Detection method of fingerprint spectrum of Xuanqitongbi capsule

Abstract

This invention discloses a method for detecting the fingerprint spectrum of Xuanqi Tongbi Capsules, comprising the following steps: 1. Preparation of a reference solution; 2. Preparation of a test solution; 3. Precisely injecting the reference solution and the test solution into a high-performance liquid chromatograph and recording the chromatograms; 4. Importing the fingerprint spectrum of the test solution obtained in step 3 into a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, selecting chromatographic peaks present in the chromatograms of different batches of Xuanqi Tongbi Capsules as common peaks; generating a reference fingerprint spectrum of Xuanqi Tongbi Capsules using the average value calculation method, calculating the relative retention time and relative peak area of ​​each common peak; and labeling the chemical components of the peaks in the reference fingerprint spectrum according to the retention time of the reference solution chromatogram. The detection method for the fingerprint spectrum of Xuanqi Tongbi Capsules provided by this invention has the advantages of good stability, high precision, and good reproducibility, and can comprehensively, objectively, and accurately detect and evaluate the quality of Xuanqi Tongbi Capsules.

Description

Technical Field

[0001] The invention relates to a detection method for a traditional Chinese medicine preparation, and in particular to a detection method for a fingerprint spectrum of Xuanqi Tongbi capsules. Background Art

[0002] Xuanqi Tongbi Capsule is a clinical prescription proven by Professor Wu Zhicheng, the pioneer of Chinese ant therapy, for 60 years. It is made from ants, Panax notoginseng, Astragalus, Paris polyphylla, Geranium and Glehnia littoralis. It has the effects of promoting blood circulation and removing blood stasis, nourishing the liver and kidneys, reducing swelling and relieving pain. It is used for joint pain, swelling, cold hands and feet, numbness of limbs, and pain in shoulders, arms, waist and legs caused by wind-cold-dampness arthritis.

[0003] Xuanqi Tongbi Capsules are made of a variety of traditional Chinese medicines with complex chemical components. Quality control is the basis for ensuring the effectiveness of clinical medication. In the prior art, there are few research reports on Xuanqi Tongbi Capsules, and there is a lack of characteristic spectra that fully reflect the effective ingredients contained in Xuanqi Tongbi Capsules. Traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method that can more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicines and their preparations, and then provide an overall description and evaluation of the quality of the medicine. Therefore, it is urgent to provide a fingerprint detection method for Xuanqi Tongbi Capsules to better control product quality. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and to provide a fingerprint spectrum detection method for Xuanqi Tongbi Capsules, which can objectively, comprehensively and accurately evaluate the quality of Xuanqi Tongbi Capsules, and is of great significance for controlling the quality of Xuanqi Tongbi Capsules and ensuring clinical efficacy.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first object of the present invention is to provide a method for detecting the fingerprint of Xuanqi Tongbi Capsule, comprising the following steps:

[0007] Step 1: Preparation of standard solution:

[0008] (1) Preparation of mixed standard solution: accurately weigh corilagin, pariflavone I, pariflavone II, pariflavone VII, calycosin, calycosin glucoside, notoginsenoside R1, ginsenoside Rb1, ginsenoside Rg1 and astragaloside IV standard products, place in a volumetric flask, dilute to volume with methanol, and shake well to prepare a mixed standard solution;

[0009] (2) Preparation of single-label solution: Accurately weigh ginsenoside Re in a volumetric flask, dilute to volume with methanol, and shake well to prepare a single-label solution;

[0010] Since the retention times of ginsenoside Rg1 and single-standard ginsenoside Re in the mixed standard solution are close, ginsenoside Re was prepared separately from the other standards.

