A characteristic spectrum detection method and quality control method for Astragalus granules

Abstract

The present invention discloses a characteristic spectrum detection method and a quality control method of Astragalus granules, and belongs to the technical field of drug analysis. The characteristic spectrum detection method disclosed in the present invention respectively adopts HPLC-ELSD and HPLC-VWD to detect the characteristic spectrum of Astragalus granules. The present invention discloses the application of the characteristic spectrum detection method in the quality control of Astragalus granules. The present invention also discloses a quality control method of Astragalus granules, adopts the above-mentioned characteristic spectrum detection method of Astragalus granules to detect the characteristic spectrum of Astragalus granules, and adopts liquid chromatography to detect the content of calycosin isoflavone glucoside in Astragalus granules. The present invention is the first to respectively adopt HPLC-ELSD and HPLC-VWD to detect the characteristic spectrum of Astragalus granules; and detect the content of calycosin isoflavone glucoside by HPLC; and realizes the comprehensive and effective control of the quality of Astragalus granules, which is of great significance for further ensuring the safety and effectiveness of drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a characteristic spectrum detection method and a quality control method for astragalus granules. Background Art

[0002] Huangqi Granules are made from a single herb, Huangqi, which has the effects of replenishing qi and strengthening the exterior, promoting diuresis, expelling toxins and pus, and promoting tissue regeneration. They are used for shortness of breath, palpitations, collapse, spontaneous sweating, edema due to physical weakness, chronic diarrhea, rectal prolapse, uterine prolapse, carbuncle that is difficult to ulcerate, and wounds that do not heal for a long time. In the existing technology, such as the "Chinese Pharmacopoeia" (2020 edition, Volume 1), in addition to the conventional properties, detection, and identification items, the quality control of Huangqi Granules only has the content determination of astragaloside IV.

[0003] Astragalus contains a variety of chemical components, including saponins, flavonoids, polysaccharides, amino acids, trace elements, alkaloids, etc. Saponins mainly include astragaloside I, astragaloside II, astragaloside III, astragaloside A, isoastragaloside I, isoastragaloside II, acetylastragaloside I, etc.; flavonoids mainly include quercetin, calycosin glucoside, formononetin, calycosin, formononetin, etc.

[0004] Flavonoid compounds are one of the main active ingredients of Astragalus. Compared with Radix Astragali, the isoflavone secondary metabolite, calycosin glucoside, is only found in Astragalus and is unique to Astragalus. According to the literature "Research Progress on the Pharmacological Effects of Calycosin Glucoside" (Li Liangliang et al., Journal of Hainan Medical College, 2020, 26(02): 156-160.DOI:10.13210 / j.cnki.jhmu.20191223.001.), the pharmacological effects of calycosin glucoside, such as antioxidant, immune regulation, antiviral and improvement of myocardial function, can play a therapeutic role in the applicable population corresponding to the functional indications of Astragalus Granules (such as bronchial pneumonia and viral myocarditis in patients with lung and spleen qi deficiency). Therefore, this compound is a major active ingredient related to the functional indications of the finished drug, and is suitable as a content determination indicator for quality control of Astragalus Granules. In addition, in the Chinese Pharmacopoeia (2020 edition, Volume 1), the quality standard of Astragalus membranaceus medicinal materials includes the content control of astragaloside I as well as the content control of calycosin glucoside. Therefore, it is necessary to introduce the content determination of calycosin glucoside into the quality control of Astragalus membranaceus granules.

[0005] The literature "Research Progress on Biosynthesis of Astragaloside Components in Traditional Chinese Medicine" (Xiao Yishu et al., Journal of Shanghai University of Traditional Chinese Medicine, 2021, 35(05): 80-88+96. DOI: 10.16306 / j.1008-861x.2021.05.013.) points out that astragaloside components with cycloflavonoid as the parent core structure have multiple pharmacological effects. The specific details are shown in Table 1: Table 1 Astragaloside components and pharmacological effects based on cycloastragenol as the core structure

[0006] The document "Modern Research Progress of Astragalus and Prediction Analysis of Its Quality Markers" (Dai Yuting et al., Chinese Journal of Traditional Chinese Medicine, 2022, 47(07): 1754-1764. DOI: 10.19540 / j.cnki.cjcmm.20211213.201.) reviews the research status of the chemical components and pharmacological effects of Astragalus, and conducts quality marker prediction analysis based on the theory of quality markers (Q-markers) of traditional Chinese medicine, traditional efficacy, chemical component effectiveness, measurability, phytopathology and pharmacokinetics, and speculates that its total polysaccharides, flavonoids such as calycosin glucoside, formononetin, calycosin, quercetin and formononetin, and saponins such as astragaloside II, astragaloside III and astragaloside IV can be used as quality markers of Astragalus. However, this document only gives the quality markers of Astragalus, and does not provide specific technical guidance on how to measure these markers, especially how to measure these markers simultaneously.

[0007] The characteristic spectrum of traditional Chinese medicine is a comprehensive identification method based on the systematic study of the chemical components of traditional Chinese medicine. It is mainly used to evaluate the quality uniformity and stability of traditional Chinese medicines and traditional Chinese medicine preparations. Since traditional Chinese medicines and their preparations are usually complex systems with multiple components, their quality evaluation requires the use of detection methods that can provide rich chemical composition information. The establishment of a characteristic spectrum of traditional Chinese medicine can comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicines and their preparations, thereby comprehensively describing and evaluating the quality of the medicines.

[0008] Therefore, establishing the characteristic spectrum of Astragalus granules is of great significance for effectively controlling the quality of Astragalus granules. Summary of the invention

[0009] One of the purposes of the present invention is to provide a characteristic spectrum detection method for Astragalus granules, which can comprehensively reflect the types and quantities of chemical components of Astragalus granules and facilitate the quality control of Astragalus granules.

[0010] The second object of the present invention is to provide an application of the characteristic spectrum detection method in the quality control of Astragalus granules.

