Construction method and detection method of fingerprint spectrum of antiviral traditional Chinese medicine formula granules

Abstract

The present application relates to the technical field of traditional Chinese medicine detection, in particular to a method for constructing and detecting the fingerprint of an antiviral traditional Chinese medicine formula granule. The method for constructing comprises the following steps: dissolving the antiviral traditional Chinese medicine formula granule in a solvent to prepare a test solution; detecting the test solution by high performance liquid chromatography tandem mass spectrometry, scanning data in positive and negative ion modes respectively, and constructing the fingerprint of the antiviral traditional Chinese medicine formula granule. The fingerprint constructed by the present application can comprehensively reflect the quality of the antiviral traditional Chinese medicine formula granule, make up for the defects in inspection and identification caused by the lack of appearance characteristics of medicinal materials and decoction pieces, and the method is efficient and convenient, and the result is objective.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine detection, in particular to a method for constructing and detecting a fingerprint of an antiviral traditional Chinese medicine formula granule. BACKGROUND

[0002] The traditional Chinese medicine formula granule is a kind of traditional Chinese medicine preparation, which has the characteristics of convenient dosage adjustment, standard specification, and consistent efficacy with the corresponding traditional decoction pieces. However, due to the loss of the characteristics of the medicinal material decoction pieces, it is impossible to check and identify them by their original shape, color, texture, cross-section, and odor.

[0003] The fingerprint of the traditional Chinese medicine formula granule can reflect the chemical characteristics of traditional Chinese medicine. Establishing the fingerprint of traditional Chinese medicine is beneficial to comprehensively reflecting the types of chemical components contained in the traditional Chinese medicine formula, and further describing and evaluating the traditional Chinese medicine formula granule as a whole.

[0004] Therefore, it is necessary to construct the fingerprint of the antiviral traditional Chinese medicine formula granule, which aims to reflect the quality of the antiviral traditional Chinese medicine formula granule. SUMMARY

[0005] Based on this, the present application provides a method for constructing and detecting the fingerprint of an antiviral traditional Chinese medicine formula granule.

[0006] The first aspect of the present application provides a method for constructing the fingerprint of an antiviral traditional Chinese medicine formula granule, and the technical solution is as follows:

[0007] A method for constructing the fingerprint of an antiviral traditional Chinese medicine formula granule, comprising the following steps:

[0008] Dissolve the antiviral traditional Chinese medicine formula granule in a solvent to prepare a test solution; wherein the antiviral traditional Chinese medicine formula granule comprises the following components by weight:

[0009] Houpu 8-18 parts, Gualou 10-20 parts, Huangqi 5-15 parts, Chaihu 5-15 parts, Cangzhu 2-10 parts, Shengjiang 2-10 parts, Beishashen 2-10 parts, and Chishao 3-11 parts;

[0010] Perform high performance liquid chromatography tandem mass spectrometry detection on the test solution, respectively in positive and negative ion modes, to construct the fingerprint of the antiviral traditional Chinese medicine formula granule;

[0011] The chromatographic conditions of the high performance liquid chromatography tandem mass spectrometry include:

[0012] The mobile phase includes mobile phase A and mobile phase B, the mobile phase A includes methanol, the mobile phase B includes an aqueous solution of formic acid, and the elution mode is gradient elution;

[0013] The gradient elution procedure includes the following procedure:

[0014] 0 min ~ 10 min, the volume fraction of the mobile phase A is raised from 10% to 30%, and the volume fraction of the mobile phase B is reduced from 90% to 70%;

[0015] 10 min ~ 25 min, the volume fraction of the mobile phase A is raised from 30% to 75%, and the volume fraction of the mobile phase B is reduced from 70% to 25%;

[0016] 25 min ~ 60 min, the volume fraction of the mobile phase A is raised from 75% to 90%, and the volume fraction of the mobile phase B is reduced from 25% to 10%;

[0017] 60 min ~ 65 min, the volume fraction of the mobile phase A is raised from 90% to 100%, and the volume fraction of the mobile phase B is reduced from 10% to 0%.

[0018] In some embodiments, the chromatographic conditions of the high performance liquid chromatography tandem mass spectrometry further include one or more of the following conditions:

[0019] 1) octadecylsilane-bonded silica gel as the stationary phase; 2) detection wavelength of 200 nm ~ 300 nm, and 3) column temperature of 32℃ ~ 38℃.