[0011] Step 2, preparation of Xuanqi Tongbi Capsule test solution:

[0012] Take different batches of Xuanqi Tongbi capsules, accurately weigh the contents of Xuanqi Tongbi capsules respectively, add methanol, reflux or ultrasonically treat, filter, take the filtrate, pass it through a microporous filter membrane, and obtain the test solution;

[0013] Step 3, respectively accurately pipette the mixed standard solution, the single standard solution and each batch of test sample solution prepared in step 1 and inject them into a high performance liquid chromatograph, use high performance liquid chromatography-ultraviolet and / or evaporative light scattering detection, and record the chromatogram;

[0014] The liquid chromatography conditions were:

[0015] Chromatographic column: octadecylsilane bonded silica gel chromatographic column;

[0016] Mobile phase: Phase A is acetonitrile, phase B is water,

[0017] Elution procedure: gradient elution with a volume fraction of 5-95% phase A and 95-5% phase B;

[0018] Column temperature: 30-40°C;

[0019] Flow rate: 0.8~1.2mL / min;

[0020] Injection volume: 10 μL;

[0021] UV detector and / or evaporative light scattering detector,

[0022] UV detection wavelength: 203nm,

[0023] Evaporative light scattering conditions:

[0024] Drift tube temperature: 90°C;

[0025] Carrier gas flow rate: 1.4SLPM;

[0026] Step 4. Import the chromatogram of the test solution of Xuanqi Tongbi Capsule obtained in step 3 into the Chinese medicine chromatographic fingerprint similarity evaluation system; select the chromatographic peaks existing in the chromatograms of different batches of Xuanqi Tongbi Capsule as common peaks; generate the control fingerprint of Xuanqi Tongbi Capsule by the average value calculation method, calculate the relative retention time and relative peak area of ​​each common peak; and mark the chemical composition of the peaks in the control fingerprint of Xuanqi Tongbi Capsule according to the retention time of the chromatogram of the standard solution to obtain the fingerprint of Xuanqi Tongbi Capsule.

[0027] Furthermore, the concentration of the mixed standard solution in step 1 is 200±20μg / mL of corilagin, 200±20μg / mL of Paris saponin I, 90±20μg / mL of Paris saponin II, 120±20μg / mL of Paris saponin VII, 40±20μg / mL of calycosin, 50±20g / mL of calycosin glucoside, 100±20μg / mL of notoginseng saponin R1, 120±20μg / mL of ginsenoside Rb1, 140±20μg / mL of ginsenoside Rg1, and 130±20μg / mL of astragaloside IV.

[0028] Preferably, the concentration of the mixed standard solution in step 1 is 200 μg / mL of corilagin, 200 μg / mL of paridis saponin I, 90 μg / mL of paridis saponin II, 120 μg / mL of paridis saponin VII, 40 μg / mL of calycosin, 50 g / mL of calycosin glucoside, 100 μg / mL of notoginseng saponin R1, 120 μg / mL of ginsenoside Rb1, 140 μg / mL of ginsenoside Rg1, and 130 μg / mL of astragaloside IV.

[0029] Furthermore, the concentration of ginsenoside Re in the single-label solution in step 1 is 150±20 μg / mL.

[0030] Preferably, the concentration of ginsenoside Re in the single-label solution in step 1 is 150 μg / mL.

[0031] Furthermore, in step 2, the ratio of the mass M of the contents of Xuanqi Tongbi Capsule to the volume V of methanol is M:V=1g:(10:15)mL; the ultrasonic time is not less than 30min; the pore size of the microporous filter membrane is 0.22μm; preferably, the ratio of the mass M of the contents of Xuanqi Tongbi Capsule to the volume V of methanol is M:V=1g:10mL.

[0032] Furthermore, the chromatographic column in step 3 is Shim-pack GIST (250 mm×4.6 mm, 5 μm).

[0033] Further, the gradient elution program in step 3 is as follows:

[0034]

[0035] The second object of the present invention is to provide a fingerprint of Xuanqi Tongbi Capsule obtained by the aforementioned establishment method, wherein the fingerprint of Xuanqi Tongbi Capsule is an ultraviolet detection fingerprint and / or an evaporative light scattering detection fingerprint.