[0011] The third object of the present invention is to provide a quality control method for Astragalus granules, which adopts the above-mentioned characteristic spectrum detection method to detect the characteristic spectrum of Astragalus granules, and adopts liquid chromatography to detect the content of calycosin glucoside in Astragalus granules.

[0012] To achieve the above purpose, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention discloses a method for detecting a characteristic spectrum of Astragalus granules, which uses HPLC-ELSD and HPLC-VWD to detect the characteristic spectrum of Astragalus granules; When HPLC-ELSD is used for detection, the chromatographic conditions are as follows: using an evaporative light scattering detector, using octadecylsilane bonded silica gel as a filler; using acetonitrile as mobile phase A, using 0.04-0.06% formic acid aqueous solution as mobile phase B, and performing gradient elution according to the following regulations: 0 min, 8-12% A, 88-92% B; 15 min, 13~17% A, 83~87% B; 45 min, 68~72% A, 28~32% B; 58 min, 8~12% A, 88~92% B; When HPLC-VWD is used for detection, the chromatographic conditions are as follows: a UV detector is used, octadecylsilane bonded silica gel is used as a filler; acetonitrile is used as a mobile phase A, 0.01-0.03% formic acid aqueous solution is used as a mobile phase B, and gradient elution is performed as follows: 0 min, 13~17% A, 83~87% B; 25 min, 53~57% A, 43~47% B; 37 min, 13~17% A, 83~87% B.

[0013] In some embodiments of the present invention, when HPLC-ELSD is used for detection, the gradient elution procedure is as follows: 0 min, 10% A, 90% B; 15 min, 15% A, 85% B; 45 min, 70% A, 30% B; 58 min, 10% A, 90% B; When HPLC-VWD is used for detection, the gradient elution procedure is as follows: 0min, 15% A, 85% B; 25min, 55% A, 45% B; 37min, 15% A, 85% B.

[0014] In some embodiments of the present invention, when HPLC-ELSD is used for detection, acetonitrile is used as mobile phase A, 0.04-0.06% formic acid aqueous solution is used as mobile phase B, the column temperature is 35-50° C., and the mobile phase flow rate is 0.8-1.2 mL / min; When HPLC-VWD was used for detection, acetonitrile was used as mobile phase A, 0.01-0.03% formic acid aqueous solution was used as mobile phase B, the column temperature was 25-35°C, and the mobile phase flow rate was 0.8-1.2 mL / min.

[0015] In some embodiments of the present invention, the HPLC-ELSD detection comprises the following steps: Preparation of HPLC-ELSD test solution: Take Astragalus granules, grind them into powder, take 3 g, weigh accurately, add 40-60 mL of water, ultrasonicate for 20-40 minutes, shake well, filter, measure the filtrate, extract with water-saturated n-butanol, evaporate the n-butanol extract, dissolve the residue with methanol, transfer to a volumetric flask, add methanol to dilute to the mark, shake well, and use as HPLC-ELSD test solution; each 1 mL of HPLC-ELSD test solution contains 0.2-0.4 g Astragalus granules; Preparation of HPLC-ELSD reference solution: astragaloside IV reference substance, astragaloside I reference substance, astragaloside II reference substance, astragaloside III reference substance, isoastragaloside I reference substance, formononetin reference substance, and isoastragaloside II reference substance, place them in the same container, and add methanol to dissolve them to prepare a mixed reference solution; each 1 mL of HPLC-ELSD reference solution contains 25-100 μg of astragaloside IV, 25-100 μg of astragaloside I, 25-100 μg of astragaloside II, 25-100 μg of astragaloside III, 25-100 μg of isoastragaloside I, 25-100 μg of formononetin, and 50-200 μg of isoastragaloside II; Determination method: Accurately pipette 5-25 μL of HPLC-ELSD reference solution and HPLC-ELSD test solution respectively, inject into liquid chromatograph, measure according to the chromatographic conditions of HPLC-ELSD detection, record the chromatogram, and compare the HPLC-ELSD test solution chromatogram with the HPLC-ELSD reference solution chromatogram.

[0016] In some embodiments of the present invention, the HPLC-VWD detection comprises the following steps: Preparation of HPLC-VWD test solution: Grind Astragalus granules into powder, take 1 g, weigh accurately, add 5-15 mL of 60-80% methanol aqueous solution, sonicate for 20-40 minutes, shake well, filter, and take the filtrate as the HPLC-VWD test solution; Preparation of HPLC-VWD reference solution: take the reference substance of calycosin glucoside, calycosin isoflavone, formononetin and formononetin, place them in the same container, and add methanol to dissolve them to prepare a mixed reference solution; each 1 mL of HPLC-VWD reference solution contains 25-90 μg of calycosin glucoside, 7-30 μg of calycosin, 25-90 μg of formononetin and 7-30 μg of formononetin.

[0017] Determination method: Take 5-25 μL of HPLC-VWD reference solution and HPLC-VWD test solution, respectively, inject them into the liquid chromatograph, determine according to the chromatographic conditions of HPLC-VWD detection, record the chromatogram, and compare the chromatogram of HPLC-VWD test solution with the chromatogram of HPLC-VWD reference solution.

[0018] In a second aspect, the present invention discloses an application of a characteristic spectrum detection method for Astragalus granules, the application being an application in the quality control of Astragalus granules, wherein 13 characteristic peaks should be present in the HPLC-ELSD test sample chromatogram, wherein peak 3, peak 6, peak 7, peak 8, peak 9, peak 11, and peak 12 should correspond to the chromatographic peak retention times of formononetin, astragaloside IV, astragaloside III, astragaloside II, isoastragaloside II, astragaloside I, and isoastragaloside I in the HPLC-ELSD reference chromatogram, respectively; the peak corresponding to the astragaloside IV reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value, and the specified value is: peak 1: 0.099, peak 2: 0.615, peak 4: 0.801, peak 5: 0.931, peak 10: 1.136, peak 13: 1.273; Among them, 7 characteristic peaks should be presented in the HPLC-VWD test sample chromatogram, among which peak 1, peak 2, peak 4, and peak 5 should correspond to the retention time of the chromatographic peaks of calycosin glucoside, formononetin, calycosin, and formononetin in the HPLC-VWD reference chromatogram, respectively. The peak corresponding to the calycosin reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: Peak 3: 0.865, Peak 6: 1.648, Peak 7: 1.711.