[0020] In some embodiments, the volume fraction of formic acid in the aqueous formic acid solution is 0.05% ~ 0.2%.

[0021] In some embodiments, the solvent includes the aqueous formic acid solution and the methanol, the volume fraction of the methanol in the solvent is 8% ~ 12%, the volume fraction of the aqueous formic acid solution in the solvent is 88% ~ 92%, and the volume fraction of formic acid in the aqueous formic acid solution is 0.05% ~ 0.2%.

[0022] In some embodiments, the mass volume ratio of the anti-viral traditional Chinese medicine formula granules and the solvent is (8 ~ 12) g: 1 mL.

[0023] In some embodiments, the mass spectrometry conditions of the high performance liquid chromatography tandem mass spectrometry include one or more of the following conditions:

[0024] 1) collision gas is helium; 2) nebulizing gas is nitrogen; 3) spray voltage is 2.5KV ± 0.5KV; 4) sheath gas pressure is 75 arb ± 2 arb; 5) auxiliary gas pressure is 20 arb ± 2 arb; 6) tail gas flow rate is 4 arb ± 0.5 arb; 7) capillary temperature is 400℃ ± 10℃; 8) full scan mass range is m / z 80-1200; 9) data-dependent scan is used for the second stage, and a parent ion with a threshold value of 1000 or above is set as a scanning object, and 10) collision-induced dissociation voltage is 35V ± 2V.

[0025] The second aspect of the present application provides a detection method for an antiviral traditional Chinese medicine formula granule, and the technical solution is as follows:

[0026] A detection method for an antiviral traditional Chinese medicine formula granule, comprising the following steps:

[0027] Dissolving the antiviral traditional Chinese medicine formula granule to be tested in a solvent to prepare a sample solution to be tested;

[0028] Performing high performance liquid chromatography tandem mass spectrometry detection on the sample solution to be tested, performing data scanning in a positive ion mode and / or a negative ion mode, and obtaining corresponding spectrograms;

[0029] Comparing and analyzing the spectrograms with the fingerprint spectrum of the antiviral traditional Chinese medicine formula granule constructed by the above method;

[0030] The chromatographic conditions of the high performance liquid chromatography tandem mass spectrometry are as described above.

[0031] In some embodiments, the solvent includes a methanol and a formic acid aqueous solution, the volume fraction of the methanol in the solvent is 8% to 12%, the volume fraction of the formic acid aqueous solution in the solvent is 88% to 92%, and the volume fraction of the formic acid in the formic acid aqueous solution is 0.05% to 0.2%.

[0032] In some embodiments, the mass-volume ratio of the antiviral traditional Chinese medicine formula granule to be tested and the solvent is (8-12) g: 1 mL.

[0033] In some embodiments, the mass spectrometry conditions of the high performance liquid chromatography tandem mass spectrometry include one or more of the following conditions:

[0034] 1) collision gas is helium; 2) atomizing gas is nitrogen; 3) spray voltage is 2.5KV±0.5KV; 4) sheath gas pressure is 75arb±2arb; 5) auxiliary gas pressure is 20arb±2arb; 6) tail gas flow rate is 4arb±0.5arb; 7) capillary temperature is 400℃±10℃; 8) first full scan mass range is m / z 80~1200; 9) second level uses data-dependent scanning, sets threshold 1000 above parent ions as scanning objects and 10) collision-induced dissociation voltage is 35V±2V.

[0035] Compared with the conventional scheme, the present application has the following beneficial effects:

[0036] The present application adopts high performance liquid chromatography tandem mass spectrometry to construct a fingerprint spectrum of the antiviral traditional Chinese medicine formula granules, and based on standard products, fragment cracking rules and literature, 72 common characteristic peaks of the chemical components of the fingerprint spectrum of the antiviral traditional Chinese medicine formula granules are identified, wherein 21 are flavonoids, 12 are alkaloids, 10 are organic acids, 8 are amino acids, 7 are phenylpropanoids, 5 are volatile oils, 4 are glycosides, 3 are nucleosides and 2 are terpenes. The fingerprint spectrum constructed by the present application can comprehensively reflect the quality of the antiviral traditional Chinese medicine formula granules, make up for the defects in inspection and identification caused by the lack of appearance characteristics of medicinal materials and decoction pieces, and the method is efficient and convenient, and the result is objective. Meanwhile, the present application also provides a reference for the research on the pharmacology and efficacy of the antiviral traditional Chinese medicine formula granules, and provides a scientific experimental basis for the research on the comprehensive quality evaluation of the antiviral traditional Chinese medicine formula granules. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the drawings needed to be used in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0038] Figure 1 Total ion current diagram of the antiviral traditional Chinese medicine formula granules in the positive ion mode;

[0039] Figure 2 Total ion current diagram of the antiviral traditional Chinese medicine formula granules in the negative ion mode. DETAILED DESCRIPTION

[0040] The present application will be further described in detail below in combination with specific embodiments. The present application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing specific embodiments only and is not intended to be limiting of the application.