[0036] Furthermore, 28 common peaks were detected by ultraviolet light and 23 common peaks were detected by evaporative light scattering in the fingerprint spectrum of Xuanqi Tongbi Capsule.

[0037] Furthermore, 11 components were identified from the 28 common peaks detected by UV, among which peak 5 was corilagin with a retention time of 27.237 min, peak 6 was calycosin glucoside with a retention time of 33.739 min, peak 8 was notoginsenoside R1 with a retention time of 38.828 min, peak 9 was ginsenoside Re with a retention time of 40.217 min, peak 10 was ginsenoside Rg1 with a retention time of 40.403 min, peak 13 was calycosin with a retention time of 44.298 min, peak 14 was ginsenoside Rb1 with a retention time of 48.166 min, peak 15 was astragaloside IV with a retention time of 51.210 min, peak 20 was parisosaponin VII with a retention time of 57.933 min, peak 24 was parisosaponin II with a retention time of 67.670 min. min, peak 25 was parisosaponin I with a retention time of 70.239 min; 9 components were identified among the 23 common peaks of evaporative light scattering, among which peak 1 was calycosin glucoside with a retention time of 33.946 min, peak 3 was notoginseng saponin R1 with a retention time of 39.041 min, peak 4 was ginsenoside Re with a retention time of 40.395 min, peak 5 was calycosin with a retention time of 44.502 min, peak 6 was ginsenoside Rb1 with a retention time of 48.373 min, peak 8 was astragaloside IV with a retention time of 51.418 min, peak 12 was parisosaponin VII with a retention time of 58.122 min, peak 18 was parisosaponin II with a retention time of 67.876 min, and peak 20 was parisosaponin I with a retention time of 70.468 min.

[0038] The third object of the present invention is to provide the application of the aforementioned Xuanqi Tongbi Capsule fingerprint in the quality control of Xuanqi Tongbi Capsule.

[0039] Optimization of fingerprint detection conditions:

[0040] 1. Optimization of sample solution preparation

[0041] The present invention experimentally compares different extraction methods (ultrasound, heating reflux) and different extraction solvents (water, methanol, 70% ethanol aqueous solution, 80% ethanol aqueous solution, 90% ethanol aqueous solution, anhydrous ethanol). The results show that the spectra obtained by ultrasonic extraction and reflux extraction are slightly different, both can meet the extraction requirements, and the ultrasonic extraction efficiency is relatively high, so the ultrasonic extraction method is preferably used; in the investigation of the extraction solvent, it is found that the methanol extract chromatogram has the most information and the highest component content, and the other extraction solvents cannot fully reflect the effective ingredients on the extraction effect, so methanol is selected for extraction.

[0042] 2. Optimization of chromatographic conditions

[0043] The present invention uses a diode array detector to investigate the detection wavelength and finds that when the detection wavelength is 203 nm, the chromatographic peak response value is relatively high and the most components are detected, so 203 nm is selected as the detection wavelength;

[0044] The present invention compares the elution effects of two different elution systems, methanol-water and acetonitrile-water, under different gradients. The results show that when acetonitrile and water are used as the mobile phase, the components in Xuanqi Tongbi Capsule can achieve a good separation effect, while when the mobile phase is methanol-water, the chromatographic peak separation is poor, so acetonitrile and water are finally selected as the mobile phase.

[0045] After determining the mobile phase, the present invention investigates the elution program and finds that isocratic elution cannot achieve good separation. A large number of gradient elutions are screened, and the best gradient elution program is screened as follows: 0.01-10 min, acetonitrile volume percentage 5-7%; 10-15 min, acetonitrile volume percentage 7-10%; 15-20 min, acetonitrile volume percentage 10-14%; 20-25 min, acetonitrile volume percentage 14-19%; 25-30 min, acetonitrile volume percentage 19-25%; 30-40 min, acetonitrile volume percentage 25-35%; 40-80 min, acetonitrile volume percentage 35-65%; 80-110 min, acetonitrile volume percentage 65-95%; 110-115 min, acetonitrile volume percentage 95-5%.

