifying qi and dredging collaterals, tonifying kidney and relieving asthma, clearing heat and reducing phlegm, and digesting food and resolving accumulation.

[0003] At present, the quality control methods of the preparation are mostly limited to the determination of the content of single index component. Such detection cannot comprehensively reflect the overall chemical characteristics and the synergistic effect of the compound, thereby restricting the scientificity and comprehensiveness of the quality evaluation. In view of the multi-component and multi-target mechanism of traditional Chinese medicine, it is of great significance to establish a quality control method which can comprehensively represent the chemical characteristics of Huanglong Cough and Asthma Capsules / Huanglong Cough Granules, so as to evaluate the quality consistency and ensure the stability of clinical efficacy. By constructing a comprehensive and systematic quality control system and realizing the synchronous monitoring of multiple components, the quality status of the product can be more accurately reflected, and a scientific and reliable basis can be provided for the quality evaluation of the drug, so as to ensure the safety and effectiveness of clinical medication. SUMMARY

[0004] In view of the fact that the existing quality control of Huanglong Cough and Asthma Capsules / Huanglong Cough Granules is mostly limited to the determination of the content of single index component, which cannot comprehensively reflect the overall chemical characteristics and the synergistic effect of the compound, thereby restricting the scientificity and comprehensiveness of the quality evaluation, the present application aims to provide a fingerprint detection method for quality control of Huanglong Cough and Asthma Capsules / Huanglong Cough Granules, so as to realize the synchronous monitoring of multiple components and comprehensively reflect the quality of the product.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solution: The application provides a construction method of a Huanglong cough and asthma capsule / Huanglong cough granule fingerprint spectrum, comprising the following steps: S1, Huanglong cough and asthma capsules / Huanglong cough granules of different batches are taken as test product solutions of Huanglong cough and asthma capsules / Huanglong cough granules; 5-hydroxymethyl furfural, vitexin, hyperoside, iridoid, asperosaponin B, icariin, iridoid, asperosaponin B, and asperosaponin I are taken as control solutions to prepare single control solution; S2, under the same conditions, high performance liquid chromatography analysis is performed on the test product solution and the control solution, and corresponding chromatograms are recorded; S3, the chromatogram of the test product solution obtained in S2 is introduced into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system and similarity analysis is performed to confirm the reliability of the results; S4, high resolution mass spectrometry analysis is performed on the test product solution obtained in S1 to obtain a total ion flow chart; data analysis is performed according to the total ion flow chart and the peak conditions of the chromatogram of the test product solution obtained in S2 to obtain mass spectrum results charts of the chemical components; S5, the chromatogram of the test product solution obtained in S2 and the chromatogram of the control solution, the total ion flow chart obtained in S4 and the mass spectrum charts of the chemical components are compared to obtain a fingerprint spectrum of Huanglong cough and asthma capsules / Huanglong cough granules composed of common characteristic peaks.

[0006] In S1, the preparation of the test product solution of Huanglong cough and asthma capsules / Huanglong cough granules is specifically as follows: test product solutions of Huanglong cough and asthma capsules / Huanglong cough granules of different batches are taken, methanol aqueous solution is added, ultrasonic extraction is performed, microporous filter membrane filtration is performed, and the test product solution of Huanglong cough and asthma capsules / Huanglong cough granules is obtained.

[0007] Preferably, the mass / volume ratios of Huanglong cough and asthma capsules / Huanglong cough granules and the methanol aqueous solution are 0.5 g:25 mL and 2.0 g:25 mL, respectively.

[0008] Preferably, the methanol aqueous solution is 80% methanol aqueous solution.

[0009] Preferably, the ultrasonic extraction time is 30 min.

[0010] Preferably, the microporous filter membrane filtration adopts a 0.45 µm microporous filter membrane.

[0011] Preferably, in S1, the preparation method of the control solution is as follows: each control substance is precisely weighed and placed in a conical flask with a plug, and then pure methanol solution is added to prepare a single control solution containing 5-hydroxymethylfurfural 88 μg per 1 mL of 5-hydroxymethylfurfural methanol solution, vitexin 93 μg per 1 mL of vitexin methanol solution, hyperoside 74 μg per 1 mL of hyperoside methanol solution, iristostemonoside 69 μg per 1 mL of iristostemonoside methanol solution, wogonoside B 85 μg per 1 mL of wogonoside B methanol solution, icariin 90 μg per 1 mL of icariin methanol solution, irilone 59 μg per 1 mL of irilone methanol solution, isoirilone 65 μg per 1 mL of isoirilone methanol solution, and baohuoside I 70 μg per 1 mL of baohuoside I methanol solution.