[0019] The ±10% range described in the present invention includes the endpoint values ​​at both ends.

[0020] In a third aspect, the present invention discloses a quality control method for Astragalus granules, including two quality control indicators: a characteristic spectrum of Astragalus granules and the content of calycosin isoflavone glucoside; The characteristic spectrum of Huangqi granules was detected by the above-mentioned characteristic spectrum detection method, wherein 13 characteristic peaks should be present in the HPLC-ELSD test sample chromatogram, wherein peak 3, peak 6, peak 7, peak 8, peak 9, peak 11, and peak 12 should correspond to the retention time of the chromatographic peaks of formononetin, astragaloside IV, astragaloside III, astragaloside II, isoastragaloside II, astragaloside I, and isoastragaloside I in the HPLC-ELSD reference chromatogram, respectively; the peak corresponding to the peak of the astragaloside IV reference is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value, and the specified value is: peak 1: 0.099, peak 2: 0.615, peak 4: 0.801, peak 5: 0.931, peak 10: 1.136, peak 13: 1.273; The HPLC-VWD test sample chromatogram should show 7 characteristic peaks, of which peak 1, peak 2, peak 4, and peak 5 should correspond to the retention time of the chromatographic peaks of calycosin glucoside, formononetin, calycosin, and formononetin in the HPLC-VWD reference chromatogram, respectively. The peak corresponding to the calycosin reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value, and the specified values ​​are: peak 3: 0.865, peak 6: 1.648, peak 7: 1.711; The content of calycosin isoflavone glucoside was detected by liquid chromatography. The chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, and 0.1-0.4% formic acid solution was used as mobile phase B for gradient elution. The gradient elution procedure was as follows: 0 min, 13~17% A, 83~87% B; 20 min, 28-32%A, 68-72%B; 30 min, 28-32%A, 68-72%B; 34 min, 13~17% A, 83~87% B; Each bag of Astragalus granules contains Astragalus isoflavone glucoside C 22 H 22 O 10 It shall not be less than 1.0mg.

[0021] In some embodiments of the present invention, the gradient elution procedure for determining the content of calycosin isoflavone glucoside is as follows: 0 min, 15% A, 85% B; 20 min, 30% A, 70% B; 30 min, 30% A, 70% B; 34 min, 15% A, 85% B.

[0022] In some embodiments of the present invention, the detection wavelength is 240-280 nm, acetonitrile is used as mobile phase A, 0.1-0.4% formic acid solution is used as mobile phase B for gradient elution, and the mobile phase flow rate is 0.8-1.2 mL / min.

[0023] In some embodiments of the present invention, the detection of the content of calycosin glucoside comprises the following steps: Preparation of test sample solution: Take appropriate amount of Astragalus granules, grind them into powder, take 1g, use 25-100mL of 30-50% methanol solution as solvent, ultrasonically extract for 10-30min, take out, cool, make up the lost weight with solvent, shake well, filter, and take the filtrate as the test sample solution; Preparation of reference substance solution: Take an appropriate amount of calycosin glucoside reference substance, accurately weigh it, and add methanol to make a solution containing 10-40 μg per 1 mL as the reference substance solution; Determination method: Take 5-20 μl of the test solution and the reference solution, respectively, and inject them into the liquid chromatograph. Determine according to the chromatographic conditions for the determination of the content of calycosin isoflavone glucoside, record the chromatogram, and calculate the content of calycosin isoflavone glucoside in the test solution by the peak area according to the external standard method.

[0024] The concentration of the formic acid aqueous solution described in the present invention is a mass concentration, such as 0.04-0.06% formic acid aqueous solution means a formic acid aqueous solution with a mass fraction of 0.04-0.06%.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention establishes a characteristic spectrum detection method for Huangqi granules for the first time. The present invention adopts high performance liquid chromatography, and adopts an evaporative light detector and an ultraviolet detector respectively, to jointly establish a characteristic spectrum of Huangqi granules; it can realize the control of two types of compounds with and without ultraviolet absorption, and the control of the substance group in Huangqi granules is more comprehensive.

[0026] In the characteristic spectrum detection of the present invention, astragaloside A, astragaloside I, astragaloside II, astragaloside III, isoastragaloside I, formononetin, isoastragaloside II, calycosin isoflavone glucoside, calycosin isoflavone, formononetin, and formononetin are used as quality indicators. The "Technical Guiding Principles for the Study of Characteristic Spectra of Traditional Chinese Medicine Preparations (Trial)" points out that the characteristic spectrum should be sufficiently representative and should be able to reflect multi-component information. If the active ingredient is unclear, the chromatographic peak that appears stably in the spectrum can be selected as the characteristic peak. Although the Astragalus granules of the present invention are single-flavor medicinal material preparations, they still have the characteristics of complex ingredients and unclear active ingredients. Since this product adopts the extraction and purification process of water extraction and alcohol precipitation, multiple types of active ingredients such as flavonoids, saponins, and polysaccharides will be retained in the preparation. The present invention is based on reports of the prior art and combines the process characteristics of Astragalus granules. The above chemical components are selected as characteristic peaks of the characteristic spectrum, and an evaporative light detector and an ultraviolet detector are respectively used to establish the characteristic spectrum of the Astragalus granules. The evaluation of compounds with ultraviolet absorption, such as glycosides and flavonoids, and compounds without ultraviolet absorption, such as saponins, can be achieved, and the control of the active substance group in the preparation is more comprehensive.