[0046] Beneficial Effects

[0047] 1. According to the structural properties of the active ingredients contained in Xuanqi Tongbi Capsules, the present invention screened out the best sample preparation method and chromatographic analysis conditions through a large number of experiments, and established the fingerprint detection method of Xuanqi Tongbi Capsules, which can comprehensively, objectively and accurately detect and evaluate the quality of Xuanqi Tongbi Capsules, which is of great significance for ensuring clinical efficacy.

[0048] 2. The detection method of the fingerprint spectrum of Xuanqi Tongbi Capsule provided by the present invention has been shown through methodological verification to have the advantages of good stability, high precision, good reproducibility, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is the chromatogram of the reference solution, where Figure 1 A is the UV detection spectrum of the reference substance, Figure 1 B is the evaporative light scattering detection spectrum of the reference substance.

[0050] Figure 2 This is the reference fingerprint of the Xuanqi Tongbi Capsule sample, among which, Figure 2 A is the UV detection spectrum of Xuanqi Tongbi Capsule sample, Figure 2 B is the evaporative light scattering detection spectrum of Xuanqi Tongbi Capsule sample.

[0051] Figure 3 These are the fingerprints of 14 batches of Xuanqi Tongbi Capsule samples, among which: Figure 3 A is the UV detection spectrum of 14 batches of Xuanqi Tongbi Capsules samples. Figure 3 B is the evaporative light scattering detection spectrum of 14 batches of Xuanqi Tongbi Capsules samples. DETAILED DESCRIPTION

[0052] The embodiments of the present invention are described in detail below with reference to the examples. If no specific conditions are specified in the examples, the reaction is carried out according to conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0053] The instruments and reagents used in the embodiments are as follows:

[0054] Instruments: High-performance liquid chromatograph from Shimadzu Corporation, Japan, including quaternary pump system LC-20AT, fully automatic sampling system SIL-20A, automatic temperature-controlled column oven CTO-20AC; evaporative light scattering detector ELSD-16; ML104 / 02 electronic analytical balance (Mettler Toledo); XPR2 electronic analytical balance (Mettler Toledo); KH-500E ultrasonic cleaner (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.).

[0055] Test drugs: The sources of the samples of Xuanqi Tongbi Capsules are shown in Table 1; Corilagin (batch number 111623-200302), Paris saponin I (batch number 111590-200402), Paris saponin II (batch number 111591-200402), Paris saponin VII (batch number 111593-202105), Calycosin glucoside (batch number 111920-201505), Notoginsenoside R1 (batch number 110745-2 00617), ginsenoside Rb1 (batch number 110704-200420), ginsenoside Re (batch number 110754-201123), ginsenoside Rg1 (batch number 110703-200424), and astragaloside IV (batch number 110781-202118) were purchased from the China Food and Drug Administration; calycosin isoflavone (batch number DSTDM001201) was purchased from Chengdu Desit Biotechnology Co., Ltd. Methanol (analytical grade); acetonitrile (chromatographic grade); and ultrapure water.

[0056] Table 1 Xuanqi Tongbi Capsule Sample Information

[0057]

[0058] Example 1 Detection of the fingerprint of Xuanqi Tongbi Capsule

[0059] Step 1: Preparation of reference solution:

[0060] (1) Preparation of mixed standard solution: accurately weigh corilagin, paris saponin I, paris saponin II, paris saponin VII, calycosin, calycosin glucoside, notoginseng saponin R1, ginsenoside Rb1, ginsenoside Rg1 and astragaloside IV reference substance, place in a volumetric flask, dilute to volume with methanol, shake well to prepare 200.40 μg / mL corilagin, 205.24 μg / mL paris saponin I, 88.24 μg / mL astragaloside IV. g / mL of Paris saponin II, 120.17μg / mL of Paris saponin VII, 42.38μg / mL of calycosin, 48.32μg / mL of calycosin glucoside, 99.84μg / mL of notoginseng saponin R1, 117.23μg / mL of ginsenoside Rb1, 140.56μg / mL of ginsenoside Rg1, and 129.52μg / mL of astragaloside I.