[0042] The term

[0043] Unless otherwise indicated or contradicted, the terms or phrases used herein have the following meanings:

[0044] In the present application, the selection range involving "and / or", "or / and", "and / or" includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, which includes any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0045] In the present application, the terms "multiple", "various", "multiple times", "multiple" and the like, if not specifically limited, refer to greater than or equal to 2 in number. For example, "one or more" means one or greater than or equal to two.

[0046] In the present application, the terms "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0047] In the present application, the term "suitable" in the terms "suitable combination", "suitable manner", "any suitable manner" and the like is subject to the technical solution of the present application, the technical problem of the present application, and the intended technical effect of the present application.

[0048] In the present application, the terms "preferably", "better", "better", "preferably" only describe the better implementation or embodiment, and it should be understood that it does not constitute a limitation on the scope of protection of the present application.

[0049] In the present application, the terms "further", "furthermore", "in particular" and the like are used for descriptive purposes only and should not be construed as limiting the scope of the present application.

[0050] In the present application, the terms "optionally", "optional" and "may" mean that either of the two alternatives is possible, i.e. either of the two alternatives is selected. If there are multiple "optionally" in a technical solution, each "optionally" is independent of each other unless otherwise specified.

[0051] In the present application, the terms "first", "second", "third", "fourth" and the like in the "first aspect", "second aspect", "third aspect", "fourth aspect" and the like are only used for descriptive purposes and should not be construed as indicating or implying relative importance or quantity, nor should it be construed as implicitly indicating the importance or quantity of the technical features indicated. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration and description, and should be understood as not constituting a closed limitation on the quantity.

[0052] In the present application, the technical features described in an open manner include both the closed technical solution consisting of the listed features and the open technical solution containing the listed features.

[0053] In the present application, the numerical interval (i.e. numerical range) is selected, and unless otherwise specified, the optional numerical distribution within the above numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e. the minimum value and the maximum value) of the numerical range, as well as every numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, including the two endpoint integers of the numerical range and every integer between the two endpoints, in this document, it is equivalent to directly listing each integer, for example, t is an integer selected from 1 to 10, which means that t is any integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this document should be understood to include any and all sub-ranges included therein.

[0054] In the present application, the temperature parameter, unless otherwise specified, allows for constant temperature treatment and allows for variation within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. It is allowed to fluctuate within the range of, for example, ±5℃, ±4℃, ±3℃, ±2℃, ±1℃.

[0055] In the present application, percentage content is involved, if not otherwise specified, mass percentage for solid-liquid mixing and solid-solid mixing, volume percentage for liquid-liquid mixing.

[0056] In the present application, percentage concentration is involved, if not otherwise specified, final concentration. The final concentration refers to the proportion of the added component in the system after adding the component.

[0057] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0058] In the present application, the antiviral traditional Chinese medicine formula granules comprise the following components by weight:

[0059] Houpu 8-18 parts, Gualou 10-20 parts, Huangqi 5-15 parts, Chaihu 5-15 parts, Cangzhu 2-10 parts, Shengjiang 2-10 parts, Beishashen 2-10 parts, and Chishao 3-11 parts.

[0060] Understandably, the weight parts of Houpu in the antiviral traditional Chinese medicine formula granules include but are not limited to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and any value in any of the above two numerical intervals.

[0061] The weight parts of Gualou in the antiviral traditional Chinese medicine formula granules include but are not limited to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and any value in any of the above two numerical intervals.

[0062] The weight parts of Huangqi in the antiviral traditional Chinese medicine formula granules include but are not limited to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and any value in any of the above two numerical intervals.

[0063] The weight parts of Chaihu in the antiviral traditional Chinese medicine formula granules include but are not limited to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and any value in any of the above two numerical intervals.