[0012] In S2, the high performance liquid chromatography conditions are as follows: the column type is Hedera ODS-2-C18 (250 mm×4.6 mm, 5 µm); the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution; the ultraviolet-visible absorption detector is used, the detection wavelength is 275 nm, the column temperature is 30℃, the flow rate is 1.0 mL / min, the injection volume is 10 μL, and the gradient elution program is as follows:

[0013] Preferably, S3 is specifically as follows: the chromatograms of different batches of Huanglong Kechuan Capsules / Huanglong Chichui Granules test solution obtained in S2 are introduced into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2004 A version); the chromatographic peaks present in the chromatograms of different batches of Huanglong Kechuan Capsules / Huanglong Chichui Granules test solution are selected as common peaks, the control chromatogram of Huanglong Kechuan Capsules / Huanglong Chichui Granules is generated by using the average value calculation method, and the retention time and peak area of each common peak are calculated; after data import, multi-point correction and data matching, similarity analysis is performed; the similarity results table between the chromatograms of different batches of Huanglong Kechuan Capsules / Huanglong Chichui Granules test solution and the common peak mode is obtained and exported; and the reliability of the results is confirmed according to the similarity results table and the chromatogram of Huanglong Kechuan Capsules / Huanglong Chichui Granules test solution.

[0014] In S4, the detection conditions of high resolution mass spectrometry are as follows: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300℃, auxiliary gas temperature 300℃, scanning mode full scan mode, and mass-to-charge ratio scanning range m / z 100-1500.

[0015] S5 is specifically: according to the chromatogram of the test sample solution and the chromatogram of the reference sample solution obtained in S2, combined with the total ion current diagram obtained by high resolution mass spectrometry in S4 and the mass spectrum result diagram of the chemical composition, 9 common characteristic peaks are identified, and the retention times of the characteristic peaks are as follows: the retention time of peak No. 3 is 5-hydroxymethylfurfural, the retention time is 15.974 min; the retention time of peak No. 9 is vitexin, the retention time is 46.433 min; the retention time of peak No. 10 is hyperoside, the retention time is 49.063 min; the retention time of peak No. 15 is iridin, the retention time is 55.484 min; the retention time of peak No. 17 is asperosaponin B, the retention time is 63.703 min; the retention time of peak No. 19 is icariin, the retention time is 65.203 min; the retention time of peak No. 21 is iridin, the retention time is 69.932 min; the retention time of peak No. 24 is hyperoside, the retention time is 76.233 min; the retention time of peak No. 25 is baohuoside I, the retention time is 78.034 min, and the fingerprint of Huanglong Cough Capsule / Huanglong Cough Granules is obtained.

[0016] The application provides the fingerprint of Huanglong Cough Capsule / Huanglong Cough Granules obtained by the method for constructing the fingerprint of Huanglong Cough Capsule / Huanglong Cough Granules.

[0017] Preferably, the method for detecting the quality of Huanglong Cough Capsule / Huanglong Cough Granules is applied to the construction of the quality standard of Huanglong Cough Capsule / Huanglong Cough Granules.

[0018] Preferably, the method for detecting the quality of Huanglong Cough Capsule / Huanglong Cough Granules is applied to the detection of drugs with the same name and prescription as Huanglong Cough Capsule / Huanglong Cough Granules.

[0019] The application provides a method for detecting the quality of Huanglong Cough Capsule / Huanglong Cough Granules, which detects the quality of a Huanglong Cough Capsule / Huanglong Cough Granule sample by using the fingerprint of Huanglong Cough Capsule / Huanglong Cough Granules, and the quality markers are one or more of 5-hydroxymethylfurfural, vitexin, hyperoside, iridin, asperosaponin B, icariin, iridin, hyperoside, and baohuoside I. The detected components include one or more of 5-hydroxymethylfurfural, vitexin, hyperoside, iridin, asperosaponin B, icariin, iridin, hyperoside, and baohuoside I.

[0020] Compared with the prior art, the technical scheme of the application has the following beneficial technical effects: The application provides a construction method of a Huanglong cough and asthma capsule / Huanglong cough granule fingerprint, which combines high-performance liquid chromatography technology, adopts gradient elution and specific chromatographic conditions, can accurately separate and detect multiple active ingredients in the Huanglong cough and asthma capsule / Huanglong cough granule, and has high precision, good reproducibility, excellent separation degree and stability. The fingerprint constructed by the method can effectively ensure the quality consistency and stability between different batches of the Huanglong cough and asthma capsule / Huanglong cough granule, and provides a reliable standard for quality control. Through similarity evaluation of the fingerprint, whether the quality of the sample meets the requirements can be quickly and accurately judged, and the quality stability and reliability of different batches of products are ensured. Compared with the traditional detection method, the HPLC fingerprint technology has higher detection efficiency, can detect a large number of samples in a short time, and automatically generates a detection report, and comprehensively, objectively and accurately evaluates the quality of the Huanglong cough and asthma capsule / Huanglong cough granule. This provides an important guarantee for ensuring the clinical efficacy and ensures the product quality stability in clinical application.