[0061] (2) Preparation of single standard solution: Accurately weigh ginsenoside Re in a volumetric flask, dilute to volume with methanol, and shake well to prepare a 153.45 μg / mL ginsenoside Re single standard solution.

[0062] Step 2, preparation of Xuanqi Tongbi Capsule test solution:

[0063] Take the above 14 batches of Xuanqi Tongbi Capsules, accurately weigh 2g of the content of Xuanqi Tongbi Capsules respectively, put them into a 100ml conical flask, add 20ml of methanol, ultrasonically treat for 30 minutes, filter, take the filtrate, pass it through a 0.22μm microporous filter membrane, and obtain the test solution.

[0064] Step 3, respectively accurately aspirate the above-mentioned reference solution and each batch of test solution, inject into the high performance liquid chromatograph, and record the chromatogram; the liquid phase conditions are: chromatographic column: Shim-pack GIST (250mm×4.6mm, 5μm); mobile phase: phase A is acetonitrile, phase B is water, gradient elution; column temperature 40°C; flow rate: 1.0mL / min; injection volume: 10μL; ultraviolet detector and evaporative light scattering detector are used together, ultraviolet detection wavelength: 203nm, evaporative conditions: drift tube temperature 90°C; carrier gas flow rate 1.4SLPM; gradient elution program is as follows:

[0065]

[0066] Step 4, export the chromatograms of the 14 batches of Xuanqi Tongbi Capsule test solutions obtained in step 3, and import them into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A; select the chromatographic peaks existing in the chromatograms of the 14 batches of Xuanqi Tongbi Capsule as common peaks; generate the control fingerprint of Xuanqi Tongbi Capsule by the average value calculation method, calculate the relative retention time and relative peak area of ​​each common peak; and mark the chemical composition of the peaks in the control fingerprint of Xuanqi Tongbi Capsule according to the retention time of the chromatograms of the mixed standard and single standard standard solutions.

[0067] See the standard chromatogram for Figure 1 , see the fingerprint for reference Figure 2 , the fingerprints of 14 batches of test products are shown in Figure 3 .

[0068] The results showed that there were 28 common peaks in the UV fingerprint of Xuanqi Tongbi Capsule. According to the retention time comparison analysis of the standard, peak 5 was determined to be corilagin with a retention time of 27.237 min, peak 6 was calycosin glucoside with a retention time of 33.739 min, peak 8 was notoginsenoside R1 with a retention time of 38.828 min, peak 9 was ginsenoside Re with a retention time of 40.217 min, and peak 10 was ginsenoside Rg1 with a retention time of 40. 403min, peak 13 is calycosin, with a retention time of 44.298min, peak 14 is ginsenoside Rb1, with a retention time of 48.166min, peak 15 is astragaloside IV, with a retention time of 51.210min, peak 20 is paridis saponin VII, with a retention time of 57.933min, peak 24 is paridis saponin II, with a retention time of 67.670min, peak 25 is paridis saponin I, with a retention time of 70.239min.

[0069] There are 23 common peaks in the evaporative light scattering fingerprint. According to the retention time comparison analysis of the standard, peak 1 is determined to be calycoside with a retention time of 33.946 min, peak 3 is notoginseng saponin R1 with a retention time of 39.041 min, peak 4 is ginsenoside Re with a retention time of 40.395 min, peak 5 is calycoside with a retention time of 44.502 min, peak 6 is ginsenoside Rb1 with a retention time of 48.373 min, peak 8 is astragaloside IV with a retention time of 51.418 min, peak 12 is parisosaponin VII with a retention time of 58.122 min, peak 18 is parisosaponin II with a retention time of 67.876 min, and peak 20 is parisosaponin I with a retention time of 70.468 min.