[0027] The quality control method of the present invention also includes the determination of the content of calycosin isoflavone glucoside. The present invention detects the characteristic spectrum of Astragalus granules by using HPLC-ELSD and HPLC-VWD respectively for the first time; detects the content of calycosin isoflavone glucoside by HPLC; and realizes the comprehensive and effective control of the quality of Astragalus granules, which is of great significance for further ensuring the safety and effectiveness of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the specific chromatogram of the evaporation light characteristic spectrum of Huangqi granules; Figure 2 This is the durability chromatogram of the evaporation light characteristic spectrum of Huangqi granules; Figure 3 This is the specific chromatogram of the ultraviolet characteristic spectrum of Huangqi granules; Figure 4 This is the durability chromatogram of the UV characteristic spectrum of Huangqi granules; Figure 5 This is the specific chromatogram of the content of calycosin isoflavone glucoside in Huangqi granules; Figure 6 This is the standard curve of the content of calycosin isoflavone glucoside in Huangqi granules; Figure 7 This is a comparison chart of different instruments for determining the content of calycosin isoflavone glucoside in Huangqi granules; Figure 8 This is the effect of different chromatographic column temperatures on the separation effect of the determination of the content of calycosin isoflavone glucoside in Huangqi granules; Fig. 9This is the effect of different mobile phase flow rates on the separation effect of the determination of the content of calycosin isoflavone glucoside in Huangqi granules; Fig.10 This is a comparison chart of different chromatographic columns for the determination of the content of calycosin isoflavone glucoside in Huangqi granules; Fig.11 This is an overlay of the evaporation light characteristic spectra of multiple batches of Huangqi granules; Fig.12 This is an overlay of UV characteristic spectra of multiple batches of Astragalus Granules. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, the conditions recommended by the conventional conditions or reagent providers are used. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0030] Example 1 This embodiment discloses the use of the method of the present invention to detect the evaporation light characteristic spectrum of Astragalus granules.

[0031] 1. Determination method of evaporative light characteristic spectrum 1.1 Instruments and reagents High performance liquid chromatograph (Agilent 1260), analytical balance (sartorius), ultrasonic cleaning machine (KQ-500DV); Chromatographic column: Waters Symmetry Shield RP18 Column (4.6×250 mm, 5 μm); Reagents: Acetonitrile and formic acid were of chromatographic grade, and water was ultrapure water; other reagents were of analytical grade, and reference substances were purchased from China Food and Drug Inspection Institute and Chengdu Pusi Biotechnology Co., Ltd. Test drug: Astragalus granules (batch number: 2211A3, manufacturer: Sichuan Baili Pharmaceutical Co., Ltd.).

[0032] 1.2 Chromatographic conditions: Use acetonitrile as mobile phase A and 0.04% formic acid aqueous solution as mobile phase B for gradient elution. The gradient elution procedure is as follows: 0 min, 10% A, 90% B; 15 min, 15% A, 85% B; 45 min, 70% A, 30% B; 58 min, 10% A, 90% B; The flow rate is 0.9 mL per minute; the column temperature is 45°C; the evaporative light scattering detector. The theoretical plate number calculated based on the astragaloside IV peak is not less than 4000.

[0033] 1.3 Blank solvent: methanol 1.4 Preparation of HPLC-ELSD reference solution: Take appropriate amount of astragaloside IV reference substance, astragaloside I reference substance, astragaloside II reference substance, astragaloside III reference substance, isoastragaloside I reference substance, formononetin reference substance, and isoastragaloside II reference substance, accurately weigh them, place them in the same container, and add methanol to dissolve them to prepare HPLC-ELSD reference solution. Each 1 mL of HPLC-ELSD reference solution contains 50 μg of astragaloside IV, 50 μg of astragaloside I, 50 μg of astragaloside II, 50 μg of astragaloside III, 50 μg of isoastragaloside I, 50 μg of formononetin, and 100 μg of isoastragaloside II.

[0034] 1.5 Preparation of HPLC-ELSD test solution: Take an appropriate amount of Astragalus granules, grind them into fine powder, take about 3 g, weigh accurately, put it in a stoppered conical flask, add 50 mL of water, stopper it tightly, and treat it with ultrasound (power 800 W, frequency 40 kHz) for 30 minutes. Shake well, filter, measure 25 mL of the filtrate, shake and extract it with water-saturated n-butanol 4 times, 25 mL each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue with methanol and transfer it to a 5 mL volumetric flask, add methanol to dilute to the scale, shake well, and use it as the test solution.

[0035] 1.6 Accurately pipette 10 μL of the reference solution and the test solution respectively, inject into the HPLC, and measure to obtain the result.

[0036] According to the Technical Guidelines for the Study of Characteristic Spectra of Traditional Chinese Medicine Preparations (Trial) issued by the Drug Evaluation Center of the State Food and Drug Administration, in the evaluation method of the characteristic spectrum of traditional Chinese medicine preparations, the specified value range of the relative retention time of the characteristic peak should generally not exceed ±10%. The present invention refers to this guideline to conduct a methodological investigation of the evaporation light characteristic spectrum of Astragalus Granules, and determines that the relative retention time of the characteristic peak does not exceed ±10%.

[0037] 2. Specificity experiment According to the method of this embodiment, blank solvent, HPLC-ELSD reference solution, and HPLC-ELSD test solution were prepared respectively. 10 μL of each of blank solvent, HPLC-ELSD reference solution, and HPLC-ELSD test solution were accurately pipetted and injected into the high performance liquid chromatograph respectively, and measured according to the chromatographic conditions of this embodiment.

[0038] The results are attached Figure 1As shown: the blank solvent has no interference on the characteristic peaks. The HPLC-ELSD test sample chromatogram includes 13 characteristic peaks. Peak 3, peak 6, peak 7, peak 8, peak 9, peak 11, and peak 12 should correspond to the retention time of the chromatographic peaks of formononetin, astragaloside IV, astragaloside III, astragaloside II, isoastragaloside II, astragaloside I, and isoastragaloside I in the HPLC-ELSD reference chromatogram, respectively; the peak corresponding to the astragaloside IV reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The results are shown in Table 2: Table 2 Specificity results of evaporative light characteristic spectrum

[0039] The theoretical plate number of astragaloside IV peak is greater than 4000. The above results show that the method of the present invention has good specificity.