[0021] The HPLC fingerprint of the Huanglong cough and asthma capsule / Huanglong cough granule constructed by the application has 25 common peaks, and 9 characteristic peaks are identified, which significantly enriches the chemical component characterization information of the preparation. The established fingerprint has strong characteristics and can comprehensively and systematically reflect the overall composition of the chemical components in the Huanglong cough and asthma capsule / Huanglong cough granule. The obtained chromatogram has a smooth baseline and good peak shape, which provides a reliable basis for comprehensively evaluating the quality of the preparation.

[0022] The method for detecting the quality of the Huanglong cough capsule / Huanglong cough granule provided by the application makes up for the deficiency of the existing Huanglong cough and asthma capsule / Huanglong cough granule execution standard in the content determination item, and overcomes the limitation that the existing detection method can only reflect the quality of a small number of components. The established fingerprint method is an effective supplement to the existing quality control system, can reflect the types and quantities of chemical components in the preparation at the same time, more scientifically represents the overall quality, and is beneficial to realize the overall quality control of the product. The method can accurately and quickly identify different batches of products, comprehensively evaluates the quality through the presence or absence of common peaks, has good reproducibility, stability and applicability, is suitable for the quality control of the preparation, and can also provide a reference for the quality standard research of other traditional Chinese medicine compound preparations. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The HPLC fingerprint of the Huanglong cough and asthma capsule of the application; Figure 2 The chromatogram obtained by optimizing the extraction method in the preparation process of the test solution of the Huanglong cough and asthma capsule of the application; Figure 3 The chromatogram obtained by optimizing the extraction solvent in the preparation process of the test solution of the Huanglong cough and asthma capsule of the application; Figure 4 The chromatogram obtained by optimizing the extraction time in the preparation process of the test solution of Huanglong Kechuan Capsules of the present application; Figure 5 The chromatogram obtained by optimizing the detection wavelength in the chromatographic conditions; Figure 6 The chromatogram obtained by optimizing the column temperature in the chromatographic conditions; Figure 7 The chromatogram obtained by optimizing the flow rate in the chromatographic conditions; Figure 8 The chromatogram obtained by optimizing the composition of the mobile phase in the chromatographic conditions; Figure 9 The chromatogram obtained by optimizing the elution program in the chromatographic conditions; Figure 10 The chromatogram and mass spectrum of 5-hydroxymethylfurfural of the present application, (A) is the chromatogram, and (B) is the mass spectrum; Figure 11 The chromatogram and mass spectrum of vitexin of the present application, (A) is the chromatogram, and (B) is the mass spectrum; Figure 12 The chromatogram and mass spectrum of hyperoside of the present application, (A) is the chromatogram, and (B) is the mass spectrum; Figure 13 The chromatogram and mass spectrum of irilone of the present application, (A) is the chromatogram, and (B) is the mass spectrum; Figure 14 The chromatogram and mass spectrum of Daurisoline B of the present application, (A) is the chromatogram, and (B) is the mass spectrum; Figure 15 The chromatogram and mass spectrum of Icariin of the present application, (A) is the chromatogram, and (B) is the mass spectrum; Figure 16 The chromatogram and mass spectrum of Irilavone of the present application, (A) is the chromatogram, and (B) is the mass spectrum; Figure 17 The chromatogram and mass spectrum of Hyporisoflavone of the present application, (A) is the chromatogram, and (B) is the mass spectrum; Figure 18 The chromatogram and mass spectrum of Baohuoside I of the present application, (A) is the chromatogram, and (B) is the mass spectrum; Figure 19 The total ion current chromatogram of Huanglong Kechuan Capsules of the present application under the positive ion mode; Figure 20 The total ion current chromatogram of Huanglong Kechuan Capsules of the present application under the negative ion mode; Figure 21 The test sample fingerprint chromatogram of 15 batches of Huanglong Kechuan Capsules of the present application; Figure 22Fingerprint of 15 batches of Huanglong cough granules for test sample of the present application. DETAILED DESCRIPTION

[0024] The following examples are used to further illustrate the present application, but these examples are only for better understanding the application and not intended to limit the scope or principles of the application, and the embodiments of the present application are not limited to the following. If not specifically indicated, the test methods used are conventional methods, and the raw materials used are commercial products, and the instruments used in the present application are shown in the following table.