[0064] The weight parts of Cangzhu in the antiviral traditional Chinese medicine formula granules include but are not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, and any value in any of the above two numerical intervals.

[0065] The weight parts of Shengjiang in the antiviral traditional Chinese medicine formula granules include but are not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, and any value in any of the above two numerical intervals.

[0066] The weight parts of Radix Hedysari in the anti-virus traditional Chinese medicine formula granules include, but are not limited to, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, and any value in any two numerical value intervals of the above.

[0067] The weight parts of Radix Hedysari in the anti-virus traditional Chinese medicine formula granules include, but are not limited to, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, and any value in any two numerical value intervals of the above.

[0068] In some preferred examples, the anti-virus traditional Chinese medicine formula granules include the following components by weight:

[0069] Magnolia officinalis 10-15 parts, Trichosanthes kirilowii 13-18 parts, Scutellaria baicalensis 8-13 parts, Bupleurum chinense 8-13 parts, Atractylodes lancea 3-8 parts, Zingiber officinale 3-8 parts, Radix Hedysari 3-8 parts, Radix Paeoniae Rubra 4-9 parts.

[0070] In some preferred examples, the anti-virus traditional Chinese medicine formula granules include the following components by weight:

[0071] Magnolia officinalis 12-13 parts, Trichosanthes kirilowii 15-16 parts, Scutellaria baicalensis 10-11 parts, Bupleurum chinense 10-11 parts, Atractylodes lancea 5-6 parts, Zingiber officinale 5-6 parts, Radix Hedysari 5-6 parts, Radix Paeoniae Rubra 6-7 parts.

[0072] In some preferred examples, the anti-virus traditional Chinese medicine formula granules include the following components by weight:

[0073] Magnolia officinalis 12 parts, Trichosanthes kirilowii 15 parts, Scutellaria baicalensis 10 parts, Bupleurum chinense 10 parts, Atractylodes lancea 5 parts, Zingiber officinale 5 parts, Radix Hedysari 5 parts, Radix Paeoniae Rubra 6 parts.

[0074] Optionally, the anti-virus traditional Chinese medicine formula granules further include a pharmaceutically acceptable excipient.

[0075] In the present application, the preparation method of the anti-virus traditional Chinese medicine formula granules includes the following steps:

[0076] Take each component of the anti-virus traditional Chinese medicine formula granules, and prepare formula granules of each component;

[0077] Mix the formula granules of each component to prepare the anti-virus traditional Chinese medicine formula granules.

[0078] It can be understood that the preparation of the formula granules of each component can be specifically referred to the method published by the State Drug Administration of the State Drug Standard.

[0079] In some examples, the method for preparing the formula granules of each component includes the following steps:

[0080] Take the medicinal materials of each component, respectively add water extraction, and obtain the extraction liquid of each component;

[0081] Concentrating the extract of each component respectively to obtain concentrated extract of each component;

[0082] Adding pharmaceutically acceptable adjuvant to the concentrated extract of each component respectively, spray drying to obtain formula granules of each component.

[0083] Optionally, the mass of water is 3-8 times of the mass of medicinal materials of the component to be extracted.

[0084] Optionally, the extraction is performed for 2-3 times, and the extract of each time is combined to obtain the extract of one component.

[0085] Optionally, the concentrated extract of each component has a relative density of 1.05-1.20.

[0086] Example 1 Construction of Fingerprint of Antiviral Traditional Chinese Medicine Formula Granules

[0087] 1 Instruments and materials

[0088] 1.1 Instruments

[0089] UtiMate 3000 HPLC-LTQ Obitrap MS (Thermo Fisher Corporation), including online degassing machine, four-element pump, automatic sampler, column temperature box, variable wavelength detector, and ESI ion source, one-millionth electronic balance (model: NBL214i; Wuhan Aide Me Heng Instrument Co., Ltd.), Thermo Scientific Xcalibur software.

[0090] 1.2 Materials

[0091] Antiviral Traditional Chinese Medicine Formula Granules (batch number: J2106001, J2106002, J2106003), methanol (chromatographic pure) and formic acid (chromatographic pure) are products of Merck, and other analytical reagents are from Tianjin Damao Reagent Co., Ltd.