[0040] 3. Precision experiment Take Huangqi granules test sample (batch number 2211A3), prepare HPLC-ELSD test sample solution according to the method of this example and detect it, inject sample 6 times continuously, and investigate the relative retention time and relative peak area of ​​characteristic peaks respectively. The results show that the RSD of relative retention time of characteristic peaks in the test sample is less than 1.0%, and the RSD of relative peak area is less than 4.0%, indicating that the precision of the instrument is good.

[0041] 4. Repeatability Experiment Take Huangqi granules test sample (batch number 2211A3), prepare HPLC-ELSD test sample solution according to the method of this example and detect it, prepare 6 parts by the same method, and investigate the relative retention time and relative peak area of ​​characteristic peaks respectively. The results show that the RSD of the relative retention time of the characteristic peaks in the test sample is less than 1.0%, and the RSD of the relative peak area is less than 5.0%, indicating good repeatability.

[0042] 5. Stability test Take the Huangqi granules test sample (batch number 2211A3), prepare the HPLC-ELSD test solution according to the method of this example and perform the test, inject and analyze at 0, 5 h, 15 h, 25 h, 30 h, 36 h, 48 h, and investigate the relative retention time and relative peak area of ​​the characteristic peaks. The results show that the RSD of the relative retention time of the characteristic peaks in the test sample is less than 1.0%, and the RSD of the relative peak area is less than 4.0%, indicating that the test solution has good stability within 48 h.

[0043] 6. Durability test Take the Astragalus granules test sample (batch number 2211A3), prepare the HPLC-ELSD test solution according to the method of this example and perform the test. By changing the flow rate, column temperature, and different chromatographic columns for sample injection and analysis, the durability of the method is investigated, and the relative retention time of the characteristic peaks is investigated.

[0044] (1) Investigation of different column temperatures The test was conducted by changing the column temperature under "1.2 Chromatographic Conditions" of this example. The column temperatures used in this test were 43°C and 47°C respectively; (2) Investigation of different mobile phase flow rates The test was conducted by changing the mobile phase flow rate under "1.2 Chromatographic Conditions" of this example. The mobile phase flow rates used in this test were 0.8 ml / min and 1.0 ml / min respectively; (3) Investigation of different chromatographic columns The same sample was tested using different chromatographic columns.

[0045] Chromatographic column 1 was Nap ChromCore C18, (4.6 × 250 mm, 5 μm); Column 2 was YMC Hydrosphere C18, (4.6 × 250 mm, 5 μm); Chromatographic column 3 is Shimadzu shim-pack GIST C18, (4.6×250 mm, 5 μm); Column 4 was Waters XBridge®Shield RP18, (4.6×250 mm, 5 μm); Chromatographic column 5 was Nap ChromCore 120 C18-T, (4.6 × 250 mm, 5 μm); The results are attached Figure 2 As shown in the figure: Under different column temperatures and flow rates, the relative retention times of peaks 1 to 2, 4 to 5, 10, 13 and S are all within the range of ±10% of the specified value, which meets the requirements. However, there is a certain impact on astragaloside III and peak 10. Therefore, strict requirements should be imposed on the flow rate and column temperature. The fixed flow rate is 0.9 ml / min and the column temperature is 45 ° C. Different chromatographic columns have large differences in the relative retention times of peaks 4, 5, 10, 13 and S, indicating that the chromatographic column model Waters Symmetry Shield RP18 Column needs to be fixed.

[0046] Example 2 This embodiment discloses the use of the method of the present invention to detect the ultraviolet characteristic spectrum of Astragalus granules.

[0047] 1. UV characteristic spectrum determination method 1.1 Instruments and reagents High performance liquid chromatograph (Shimadzu LC-2050C), analytical balance (sartorius), ultrasonic cleaning machine (KQ-500DV); Chromatographic column: Nap ChromCore C18 (4.6×250 mm, 5 μm); Reagents: Acetonitrile and formic acid were of chromatographic grade, and water was ultrapure water; other reagents were of analytical grade, and reference materials were purchased from China Food and Drug Inspection Institute and Chengdu Pusi Biotechnology Co., Ltd. Test drug: Astragalus granules (batch number: 2211A3), manufacturer: Sichuan Baili Pharmaceutical Co., Ltd.

[0048] 1.2 Chromatographic conditions: acetonitrile as mobile phase A, 0.02% formic acid aqueous solution as mobile phase B for gradient elution, mobile phase flow rate of 1.1 mL / min; column temperature of 30°C; detection wavelength of 230 nm. The theoretical plate number calculated based on the peak of calycosin isoflavone glucoside is not less than 3000.

[0049] The gradient elution program is as follows: 0min, 15% A, 85% B; 25min, 55% A, 45% B; 37min, 15% A, 85% B.

[0050] 1.3 Blank solvent: 70% methanol.

[0051] 1.4 Preparation of HPLC-VWD reference solution: Take appropriate amounts of calycosin glucoside reference substance, calycosin, formononetin, and formononetin, weigh them accurately, put them in the same container, and add methanol to make a mixed solution containing 45 μg of calycosin glucoside, 15 μg of calycosin, 45 μg of formononetin, and 15 μg of formononetin per 1 mL, as the HPLC-VWD reference solution.

[0052] 1.5 Preparation of HPLC-VWD test solution: Take an appropriate amount of Astragalus granules, grind them into powder, take about 1.0 g, weigh accurately, put it in a stoppered conical flask, add 10 mL of 70% methanol solution, stopper it tightly, and treat it with ultrasound (power 800 W, frequency 40 kHz) for 30 min. Cool it, shake it well, filter it, and take the filtrate as the HPLC-VWD test solution.

[0053] 1.6 Accurately pipette 10 μL of HPLC-VWD reference solution and HPLC-VWD test solution respectively, inject into high performance liquid chromatograph, and measure to obtain the result.