[0070] The generated reference fingerprint R was used to establish the common chromatographic peak pattern. The similarities between the 14 batches of Xuanqi Tongbi Capsules and the common pattern were all above 0.95, indicating that the established fingerprints can be used to evaluate the quality of Xuanqi Tongbi Capsules. In actual testing, the ultraviolet fingerprint and evaporative light scattering fingerprint can be used alone or together. The similarity results are shown in Tables 2 and 3.

[0071] Table 2 Similarity results of UV fingerprints of 14 batches of Xuanqi Tongbi Capsules

[0072] S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 R S1 1.000 0.961 0.996 0.949 0.931 0.964 0.977 0.962 0.97 0.947 0.946 0.94 0.952 0.954 0.981 S2 0.961 1.000 0.956 0.975 0.954 0.965 0.949 0.966 0.946 0.93 0.92 0.967 0.966 0.981 0.984 S3 0.996 0.956 1.000 0.944 0.925 0.961 0.969 0.955 0.96 0.952 0.936 0.933 0.945 0.949 0.976 S4 0.949 0.975 0.944 1.000 0.986 0.951 0.948 0.945 0.948 0.931 0.933 0.939 0.939 0.944 0.978 S5 0.931 0.954 0.925 0.986 1.000 0.945 0.94 0.936 0.933 0.924 0.927 0.937 0.929 0.924 0.968 S6 0.964 0.965 0.961 0.951 0.945 1.000 0.972 0.976 0.941 0.956 0.917 0.975 0.97 0.969 0.984 S7 0.977 0.949 0.969 0.948 0.94 0.972 1.000 0.975 0.98 0.958 0.953 0.957 0.968 0.956 0.985 S8 0.962 0.966 0.955 0.945 0.936 0.976 0.975 1.000 0.962 0.931 0.931 0.983 0.993 0.983 0.986 S9 0.97 0.946 0.96 0.948 0.933 0.941 0.98 0.962 1.000 0.933 0.985 0.938 0.951 0.949 0.977 S10 0.947 0.93 0.952 0.931 0.924 0.956 0.958 0.931 0.933 1.000 0.92 0.932 0.931 0.937 0.961 S11 0.946 0.92 0.936 0.933 0.927 0.917 0.953 0.931 0.985 0.92 1.000 0.918 0.922 0.923 0.957 S12 0.94 0.967 0.933 0.939 0.937 0.975 0.957 0.983 0.938 0.932 0.918 1.000 0.987 0.986 0.979 S13 0.952 0.966 0.945 0.939 0.929 0.97 0.968 0.993 0.951 0.931 0.922 0.987 1.000 0.988 0.982 S14 0.954 0.981 0.949 0.944 0.924 0.969 0.956 0.983 0.949 0.937 0.923 0.986 0.988 1.000 0.982 R 0.981 0.984 0.976 0.978 0.968 0.984 0.985 0.986 0.977 0.961 0.957 0.979 0.982 0.982 1.000

[0073] Table 3 Similarity results of evaporation fingerprints of 14 batches of Xuanqi Tongbi Capsules

[0074]

[0075]

[0076] Example 2 Methodological study of fingerprint detection method:

[0077] 1. Precision study

[0078] The test solution of sample numbered S1 prepared according to the method of Example 1 was analyzed according to the detection method of Example 1, and parallel injection was performed 6 times. The UV chromatographic peak of No. 13 isoflavones was used as the reference peak, and the RSD values ​​of the relative retention time of each common peak were calculated to be less than 0.26%, and the RSD values ​​of the relative peak area were less than 1.92%. The evaporation took No. 8 astragaloside I as the reference peak, and the RSD values ​​of the relative retention time of each common peak were calculated to be less than 0.18%, and the RSD values ​​of the relative peak area were less than 1.57%. The results showed that the parallel injection precision of the equipment was good.