[0092] 2 Experimental method

[0093] 2.1 Chromatographic conditions

[0094] The chromatographic column is Thermo Hypersil ODS C18 (4.6 mm x 250 mm, 5 μm); the mobile phase is composed of methanol (A) and 0.1% formic acid aqueous solution (B) and gradient elution (0-10 min, 10% A→30% A; 10-25 min, 30% A→75% A; 25-60 min, 75% A→90% A; 60-65 min, 90% A→100% A); the detection wavelength is 280 nm; the column temperature is 35℃, and the injection amount is 10 μL.

[0095] 2.2 Mass spectrometry conditions

[0096] Thermo LTQ-Obitrap mass spectrometer, ionization in positive and negative ion modes respectively, high-purity helium as collision gas, high-purity nitrogen as atomization gas, and the detector was optimized to obtain the maximum yield of [M-H] + , [M-H] - ions. The parameters were optimized as follows: spray voltage 2.5 kV, sheath gas (N2) pressure 75 arb, auxiliary gas (N2) pressure 20 arb, tail gas flow rate 4 arb, and capillary temperature 400℃. All sample data acquisition was performed using full scan mode with a resolution of 70000 FWHM. The first full scan mass number range was m / z 80-1200; the second was data-dependent scan, with parent ions above 1000 set as scan objects, dynamic exclusion time set to 15 s, repeat scan number set to 2, repeat scan time set to 15 s, and collision-induced dissociation voltage 35 V.

[0097] 2.3 Preparation of test solution

[0098] An appropriate amount of anti-viral traditional Chinese medicine formula granules was weighed, the initial mobile phase was used as the solvent (including 10% by volume methanol and 90% by volume formic acid aqueous solution, wherein the volume fraction of formic acid in the formic acid aqueous solution was 0.1%), which was dissolved to prepare a 10 g / mL solution, filtered with a 0.22 μm microporous filter, and the filtered solution was used as the test solution.

[0099] 2.4 Determination

[0100] The test solution under item “2.3” was injected into the UtiMate 3000 HPLC-LTQ Obitrap MS, and determined according to the chromatographic conditions under item “2.1” and the mass spectrometry conditions under item “2.2”.

[0101] 2.5 Data processing

[0102] The chemical substances of 8 traditional Chinese medicines contained in the antiviral traditional Chinese medicine dispensing granules were searched through databases such as Wanfang Database, VIP Database, China National Knowledge Infrastructure, Pubmed and Science, and an antiviral traditional Chinese medicine dispensing granule chemical component database was established, including the name, relative molecular mass and chemical formula of the chemical substances. There were 2151 chemical components in the self-built antiviral traditional Chinese medicine dispensing granule chemical database, including 439 Magnoliae officinalis, 328 Bupleuri, 310 Trichosanthis, 133 Radicis Paeoniae Rubrae, 224 Scophulariae, 215 Zingiberis, 146 Adenophorae, and 331 Atractylodis. The liquid-mass spectrometry chromatograph was used for analyzing the antiviral traditional Chinese medicine dispensing granules, and the total ion chromatograms in positive and negative modes were obtained, respectively. The data were analyzed and processed by Xcalibur workstation, and the molecular ion peaks corresponding to the chromatographic peaks were preliminarily judged by the first mass spectrometry information of the chromatographic peaks at the same retention time in positive and negative ion modes. The compounds of the chromatographic peaks were preliminarily inferred by comparing the corresponding relative molecular mass and possible molecular formula with the self-built chemical component database. In addition, the compounds can also be inferred by the addition rule of molecular ion peaks in positive or negative ion mode. Generally, the molecular ion peaks in positive ion mode usually exist in the form of [M+H] + , and there may also be [M+Na] + , [M+K] + , [M+NH4] + , [2M+H] + , [2M+Na] + , etc. The molecular ion peaks in negative ion mode usually exist in the form of [M-H] - , and there may also be [M-H] - , [M+Ac] - , [2M-H] - , [M+Cl] - , etc. The secondary mass spectrometry data of the molecular ion peaks can be obtained by selecting the secondary chromatogram data with better signal-to-noise ratio, and the secondary fragmentation of the molecular ion peaks is further analyzed. The compounds can be accurately inferred by comparing the accurate relative molecular mass and chemical formula provided by Xcalibur workstation with the data in related literatures, and the fragmentation characteristics of the same type of chemical components are induced by the secondary fragmentation ions, the analyzed chemical substances are classified, and the medicinal materials are attributed.