[0054] According to the Technical Guidelines for the Study of Characteristic Spectra of Traditional Chinese Medicine Preparations (Trial) issued by the Drug Evaluation Center of the State Food and Drug Administration, in the evaluation method of the characteristic spectrum of traditional Chinese medicine preparations, the specified value range of the relative retention time of the characteristic peak should generally not exceed ±10%. The present invention refers to this guideline to conduct a methodological investigation of the ultraviolet characteristic spectrum of Huangqi granules, and determines that the relative retention time of the characteristic peak does not exceed ±10%.

[0055] 2. Specificity experiment According to the method of this embodiment, blank solvent, HPLC-VWD reference solution, and HPLC-VWD test solution were prepared respectively, and 10 μL of each of blank solvent, HPLC-VWD reference solution, and HPLC-VWD test solution were accurately pipetted and injected into high performance liquid chromatograph respectively, and the chromatographic conditions were determined according to the method of this embodiment.

[0056] The results are attached Figure 3 As shown: the blank solvent has no interference with the characteristic peaks; the HPLC-VWD test sample chromatogram includes 7 characteristic peaks, of which peak 1, peak 2, peak 4, and peak 5 should correspond to the retention times of the chromatographic peaks of calycosin glucoside, formononetin, calycosin, and formononetin in the HPLC-VWD reference chromatogram, respectively. The peak corresponding to the calycosin reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, as shown in Table 3: Table 3 UV characteristic spectrum specificity detection results

[0057] The theoretical plate number of the peak of calycosin isoflavone glucoside is greater than 3000. The results show that the method of the present invention has good specificity.

[0058] 3. Precision experiment Take the Huangqi granules test sample (batch number 2211A3), prepare the HPLC-VWD test sample solution according to the method of this example and detect it, inject the sample 6 times continuously, and investigate the relative retention time and relative peak area of ​​the characteristic peaks respectively. The results show that the RSD of the relative retention time of the characteristic peaks in the test sample is less than 1.0%, and the RSD of the relative peak area is less than 3.0%, indicating that the instrument has good precision.

[0059] 4. Repeatability Experiment Take Huangqi granules test sample (batch number 2211A3), prepare HPLC-VWD test sample solution according to the method of this example and detect it, prepare 6 copies by the same method, and investigate the relative retention time and relative peak area of ​​the characteristic peaks respectively. The results show that the RSD of the relative retention time of the characteristic peaks in the test sample is less than 1.0%, and the RSD of the relative peak area is less than 5.0%, indicating good repeatability.

[0060] 5. Stability test Take the Huangqi granules test sample (batch number 2211A3), prepare the HPLC-VWD test solution according to the method of this example and detect it, inject and analyze at 0, 6 h, 12 h, 18 h, 24 h, 36 h, and 48 h, and investigate the relative retention time and relative peak area of ​​the characteristic peaks. The results show that the RSD of the relative retention time of the characteristic peaks in the HPLC-VWD test sample chromatogram is less than 1.0%, and the RSD of the relative peak area is less than 3.0%, indicating that the test solution has good stability within 48 h.

[0061] Durability Test: Astragalus granules (batch number 2211A3) were used as test samples, and HPLC-VWD test solution was prepared and tested according to the method of this embodiment. The flow rate, column temperature, and different chromatographic columns were used for sample analysis to examine the durability of the method and the relative retention time of the characteristic peaks.

[0062] (1) Investigation of different column temperatures The test was conducted by changing the column temperature under "1.2 Chromatographic Conditions" of this example. The column temperatures used in this test were 25°C and 35°C respectively; (2) Investigation of different mobile phase flow rates The test was conducted by changing the mobile phase flow rate under "1.2 Chromatographic Conditions" of this example. The mobile phase flow rates used in this test were 1.0 ml / min and 1.2 ml / min respectively; (3) Investigation of different chromatographic columns The same sample was tested using different chromatographic columns.

[0063] Chromatographic column 1 is Shimadzu Shim-pack GIST C18, (4.6×250 mm, 5 μm); Chromatographic column 2 is Yuexu Ultimate® plus C18 HPLC Columin, (4.6×250 mm, 5 μm); The results are attached Figure 4 As shown: under different column temperatures, flow rates and different chromatographic column conditions, the relative retention times of peaks 3, 6~7 and S peak are all within ±10% of the specified value, which meets the requirements, indicating that this method has good durability.

[0064] Example 3 This embodiment discloses a method for investigating the content of calycosin glucoside in the Astragalus granules of the present invention.

[0065] 1. Determination method 1.1 Instruments and reagents High performance liquid chromatograph (Shimadzu LC-2050C), analytical balance (sartorius), ultrasonic cleaning machine (KQ-500DV); Chromatographic column: Waters XBridge® Shield RP18 (4.6×250 mm, 5 μm); Reagents: Acetonitrile and formic acid were of chromatographic grade, and water was ultrapure water; other reagents were of analytical grade, and reference materials were purchased from the China Food and Drug Inspection Institutes; Test drug: Huangqi granules (batch number: 2211A3), manufacturer: Sichuan Baili Pharmaceutical Co., Ltd.; 1.2 Chromatographic conditions: acetonitrile as mobile phase A, 0.2% formic acid solution as mobile phase B, elution gradient; mobile phase flow rate 1.0 mL / min, column temperature 30°C, detection wavelength 260 nm, theoretical plate number calculated based on the calycosin glucoside peak should be no less than 3000.

[0066] The gradient elution procedure for the determination of the content of calycosin isoflavone glucoside is as follows: 0 min, 15% A, 85% B; 20 min, 30% A, 70% B; 30 min, 30% A, 70% B; 34 min, 15% A, 85% B; 1.3 Blank solution: 50% methanol solution; 1.4 Preparation of reference substance solution: Take an appropriate amount of calycosin glucoside reference substance, weigh accurately, and add methanol to make a solution containing 20 μg per 1 mL as the reference substance solution; 1.5 Preparation of test solution: Take an appropriate amount of Astragalus granules, grind them into fine powder, take about 1.0 g, weigh it accurately, put it in a stoppered conical flask, accurately add 50 mL of 50% methanol solution by mass, stopper it tightly, weigh it, treat it ultrasonically (power 800 W, frequency 40 kHz) for 15 minutes, take it out, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate as the test solution.