[0025]

[0026] The different batches of Huanglong cough capsule samples and Huanglong cough granule samples used in the present application were purchased from the market, and the reagents used in the production of Huanglong cough capsule / Huanglong cough granule by Shaanxi Dongke Pharmaceutical Co., Ltd. are shown in the following table.

[0027]

[0028] The reference substances: 5-hydroxymethylfurfural reference substance (batch number: 111626-202417, purity 99.8%), vitexin reference substance (batch number: 111687-202306, purity: 99.5%), hyperoside reference substance (batch number: 111521-202310, purity: 94.7%), icariin reference substance (batch number: 110737-202418, purity: 98.4%), isoorientin reference substance (batch number: 111557-202104, purity: 99.4%), baohuoside I reference substance (batch number: 111852-202505, purity: 97.2%) were purchased from China Institute for Drug Control; Asperuloside B (batch number: 110623-73-9, purity: 98%) was purchased from Chengdu Pusai Biological Technology Co., Ltd.; Irisolidon reference substance (batch number: 103997, purity: 99%), irilone reference substance (batch number: 102415, purity: 98%) were purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd.

[0029] The present application provides a fingerprint of Huanglong cough capsule / Huanglong cough granule, and the specific construction method is as follows: S1, different batches of Huanglong cough capsule / Huanglong cough granule were used as test sample to prepare test sample solution; 5-hydroxymethylfurfural, vitexin, hyperoside, irilone, asperuloside B, icariin, irilone, isoorientin, baohuoside I were used as reference substance to prepare single reference substance solution; S2, under the same conditions, the test sample solution and the reference substance solution of Huanglong cough capsule / Huanglong cough granule were analyzed by high performance liquid chromatography, and the corresponding chromatogram was recorded; S3, the chromatogram of the test sample solution obtained in S2 is introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system and similarity analysis is performed to confirm the reliability of the results; S4, high resolution mass spectrometry is performed on the test sample solution obtained in S1 to obtain a total ion current diagram; data analysis is performed according to the total ion current diagram and the peak conditions of the chromatogram of the test sample solution obtained in S2 to obtain mass spectrum results diagrams of the chemical components; S5, the chromatogram of the test sample solution obtained in S2 and the chromatogram of the control sample solution, the total ion current diagram obtained in S4 and the mass spectrum diagrams of the chemical components are compared to obtain an HPLC fingerprint of Huanglong Cough Capsule / Huanglong Cough Granules composed of common characteristic peaks.

[0030] The preparation method of the Huanglong Cough Capsule test sample solution in S1 is preferably as follows: 0.5 g of the content of Huanglong Cough Capsules of different batches is respectively taken and placed in a conical flask with a stopper, 25 mL of 80% methanol solution is added, ultrasonic extraction is performed for 30 min, filtration is performed, the filtrate is filtered through a 0.45 µm microporous filter membrane to obtain the Huanglong Cough Capsule test sample solution.

[0031] The preparation method of the Huanglong Cough Granule test sample solution is as follows: 2.0 g of fine powder of Huanglong Cough Granules of different batches is respectively taken and placed in a conical flask with a stopper, 25 mL of 80% methanol solution is added, ultrasonic extraction is performed for 30 min, filtration is performed, and then filtration is performed through a 0.45 µm microporous filter membrane to obtain the Huanglong Cough Granule test sample solution.

[0032] The preparation method of the standard sample solution in S2 is preferably as follows: each control sample is precisely weighed and placed in a conical flask with a stopper, and a pure methanol solution is added to prepare a single control sample solution containing 5-hydroxymethylfurfural 88 µg, vitexin 93 µg, hyperoside 74 µg, irilone 69 µg, asperosaponin B 85 µg, icariin 90 µg, irilone 59 µg, hypoglycoside 65 µg, and baohuoside I 70 µg per 1 mL.