[0103] 3 Identification results

[0104] Figure 1 The total ion chromatogram of the antiviral traditional Chinese medicine dispensing granules (batch number: J2106001) in positive ion mode is shown in Figure 1, Figure 2 and the total ion chromatogram of the antiviral traditional Chinese medicine dispensing granules (batch number: J2106001) in negative ion mode is shown in Figure 2.

[0105] A total of 72 chemical components were identified, including 21 flavonoids, 12 alkaloids, 10 organic acids, 8 amino acids, 7 phenylpropanoids, 5 volatile oils, 4 glycosides, 3 nucleosides, and 2 terpenes. See Table 1.

[0106] Table 1

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116] 4. Construction of fingerprint chromatograms under different ion modes

[0117] 4.1 Construction of fingerprint chromatograms under positive ion mode

[0118] The antiviral traditional Chinese medicine granules with batch numbers J2106001, J2106002, and J2106003 were prepared into sample solutions according to the preparation method under item “2.3”. The sample solutions were injected into UtiMate 3000 HPLC-LTQ Obitrap MS, and determined according to the chromatographic conditions under item “2.1” and the mass spectrometric conditions under item “2.2”. The positive ion mode was scanned to obtain three chromatograms. The chromatograms were introduced into the similarity evaluation system for chromatographic peak matching to generate the control fingerprint chromatogram and the superimposed chromatogram, and the 72 common peaks were marked.

[0119] The chromatograms of the above three batches were compared with the control chromatogram, and the similarity values of the chromatograms of the above three batches with the control chromatogram were calculated using the similarity evaluation system. The similarity values of all the chromatograms were greater than 0.950.

[0120] Precision examination

[0121] Take the batch number J2106001 test sample solution, repeat the sample 6 times according to the above chromatographic conditions, and count the peak retention time and peak area. The results show that the RSD is less than 0.5%, which meets the requirements.

[0122] Repeatability investigation

[0123] Take the same batch number of antiviral formula granules (J2106001), prepare 6 parallel samples according to the test sample preparation method, and repeat the sample 6 times according to the above chromatographic conditions, and count the peak retention time and peak area. The results show that the RSD is less than 0.5%. It meets the requirements.

[0124] Stability investigation

[0125] Take the batch number J2106001 antiviral formula granules, and sample at 0, 2, 4, 8, 10, 12, and 24 hours according to the above chromatographic conditions. Count the peak retention time and peak area. The results show that the RSD is less than 0.5%. It meets the requirements.

[0126] 4.2 Fingerprint spectrum construction under negative ion mode

[0127] Take the batch numbers J2106001, J2106002, and J2106003 antiviral traditional Chinese medicine formula granules, prepare the test sample solution according to the preparation method in item 2.3, and sample into UtiMate 3000 HPLC-LTQ Obitrap MS. Determine according to the chromatographic conditions in item 2.1 and the mass spectrometry conditions in item 2.2. Scan under negative ion mode. Get three spectra. Import them into the similarity evaluation system for chromatographic peak matching, and generate the control fingerprint spectrum and the superimposed spectrum. Mark the 72 common peaks.

[0128] Take the control spectrum as the reference spectrum, compare the above three batch numbers with the control spectrum, and calculate the similarity values of the above three batch numbers with the control spectrum using the similarity evaluation system. The similarity of all spectra is greater than 0.950.

[0129] Precision investigation

[0130] Take the batch number J2106001 test sample solution, repeat the sample 6 times according to the above chromatographic conditions, and count the peak retention time and peak area. The results show that the RSD is less than 0.5%, which meets the requirements.

[0131] Repeatability investigation

[0132] Take the same batch number of antiviral formula granules (J2106001), prepare 6 parallel samples according to the test sample preparation method, and repeat the sample 6 times according to the above chromatographic conditions, and count the peak retention time and peak area. The results show that the RSD is less than 0.5%. It meets the requirements.

[0133] Stability study

[0134] Take the batch number as J2106001 antiviral formula granules, according to the above chromatographic conditions, respectively at 0, 2, 4, 8, 10, 12, 24 hours, statistics of each peak retention time and peak area. The results show that the RSD is less than 0.5%. It meets the requirements.

[0135] Example 2 Detection method of antiviral traditional Chinese medicine formula granules

[0136] 1 Instruments and materials

[0137] 1.1 Instrument same as example 1.

[0138] 1.2 Materials

[0139] The antiviral traditional Chinese medicine formula granules to be tested, and the others are the same as example 1.