[0067] 1.6 Determination method: Accurately pipette 10 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine.

[0068] 2. Specificity experiment See also Figure 5As shown, a blank solvent, a reference solution, and a test solution were prepared according to the method in this embodiment and tested, and the characteristic peak retention time and theoretical plate number were investigated. The results showed that the blank solvent had no interference, the retention time of the reference solution and the test solution was consistent, and the theoretical plate number of the calycosin glucoside peak was not less than 3000, indicating that the method had good specificity.

[0069] 3. Precision experiment Take the Huangqi granules test sample (batch number 2211A3), prepare the test sample solution according to the method in this example and perform the test, inject the sample 6 times continuously, and investigate the retention time and peak area of ​​calycosin isoflavone glucoside. The results show that the RSD of the retention time of the 6 injections in the test sample is less than 1.0%, and the RSD of the peak area is less than 2.0%, indicating that the instrument has good precision.

[0070] 4. Linear investigation Take an appropriate amount of the reference substance of calycosin isoflavone glucoside (purity 96.8%), place it in a 25 mL volumetric flask, dissolve it in methanol to make a solution containing 437.54 μg of calycosin isoflavone glucoside per 1 mL, and then dilute it to a concentration of 4.38 μg / mL, 21.88 μg / mL, 43.75 μg / mL, 65.63 μg / mL, and 87.51 μg / mL. Accurately draw 10 μL respectively, inject it into the liquid chromatograph, measure it, obtain the peak area, and draw a standard curve with concentration (X, μg / mL) as the horizontal axis and peak area (Y) as the vertical axis.

[0071] The results are attached Figure 6 As shown, the linear regression equation is y = 34.570x + 16.991, and the correlation coefficient r = 1.000, which is greater than 0.999 and meets the requirements. This shows that the linear relationship of calycosin isoflavone glucoside is good in the range of 4.38 μg / mL-87.51 μg / mL.

[0072] 5. Repeatability Experiment Take Huangqi granules test sample (batch number 2211A3), prepare test sample solution according to the method in this example and test, prepare 6 samples by the same method, and examine the RSD of the 6 results. The results show that the RSD of the test sample content is less than 3.0%, indicating good repeatability.

[0073] 6. Intermediate precision experiment (another operator measures the sample content on a different instrument at a different time) Take the Huangqi granules test sample (batch number 2211A3), prepare the test sample solution according to the method in this example and test it. Prepare 6 copies by the same method, and examine the RSD of the 6 copies and the RSD of the 12 copies (including the 6 copies of repeatability). The results are shown in the attached Figure 7As shown, the intermediate precision RSD of the test sample content is less than 3.0%, and the RSD of the content of 12 test sample solutions is less than 6.0%, indicating that the intermediate precision is good.

[0074] 7. Accuracy Accuracy is evaluated by the sample addition recovery test. A reference substance of the component to be tested with a known purity of 100% of the sample content is precisely added to the test sample with a known content of the component to be tested, and the test is performed according to the law. The recovery rate is calculated by dividing the difference between the measured value and the content in the test sample by the amount of the reference substance added.

[0075] Take the Astragalus granules test sample (batch number 2211A3), prepare the test sample solution according to the method in this embodiment (the sample amount is halved, and a reference substance of the measured component with a known purity of 100% of the sample content is added), and test, prepare 6 portions by the same method, and investigate the recovery rate and the RSD of the 6 contents; the recovery rates of the added samples of the isoflavone glucosides are all in the range of 85%-110%, the average recovery rate is 93.4%, and the RSD of the 6 portions is <4.0%, indicating that the method has good accuracy and the accuracy verification is qualified.

[0076] 8. Stability test Take the Huangqi granules test sample (batch number 2211A3), prepare the test sample solution and the reference solution according to the method in this embodiment, and test them. The samples are injected and analyzed at 0, 6 h, 12 h, 18 h, 24 h, 30 h, and 36 h, and the RSD of the peak area of ​​the reference and test samples injected 8 times is investigated. The results show that the RSD of the reference solution and the test solution is less than 2.0% within 36 hours, indicating that the reference solution and the test solution have good stability within 36 hours.

[0077] Table 4 Results of stability of calycosin glucoside content

[0078] 9. Durability test The durability of the method was investigated by changing the flow rate, column temperature, and different chromatographic columns. Astragalus granules (batch number 2211A3) were taken, and the test sample solution and the reference sample solution were prepared according to the method in this example and tested.

[0079] (1) Investigation of different column temperatures The test was performed by changing the column temperature under "1.2 Chromatographic Conditions" of this example. The results are shown in the attached Figure 8 There was no significant difference in the content at different column temperatures, indicating that the method had good durability within the column temperature range of 25℃-35℃.

[0080] (2) Investigation of different mobile phase flow rates The test was performed by changing the flow rate of the mobile phase under "1.2 Chromatographic Conditions" of this example. The results are shown in the attached figure. Fig. 9 When the flow rate was 0.9-1.1 mL / min, the RSD of the content of calycosin isoflavone glucoside was 3.9%, which was less than 4.0%, meeting the requirements.

[0081] (3) Investigation of different chromatographic columns The same sample was tested using different chromatographic columns. The results are shown in the attached figure. Fig.10 The RSD of the content of calycosin glucoside in different chromatographic columns was 3.9%, less than 4.0%, which met the requirements.

[0082] Chromatographic column 1 is Waters XBridge® Shield RP18, (4.6×250 mm, 5 μm); Chromatographic column 2 was Nanospect ChromCore C18, (4.6 × 250 mm, 5 μm); Chromatographic column 3 is Shimadzu shim-pack GIST C18, (4.6×250 mm, 5 μm); Chromatographic column 4 is a Waters Symmetry Shield RP18 Column, (4.6×250 mm, 5 μm); Example 4 This embodiment discloses that the method of the present invention is used to detect the evaporation light characteristic spectrum, ultraviolet characteristic spectrum and calycosin glucoside content of multiple batches of marketed Huangqi granules.