[0033] The liquid chromatography conditions in S3 are as follows: a Hedera ODS-2-C18 (250 mm×4.6 mm, 5 µm) chromatographic column is used; an ultraviolet-visible absorption detector is used; the detection wavelength is 275 nm; the flow rate is 1.0 mL / min; the injection amount is 10 µL; the column temperature is 30℃; and the mobile phase is acetonitrile (A)-0.1% phosphoric acid aqueous solution (B) for gradient elution, and the elution program is shown in Table 1: Table 1: Liquid chromatography elution program

[0034] The present application provides an optimized process for detecting the above-mentioned HPLC fingerprint of Huanglong Cough Capsule / Huanglong Cough Granule: (1) Preparation of test solution optimization This example is to investigate the different extraction methods (ultrasonic, reflux, immersion) by experiment, see attached Figure 2 , the results found that the composition and content of the chromatogram of the three methods are similar. Given the ultrasonic extraction method is more simple and efficient, so the final choice of it as the preparation method of test solution; This example compares the extraction effect of different extraction solvents (water, pure ethanol solution, 80% methanol solution and pure methanol solution), see attached Figure 3 , the results found that when the 80% methanol solution as the extraction solvent, the chromatogram information of the extract is more and the content of the components is higher, so the 80% water methanol solution is selected for extraction; This example compares the extraction effect of different extraction time (30 min, 40 min, 60 min), see attached Figure 4 , the results found that the types and contents of each component in the chromatogram of the three extraction times are basically the same, among which the ultrasonic extraction time is shorter and can meet the experimental requirements. Based on the simplicity and efficiency of the method, the ultrasonic extraction of 30 min is finally selected as the sample pretreatment method; (2) Chromatographic condition optimization This example investigates the detection wavelength by using ultraviolet-visible absorption detector, extracts the chromatogram at 210 nm, 245 nm, 275 nm, 310 nm, see attached Figure 5 , the results found that the information contained in the chromatogram is most comprehensive and the baseline is smooth when the detection wavelength is 275 nm, so this method is selected as the detection wavelength condition; This example selects the column temperature (28℃, 30℃, 32℃), see attached Figure 6 , the results found that the peak situation is best when the column temperature is kept at 30℃, and the separation effect of each component is better, so the column temperature is finally selected as 30℃; This example selects the flow rate (0.6 mL / min, 0.8 mL / min, 1.0 mL / min), see attached Figure 7 , the results found that the peak situation is best when the flow rate is 1.0 mL / min, and the separation effect of each component is better, so the flow rate is finally selected as 1.0 mL / min; This example selects a variety of mobile phase systems (acetonitrile-water, acetonitrile-0.1% phosphoric acid, methanol-water, methanol-0.1% phosphoric acid), see attached Figure 8, it is found that the number of detectable chromatographic peaks in the chromatogram is significantly more than other mobile phase systems, and the separation degree of each component is good. Based on the comprehensive evaluation of the number of detected chromatographic peaks and the separation effect, acetonitrile-0.1% phosphoric acid is finally selected as the optimal mobile phase system; after determining the optimal detection wavelength, column temperature and flow rate, the application selects acetonitrile-0.1% phosphoric acid as the mobile phase through a large number of experimental screening of the mobile phase, and optimizes the elution program, and part of the elution program is as follows: Table 2: Elution program 1

[0035] Table 3: Elution program 2

[0036] Table 4: Elution program 3

[0037] Table 5: Elution program 4

[0038] Table 6: Elution program 5

[0039] The detection results are shown in Figure 9 . It can be known from Figure 9 that the elution program 5 has good separation degree, high peak height, stable baseline and complete chromatographic information, therefore, the elution program 5 is selected as the optimal elution program.

[0040] The application will be further described in combination with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. In addition, it should be understood that after reading the content taught by the application, those skilled in the art can make various modifications or changes to the application, and these equivalent forms also fall within the scope defined by the appended claims of the application.

[0041] Example 1 This embodiment provides a method for constructing the fingerprint spectrum of Huanglong Cough Capsules (see the attached Figure 1 ) / Huanglong Cough Granules based on the above-mentioned optimized conditions.

[0042] S1, preparation of test sample solution: Different batches of Huanglong Cough Capsule contents 0.5 g were weighed and placed in a conical flask with a stopper, 80% methanol solution 25 mL was added, ultrasonic extraction was carried out for 30 min, filtration was carried out, the filtrate was filtered through a 0.45 µm microporous filter membrane, and the Huanglong Cough Capsule test sample solution was obtained.

[0043] Take 2.0 g of Huanglong Cough Granules of different batches, respectively, into a conical flask with a stopper, add 80% methanol solution 25 mL, ultrasonic extraction for 30 min, filter, and then filter through a 0.45 µm microporous filter to obtain the Huanglong Cough Granules test solution.

[0044] Preparation of reference solution: Take each reference substance into a conical flask with a stopper, respectively, add pure methanol solution to prepare a single reference solution containing 5-hydroxymethylfurfural 88 µg, vitexin 93 µg, hyperoside 74 µg, irilone 69 µg, asperosaponin B 85 µg, icariin 90 µg, irilone 59 µg, hypopiunidin 65 µg, and baohuoside I 70 µg per 1 mL.