[0079] 2. Reproducibility study

[0080] Six samples numbered S1 were accurately weighed in parallel, and 6 test solutions were prepared according to the method of Example 1. Referring to the chromatographic conditions of Example 1, the UV chromatographic peak of No. 13 calycosin was used as the reference peak, and the RSD values ​​of the relative retention time of each common peak were calculated to be less than 0.35%, and the RSD values ​​of the relative peak area were less than 2.34%. The evaporation took No. 8 astragaloside I as the reference peak, and the RSD values ​​of the relative retention time of each common peak were calculated to be less than 0.23%, and the RSD values ​​of the relative peak area were less than 1.94%. The results showed that the method had good repeatability.

[0081] 3. Stability study

[0082] The test solution of sample numbered S1 prepared according to the method of Example 1 was analyzed according to the detection method of Example 1, using injection analysis at different times of 0, 2, 4, 8, 12, and 24 hours, using the No. 13 chromatographic peak of isoflavones in UV as the reference peak, and calculating the RSD values ​​of the relative retention time of each common peak was less than 0.23%, and the RSD values ​​of the relative peak area were less than 1.86%, using No. 8 astragaloside I as the reference peak in evaporation, and calculating the RSD values ​​of the relative retention time of each common peak was less than 0.29%, and the RSD values ​​of the relative peak area were less than 1.96%, indicating that the chromatographic peak of the test solution of Xuanqi Tongbi Capsules within 24 hours had almost no change, and the stability was good.

[0083] The above experimental results show that the fingerprint spectrum detection method of Xuanqi Tongbi Capsule provided by the present invention has good stability, high precision and good repeatability, and can comprehensively and objectively evaluate the quality of Xuanqi Tongbi Capsule, which is of great significance for ensuring clinical efficacy.

Claims

1. A method for detecting the fingerprint of Xuanqi Tongbi Capsule, characterized in that: The following steps are involved: Step 1: Preparation of standard solution: (1) Preparation of mixed standard solution: accurately weigh corilagin, pariflavone I, pariflavone II, pariflavone VII, calycosin, calycosin glucoside, notoginsenoside R1, ginsenoside Rb1, ginsenoside Rg1 and astragaloside IV standard products, place in a volumetric flask, dilute to volume with methanol, and shake well to prepare a mixed standard solution; (2) Preparation of single-label solution: Accurately weigh ginsenoside Re in a volumetric flask, dilute to volume with methanol, and shake well to prepare a single-label solution; Step 2, preparation of Xuanqi Tongbi Capsule test solution: Take different batches of Xuanqi Tongbi capsules, accurately weigh the contents of Xuanqi Tongbi capsules respectively, add methanol, reflux or ultrasonically treat, filter, take the filtrate, pass it through a microporous filter membrane, and obtain the test solution; Step 3, respectively accurately pipette the mixed standard solution, the single standard solution and each batch of test sample solution prepared in step 1 and inject them into a high performance liquid chromatograph, use high performance liquid chromatography-ultraviolet and / or evaporative light scattering detection, and record the chromatogram; The liquid chromatography conditions were: Chromatographic column: octadecylsilane bonded silica gel chromatographic column; Mobile phase: Phase A is acetonitrile, phase B is water, Elution procedure: gradient elution with a volume fraction of 5-95% phase A and 95-5% phase B; Column temperature: 30-40°C; Flow rate: 0.8~1.2mL / min; Injection volume: 10 μL; UV detector and / or evaporative light scattering detector detection, UV detection wavelength: 203nm, Evaporative light scattering conditions: Drift tube temperature: 90°C; Carrier gas flow rate: 1.4SLPM; Step 4, importing the chromatogram of the Xuanqi Tongbi Capsule test solution obtained in step 3 into the Chinese medicine chromatographic fingerprint similarity evaluation system; selecting the chromatographic peaks existing in the chromatograms of different batches of Xuanqi Tongbi Capsule as common peaks; generating a control fingerprint of Xuanqi Tongbi Capsule by using the average value calculation method, calculating the relative retention time and relative peak area of ​​each common peak; and marking the chemical composition of the peaks in the control fingerprint of Xuanqi Tongbi Capsule according to the retention time of the chromatogram of the standard solution, to obtain the fingerprint of Xuanqi Tongbi Capsule; The gradient elution program in step 3 is as follows:

2. The method for detecting the fingerprint of Xuanqi Tongbi Capsule according to claim 1, characterized in that: The concentration of the mixed standard solution in step 1 is 200±20μg / mL of corilagin, 200±20μg / mL of paris saponin I, 90±20μg / mL of paris saponin II, 120±20μg / mL of paris saponin VII, 40±20μg / mL of calycosin, 50±20g / mL of calycosin glucoside, 100±20μg / mL of notoginseng saponin R1, 120±20μg / mL of ginsenoside Rb1, 140±20μg / mL of ginsenoside Rg1, 130±20μg / mL of astragaloside IV.

3. The method for detecting the fingerprint of Xuanqi Tongbi Capsule according to claim 1, characterized in that: The concentration of ginsenoside Re in the single-label solution in step 1 is 150±20 μg / mL.

4. The method for detecting the fingerprint of Xuanqi Tongbi Capsule according to claim 1, characterized in that: In step 2, the ratio of the mass M of the content of Xuanqi Tongbi Capsule to the volume V of methanol is M:V=1g:(10-15)mL; the ultrasonic time is not less than 30min; and the pore size of the microporous filter membrane is 0.22μm.

5. The method for detecting the fingerprint of Xuanqi Tongbi Capsule according to claim 4, characterized in that: In step 2, the ratio of the mass M of the content of Xuanqi Tongbi Capsule to the volume V of methanol is M:V=1g:10mL.

6. The method for detecting the fingerprint of Xuanqi Tongbi Capsule according to claim 1, characterized in that: The chromatographic column in step 3 is Shim-pack GIST, 250 mm×4.6 mm, 5 μm.

7. The method for detecting the fingerprint of Xuanqi Tongbi Capsule according to claim 1, characterized in that: In the fingerprint spectrum of Xuanqi Tongbi Capsule, 28 common peaks were detected by ultraviolet light and 23 common peaks were detected by evaporative light scattering.

8. The method for detecting the fingerprint of Xuanqi Tongbi Capsule according to claim 1, characterized in that: Eleven components were identified among the 28 common peaks detected by UV, among which peak 5 was corilagin with a retention time of 27.237 min, peak 6 was calycosin glucoside with a retention time of 33.739 min, peak 8 was notoginseng saponin R1 with a retention time of 38.828 min, peak 9 was ginsenoside Re with a retention time of 40.217 min, peak 10 was ginsenoside Rg1 with a retention time of 40.403 min, peak 13 was calycosin with a retention time of 44.298 min, peak 14 was ginsenoside Rb1 with a retention time of 48.166 min, peak 15 was astragaloside IV with a retention time of 51.210 min, peak 20 was parisosaponin VII with a retention time of 57.933 min, peak 24 was parisosaponin II with a retention time of 67.670 min. n, peak 25 is pariflavone I, with a retention time of 70.239 min; 9 components were identified among the 23 common peaks of evaporative light scattering, among which peak 1 is calycosin glucoside, with a retention time of 33.946 min, peak 3 is notoginseng saponin R1, with a retention time of 39.041 min, peak 4 is ginsenoside Re, with a retention time of 40.395 min, peak 5 is calycosin, with a retention time of 44.502 min, peak 6 is ginsenoside Rb1, with a retention time of 48.373 min, peak 8 is astragaloside IV, with a retention time of 51.418 min, peak 12 is pariflavone VII, with a retention time of 58.122 min, peak 18 is pariflavone II, with a retention time of 67.876 min, and peak 20 is pariflavone I, with a retention time of 70.468 min.

9. Application of the fingerprint spectrum of Xuanqi Tongbi Capsules described in claim 1 in the quality control of Xuanqi Tongbi Capsules.

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