[0140] 2 Experimental method

[0141] 2.1 Chromatographic conditions same as example 1.

[0142] 2.2 Mass spectrometry conditions same as example 1.

[0143] 2.3 Preparation of sample solution to be tested

[0144] An appropriate amount of antiviral traditional Chinese medicine formula granules to be tested is weighed, and the initial mobile phase is used as the solvent (including 10% methanol and 90% formic acid aqueous solution, wherein the volume fraction of formic acid in the formic acid aqueous solution is 0.1%), which is dissolved to prepare a 10 g / mL solution, filtered with a 0.22 μm microporous filter, and the filtered solution is used as the test sample solution.

[0145] 2.4 Determination

[0146] The sample solution to be tested under item 2.3 is injected into UtiMate 3000 HPLC-LTQ Obitrap MS, and the chromatographic conditions under item 2.1 and the mass spectrometry conditions under item 2.2 are used for determination. The spectrum obtained in the positive ion mode is introduced into the similarity evaluation system and the fingerprint spectrum constructed in the above positive ion mode for similarity evaluation; the spectrum obtained in the negative ion mode is introduced into the similarity evaluation system and the fingerprint spectrum constructed in the above negative ion mode for similarity evaluation. When the similarity of the spectrum obtained in the positive ion mode and the fingerprint spectrum constructed in the above positive ion mode is greater than 0.9, or the similarity of the spectrum obtained in the negative ion mode and the fingerprint spectrum constructed in the above negative ion mode is greater than 0.9, it indicates that the quality of the sample solution to be tested is qualified.

[0147] Any combination of the technical features in the above-described embodiments can be made, and for the sake of brevity, not all possible combinations are described, however, as long as the combination of the technical features does not exist in contradiction, it shall be considered within the scope of the present disclosure.

[0148] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it shall not be understood as a limitation on the patent scope of the present application. It shall be pointed out that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these shall be within the protection scope of the present application. Therefore, the protection scope of the present application patent shall be subject to the appended claims.