[0045] S2, chromatographic analysis: Inject the test solution in S1 and the reference solution in S2 into a high performance liquid chromatograph for chromatographic analysis, detect under the same conditions, use Shimadzu LC-20 high performance liquid chromatograph, the liquid chromatography conditions are as follows: chromatographic column: Hedera ODS-2-C18 (250 mm x 4.6 mm, 5 µm) chromatographic column; detector: ultraviolet-visible absorption detector, detection wavelength is 275 nm; flow rate: 1.0 mL / min; injection volume: 10 µL; column temperature: 30℃; mobile phase: acetonitrile (A)-0.1% phosphoric acid aqueous solution (B), gradient elution, use the elution program 5 parameters in Example 1.

[0046] S3, reliability confirmation Import the chromatograms of the test solutions of different batches of Huanglong Cough Capsules in S3 into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2004 A version); select the chromatographic peaks existing in the chromatograms of the test solutions of different batches of Huanglong Cough Capsules / Huanglong Cough Granules as common peaks, generate the control chromatogram of Huanglong Cough Capsules by using the average value calculation method, calculate the retention time and peak area of each common peak; after data import, multi-point correction and data matching, perform similarity analysis; obtain the similarity results table between the chromatograms of the test solutions of different batches of Huanglong Cough Capsules and the common peak mode and export; according to the similarity results table and the chromatogram of the test solution of Huanglong Cough Capsules, confirm the reliability of the results.

[0047] S4, high performance liquid chromatography-mass spectrometry analysis According to the chromatogram of the Huanglong Cough Capsule / Huanglong Cough Granule test sample solution obtained in S3 and the chromatogram of the single control sample solution, high-resolution mass spectrometry analysis is performed on the Huanglong Cough Capsule / Huanglong Cough Granule test sample solution, and the high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300 DEG C, auxiliary gas temperature 300 DEG C, scanning mode is full scan mode, mass-to-charge ratio scanning range m / z is 100-1500, and a total ion current chromatogram is obtained. The total ion current chromatogram is imported into Xcalibur software, data analysis is performed according to the peak condition of the chromatogram of the test sample solution obtained in S3, and the mass spectrum result diagram of each chemical component is obtained.

[0048] S5, Huanglong Cough Capsule / Huanglong Cough Granule Fingerprint See the accompanying Figure 10 See the accompanying Figure 20 According to the chromatogram of the Huanglong Cough Capsule / Huanglong Cough Granule test sample solution obtained in S3 and the chromatogram of the single control sample solution, and in combination with the total ion current chromatogram obtained by high-resolution mass spectrometry and the mass spectrum result diagram of the chemical components, 9 common characteristic peaks are identified, and the retention times of the characteristic peaks are: the retention time of peak No. 3 is 5-hydroxymethylfurfural, 15.974 min; the retention time of peak No. 9 is vitexin, 46.433 min; the retention time of peak No. 10 is hyperoside, 49.063 min; the retention time of peak No. 15 is irilone, 55.484 min; the retention time of peak No. 17 is asperosaponin B, 63.703 min; the retention time of peak No. 19 is icariin, 65.203 min; the retention time of peak No. 21 is irilone, 69.932 min; the retention time of peak No. 24 is hyporein, 76.233 min; and the retention time of peak No. 25 is baohuoside I, 78.034 min, so as to obtain the fingerprint of the Huanglong Cough Capsule / Huanglong Cough Granule, see the accompanying Figure 21 See the accompanying Figure 22 .

[0049] Example 2 Methodological study of the fingerprint detection method (1) Similarity study The present application uses the automatically generated control spectrum R to generate a common chromatographic peak mode, and the analysis and calculation obtain a relatively good similarity between the common chromatographic peaks of different batches of Huanglong Cough Capsule and Huanglong Cough Granule test samples, which indicates that the fingerprint of the Huanglong Cough Capsule and the Huanglong Cough Granule established by the method can well detect the quality of the Huanglong Cough Capsule and the Huanglong Cough Granule, and the results are shown in Tables 8-9.

[0050] Table 8: Similarity between each batch of Huanglong Cough Capsule sample and common chromatographic peak mode

[0051] Table 9: Similarity between each batch of Huanglong cough granule sample and common chromatographic peak pattern

[0052] (2) Precision The sample solution prepared by the method of Example 1 was analyzed according to the detection method of Example 1, 6 times of parallel injection, the injection amount was 10 μL, by analyzing the peak area and retention time of the common characteristic peak and calculating the RSD value, see Table 10 for details. As shown in Table 10, the RSD of the retention time is less than 0.845%, and the RSD of the peak area is less than 1.903%, indicating that the parallel injection precision of the equipment is good.

[0053] Table 10: Peak area and retention time of common characteristic peaks of each batch of Huanglong cough capsule sample and calculation of RSD value

[0054] (3) Stability study The sample solution prepared by the method of Example 1 was analyzed according to the detection method of Example 1, 6 times of parallel injection, the injection amount was 10 μL, by analyzing the peak area and retention time of the common characteristic peak and calculating the RSD value, see Table 11 for details. As shown in Table 11, the RSD of the retention time is less than 0.635%, and the RSD of the peak area is less than 2.075%, indicating that the sample solution of Huanglong cough capsule has almost no change within 24 h, and the stability is good.

[0055] Table 11: Peak area and retention time of common characteristic peaks of Huanglong cough capsule sample at different times and calculation of RSD value

[0056] (4) Reproducibility study According to the sample solution method in Example 1, 6 batches of sample solutions were prepared, and the injection amount was 10 μL according to the chromatographic conditions of Example 1. By analyzing the peak area and retention time of the common characteristic peak of the sample HPLC fingerprint and calculating the RSD value, see Table 12 for details. As shown in Table 12, the RSD of the retention time is less than 0.753%, and the RSD of the peak area is less than 1.733%, indicating that the sample chromatographic peak has good reproducibility, and the method has good reproducibility.

[0057] Table 12: Peak area and retention time of common characteristic peaks of each batch of Huanglong cough capsule sample and calculation of RSD value

[0058] The above experimental results show that the fingerprint spectrum construction method of the Huanglong Cough and Asthma Capsules / Huanglong Cough Pellets provided by the application has the characteristics of good stability, high precision and good repeatability, can comprehensively and objectively evaluate the quality of the Huanglong Cough and Asthma Capsules / Huanglong Cough Pellets, and provides quality assurance for clinical efficacy.

[0059] The above content is only an example and description of the concept of the application, and those skilled in the art can make various modifications or supplements to the described specific embodiments or replace them with similar ways, as long as they do not deviate from the concept of the application or exceed the scope defined by the claims, which shall belong to the protection scope of the application.

Claims

1. A method for constructing a fingerprint spectrum of Huanglong Cough and Asthma Capsules / Huanglong Cough Granules, characterized in that, Comprise the following steps: S1, with different batches of Huanglong cough capsule / Huanglong cough granules as test preparation to prepare Huanglong cough capsule / Huanglong cough granules test solution; with 5-hydroxymethyl furfural, vitexin, hyperoside, iridoid, doriden B, icariin, irilone, secondary irilone, baohuoside I as control, to prepare single control solution; S2, under the same conditions, the test solution and control solution of Huanglong cough capsule / Huanglong cough granules were analyzed by high performance liquid chromatography, and the corresponding chromatogram was recorded; S3, the chromatogram of different batches of test solution obtained in S2 was introduced into traditional Chinese medicine chromatographic fingerprint similarity evaluation system and similarity analysis was carried out to confirm the reliability of the results; S4, according to the test solution obtained in S1, mass spectrum detection was carried out, and total ion flow chart was obtained; According to the total ion flow chart and the peak situation of the test solution chromatogram obtained in S2, data analysis was carried out, and the mass spectrum result chart of each chemical component was obtained; S5, comparing the test solution and control solution chromatogram obtained in S2, the total ion flow chart and mass spectrum chart obtained in S4, the common characteristic peak was determined, and the fingerprint of Huanglong cough capsule / Huanglong cough granules was obtained.

2. The method for establishing the fingerprint of Huanglong Kechuan Capsules / Huanglong Chichui Granules according to claim 1, characterized in that, In S1, the preparation of Huanglong cough capsule / Huanglong cough granules test solution is as follows: taking different batches of Huanglong cough capsule / Huanglong cough granules test solution, adding methanol water solution, ultrasonic extraction, microporous filter membrane filtration, and obtaining Huanglong cough capsule / Huanglong cough granules test solution.

3. The method for establishing the fingerprint of Huanglong Kechuan capsules / Huanglong Chiqi granules according to claim 1, characterized in that, In S1, the preparation method of control solution is as follows: weighing each control, adding pure methanol solution, and preparing single control solution containing 5-hydroxymethyl furfural 88 μg per 1 mL 5-hydroxymethyl furfural methanol solution, vitexin 93 μg per 1 mL vitexin methanol solution, hyperoside 74 μg per 1 mL hyperoside methanol solution, iridoid 69 μg per 1 mL iridoid methanol solution, doriden B 85 μg per 1 mL doriden B methanol solution, icariin 90 μg per 1 mL icariin methanol solution, irilone 59 μg per 1 mL irilone methanol solution, secondary irilone 65 μg per 1 mL secondary irilone methanol solution, and baohuoside I 70 μg per 1 mL baohuoside I methanol solution.