Claims

1. A method for constructing a fingerprint of an anti-virus traditional Chinese medicine formula granule, characterized in that, The method comprises the following steps: Dissolving the anti-virus traditional Chinese medicine formula granules in a solvent to prepare a test solution; wherein the anti-virus traditional Chinese medicine formula granules comprise the following components by weight: Houpu 8-18 parts, Gualou 10-20 parts, Huangqi 5-15 parts, Chaihu 5-15 parts, Cangzhu 2-10 parts, Shengjiang 2-10 parts, Beishashen 2-10 parts, and Chidao 3-11 parts; The solvent comprises methanol and a methanoic acid aqueous solution, the volume fraction of the methanol in the solvent is 8-12%, the volume fraction of the methanoic acid aqueous solution in the solvent is 88-92%, and the volume fraction of the methanoic acid in the methanoic acid aqueous solution is 0.05-0.2%; Performing high performance liquid chromatography tandem mass spectrometry on the test solution to scan data in positive and negative ion modes respectively, and constructing a fingerprint of the anti-virus traditional Chinese medicine formula granules; The chromatographic conditions of the high performance liquid chromatography tandem mass spectrometry comprise: Using octadecylsilane-bonded silica gel as a stationary phase, and a mobile phase comprising mobile phase A and mobile phase B, the mobile phase A comprising methanol, the mobile phase B comprising a methanoic acid aqueous solution, the volume fraction of the methanoic acid in the methanoic acid aqueous solution being 0.05-0.2%, gradient elution, and a detection wavelength of 200-300 nm; The gradient elution program comprises the following procedures: 0-10 min, the volume fraction of the mobile phase A increasing from 10% to 30%, and the volume fraction of the mobile phase B decreasing from 90% to 70%; 10-25 min, the volume fraction of the mobile phase A increasing from 30% to 75%, and the volume fraction of the mobile phase B decreasing from 70% to 25%; 25-60 min, the volume fraction of the mobile phase A increasing from 75% to 90%, and the volume fraction of the mobile phase B decreasing from 25% to 10%; 60-65 min, the volume fraction of the mobile phase A increasing from 90% to 100%, and the volume fraction of the mobile phase B decreasing from 10% to 0%; The mass spectrometry conditions of the high performance liquid chromatography tandem mass spectrometry comprise: 1) the collision gas being helium; 2) the atomization gas being nitrogen; 3) the spray voltage being 2.5KV±0.5KV; 4) the sheath gas pressure being 75arb±2arb; 5) the auxiliary gas pressure being 20arb±2arb; 6) the tail gas flow rate being 4arb±0.5arb; 7) the capillary temperature being 400℃±10℃; 8) the first full scan mass number range being m / z 80-1200; 9) the second level using data-dependent scanning, setting the threshold value of the parent ion above 1000 as the scanning object; and 10) the collision-induced dissociation voltage being 35V±2V. The fingerprint spectrum has 72 common peaks, and the substances corresponding to the common peaks include arginine, Acetyl-beta-methylcholin, malic acid, valine, pyroglutamic acid, 1-Carbamimidoyl-4-piperidine carboxylic acid, isoleucine, (4S)-4-Isopropyl-3-propioloyl-1,3-oxazolidin-2-one, leucine, hypoxanthine, 6-Isoguanosine, guanosine, 3,4,5-trimethoxyphenol-beta-D-apiofuranosyl(1→6)-beta-D-glucopyranoside, tyrosine, phenylalanine, vanillic acid-4-O-beta-D-glucoside, protocatechuic acid, magnolol, DL-tryptophan, 3,4,5-trimethoxyphenyl-beta-D-glucopyranoside, p-hydroxybenzoic acid, 1,7-diphenyl-4-en-3-heptanone, N-feruloyl-1,4-butanediamine, tert-Butyl(4-hydroxyphenyl)carbamate, corydalis tuber base, isocorydine, umbelliferone, norlaudanosine, chlorogenic acid, nuciferine, caffeic acid, Syringic acid-4-O-alpha-L-rhamnopyranoside, laudanosine, Glehlinoside C, paeonol glycoside, 6-arabinosyl-8-glucosylchrysin, 8-arabinosyl-6-glucosyl-5,7-dihydroxyflavone, verbascoside, ferulic acid, magnolol lignan B, azelaic acid, trichodesmin-7-O-beta-D-glucuronide, apigenin-7-O-beta-D-glucoside, baicalin, wogonoside-5-O-glucoside, chrysin-7-O-glucuronide, 5,7,8-trihydroxy-6-methoxyflavone-7-O-glucuronide, wogonoside, 5,7-dihydroxy-6-methoxyflavone-7-O-glucuronide, 5,7-dihydroxy-6,8-dimethoxyflavone-7-O-glucuronide, chrysoeridol, trihydroxy-dimethoxyflavone, baicalein II, 14-methyl-butyryl-2E,8EZ,10Es-atractylentriol, trichosanic acid, wogonin, divaricatic acid, fomesone A, chrysin, dihydroxy-trimethoxyflavone, isoxepoxymestrand, hesperetin, n-butyl isooctyl phthalate, and dibutyl phthalate.

2. The method according to claim 1, characterized in that, The column temperature is 32-38°C. 3.The method for constructing the fingerprint of the anti-virus traditional Chinese medicine granules according to claim 1, characterized in that, The mass-volume ratio of the antiviral traditional Chinese medicine formula granules and the solvent is (8-12) g: 1 mL.

4. A detection method of an antiviral traditional Chinese medicine formula granule, characterized in that, The method comprises the following steps: dissolving the anti-virus traditional Chinese medicine formula granules to be tested in a solvent to prepare a sample solution to be tested, wherein the solvent comprises methanol and a formic acid aqueous solution, the volume fraction of the methanol in the solvent is 8-12%, the volume fraction of the formic acid aqueous solution in the solvent is 88-92%, and the volume fraction of the formic acid in the formic acid aqueous solution is 0.05-0.2%; performing high performance liquid chromatography tandem mass spectrometry detection on the sample solution to be tested, performing data scanning in a positive ion mode and / or a negative ion mode, and obtaining corresponding spectrograms; comparing and analyzing the spectrograms with the fingerprint spectrum of the anti-virus traditional Chinese medicine formula granules constructed by the method according to any one of claims 1-3; the chromatographic conditions of the high performance liquid chromatography tandem mass spectrometry are as described in any one of claims 1-3.

5. The method for detecting antiviral traditional Chinese medicine formula granules according to claim 4, characterized in that, the mass-volume ratio of the anti-virus traditional Chinese medicine formula granules to be tested and the solvent is (8-12) g: 1 mL.

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