4. The method for establishing the fingerprint spectrum of Huanglong Kechuan capsules / Huanglong Chiqi granules according to claim 1, characterized in that, In S2, the high performance liquid chromatography determination conditions are as follows: Hedera ODS-2-C18 chromatographic column; mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, detection wavelength is 275 nm, column temperature is 30℃, flow rate is 1.0 mL / min, injection volume is 10 μL, and gradient elution program is as follows: 。 5. The method for establishing the fingerprint of Huanglong Kechuan capsules / Huanglong Chexie granules according to claim 1, characterized in that, S3 is specifically: the chromatogram of the test solution of different batches of Huanglong Cough and Asthma Capsules / Huanglong Cough Pellets obtained in S2 is introduced into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System; the chromatographic peaks existing in the chromatograms of the test solutions of different batches of Huanglong Cough and Asthma Capsules / Huanglong Cough Pellets are selected as common peaks, and the control chromatogram of Huanglong Cough and Asthma Capsules / Huanglong Cough Pellets is generated by using the average value calculation method to calculate the retention time and peak area of each common peak; after data import, multi-point correction and data matching, similarity analysis is performed; the similarity results table between the chromatograms of the test solutions of different batches of Huanglong Cough and Asthma Capsules / Huanglong Cough Pellets and the common peak mode is obtained and exported; and the reliability of the results is confirmed according to the similarity results table and the chromatogram of the test solution of Huanglong Cough and Asthma Capsules / Huanglong Cough Pellets.

6. The method for establishing the fingerprint spectrum of Huanglong Kechuan capsules / Huanglong Chiqi granules according to claim 1, characterized in that, In S4, the detection conditions of the high-resolution mass spectrometry are: electrospray ionization, a spray voltage of 3500 V, a sheath gas flow rate of 40 arb, an auxiliary gas flow rate of 10 arb, a capillary temperature of 300℃, an auxiliary gas temperature of 300℃, a scanning mode of full scan mode, and a mass-to-charge ratio scanning range m / z of 100-1500.

7. The method for establishing the fingerprint spectrum of Huanglong Kechuan capsules / Huanglong Chiqi granules according to claim 1, characterized in that, S5 is specifically: according to the chromatogram of the test solution and the chromatogram of the control solution obtained in S2, the total ion current diagram obtained by high-resolution mass spectrometry in S4 and the mass spectrum results diagram of the chemical components are compared, and 9 common characteristic peaks are identified, and the retention times of the characteristic peaks are: the retention time of peak No. 3 is 5-hydroxymethylfurfural, 15.974 min; the retention time of peak No. 9 is vitexin, 46.433 min; the retention time of peak No. 10 is hyperoside, 49.063 min; the retention time of peak No. 15 is irilone, 55.484 min; the retention time of peak No. 17 is damianol B, 63.703 min; the retention time of peak No. 19 is icariin, 65.203 min; the retention time of peak No. 21 is irilone, 69.932 min; the retention time of peak No. 24 is hyporetin, 76.233 min; the retention time of peak No. 25 is baohuoside I, 78.034 min, to obtain the fingerprint of Huanglong Cough and Asthma Capsules / Huanglong Cough Pellets.

8. The fingerprint of Huanglong Cough and Asthma Capsules / Huanglong Cough Pellets obtained by the method of any one of claims 1-7.

9. A method for detecting the quality of Huanglong Kechuan Capsules / Huanglong Chichui Granules, characterized in that, The fingerprint of Huanglong Cough and Asthma Capsules / Huanglong Cough Pellets in claim 8 is used to detect the quality of Huanglong Cough and Asthma Capsules / Huanglong Cough Pellets or other drugs with the same name and prescription, and one or more of 5-hydroxymethylfurfural, vitexin, hyperoside, irilone, damianol B, icariin, irilone, hyporetin and baohuoside I is used as a quality marker.

10. The method for detecting the quality of Huanglong Cough and Asthma Capsules / Huanglong Cough Pellets according to claim 9, characterized in that, The basic components of Huanglong Cough and Asthma Capsules / Huanglong Cough Pellets or other drugs with the same name and prescription include one or more of 5-hydroxymethylfurfural, vitexin, hyperoside, irilone, damianol B, icariin, irilone, hyporetin and baohuoside I.

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