[0083] The Astragalus granules in this embodiment are all from Sichuan Baili Pharmaceutical Co., Ltd.

[0084] 1. Evaporative light characteristic spectrum detection Fifteen batches of Astragalus granules were tested according to the conditions in "1. Determination method" of Example 1. The results are shown in the attached figure. Fig.11 shown.

[0085] Depend on Fig.11 It can be seen that the evaporation light characteristic spectra of the 15 batches of Astragalus granules tested all showed 13 characteristic peaks, among which peak 3, peak 6, peak 7, peak 8, peak 9, peak 11, and peak 12 should correspond to the chromatographic peak retention times of formononetin, astragaloside A, astragaloside III, astragaloside II, isoastragaloside II, astragaloside I, and isoastragaloside I in the HPLC-ELSD reference chromatogram, respectively; the relative retention times of peak 1, peak 2, peak 4, peak 5, peak 10, peak 13 and peak S (peak 6) were within ±10% of the specified value.

[0086] 2. UV characteristic spectrum detection 15 batches of Astragalus granules were tested according to the conditions in "1. Determination method" of Example 2. The results are shown in the attached figure. Fig.12 shown.

[0087] Depend on Fig.12 It can be seen that the UV characteristic chromatograms of the 15 batches of Astragalus granules tested all showed 7 characteristic peaks, among which Peak 1, Peak 2, Peak 4, and Peak 5 should correspond to the retention times of the chromatographic peaks of calycosin glucoside, formononetin, calycosin and formononetin in the HPLC-VWD reference material chromatogram, respectively, and the relative retention times of Peak 3, Peak 6, Peak 7 and S peak (Peak 1) were within the range of ±10% of the specified value.

[0088] 3. Detection of the content of calycosin isoflavone glucoside 15 batches of Astragalus granules were tested according to the conditions in "1. Determination method" of Example 3. The results are shown in Table 5: Table 5 Results of the content of calycosin isoflavone glucoside in the test samples (15 batches)

[0089] From the above results, it can be seen that the content of calycosin glucoside in multiple batches of Astragalus granules ranges from 1.2 to 3.2 mg / bag, and the average content of 15 bags is 2.4 mg / bag.

[0090] The content of index components of traditional Chinese medicine preparations is related to various factors such as the origin of medicinal materials, harvesting years, processing, and preparation production technology. It can be seen from the above results that the range of the content of calycosin isoflavone glucoside in multiple batches of Astragalus granules fluctuates greatly, and only using a single calycosin isoflavone glucoside content as a quality indicator cannot accurately and comprehensively reflect the quality of Astragalus granules. On the basis of determining the content of calycosin isoflavone glucoside, the present invention uses an evaporative light detector and an ultraviolet detector to establish a characteristic spectrum of Astragalus granules, which can realize the evaluation of compounds with ultraviolet absorption such as glycosides, flavonoids and compounds without ultraviolet absorption such as saponins, thereby more comprehensively controlling the active substance group in the preparation; achieving comprehensive and effective control of the quality of Astragalus granules, which is of great significance for further ensuring the safety and effectiveness of drugs.

[0091] Finally, it should be noted that the above embodiments are only preferred embodiments of the present invention to illustrate the technical solution of the present invention, but not to limit it, and certainly not to limit the patent scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention without any substantial meaning, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention; in addition, the direct or indirect application of the technical solution of the present invention in other related technical fields is also included in the patent protection scope of the present invention.

Claims

1. A characteristic spectrum detection method for Astragalus granules, characterized in that: HPLC-ELSD and HPLC-VWD were used to detect the characteristic spectrum of Huangqi granules; When HPLC-ELSD is used for detection, the chromatographic conditions are as follows: using an evaporative light scattering detector, using octadecylsilane bonded silica gel as a filler; using acetonitrile as mobile phase A, using 0.04-0.06% formic acid aqueous solution as mobile phase B, and performing gradient elution according to the following regulations: 0 min, 8-12% A, 88-92% B; 15 minutes, 13~17% A, 83~87% B; 45 min, 68~72% A, 28~32% B; 58 min, 8~12% A, 88~92% B; When HPLC-VWD is used for detection, the chromatographic conditions are as follows: a UV detector is used, octadecylsilane bonded silica gel is used as a filler; acetonitrile is used as mobile phase A, 0.01-0.03% formic acid aqueous solution is used as mobile phase B, and gradient elution is performed as follows: 0 min, 13~17% A, 83~87% B; 25 min, 53~57% A, 43~47% B; 37 min, 13~17% A, 83~87% B.

2. The characteristic spectrum detection method of Astragalus granules according to claim 1, characterized in that: When HPLC-ELSD is used for detection, the gradient elution procedure is as follows: 0 min, 10% A, 90% B; 15 min, 15% A, 85% B; 45 min, 70% A, 30% B; 58 min, 10% A, 90% B; When HPLC-VWD is used for detection, the gradient elution procedure is as follows: 0min, 15% A, 85% B; 25min, 55% A, 45% B; 37min, 15% A, 85% B.

3. The characteristic spectrum detection method of Astragalus granules according to claim 1 or 2, characterized in that: When using HPLC-ELSD detection, acetonitrile was used as mobile phase A, 0.04-0.06% formic acid aqueous solution was used as mobile phase B, the column temperature was 35-50°C, and the mobile phase flow rate was 0.8-1.2 mL / min; When HPLC-VWD was used for detection, acetonitrile was used as mobile phase A, 0.01-0.03% formic acid aqueous solution was used as mobile phase B, the column temperature was 25-35°C, and the mobile phase flow rate was 0.8-1.2 mL / min.

4. The characteristic spectrum detection method of Astragalus granules according to claim 1 or 2, characterized in that: The following steps are included when using HPLC-ELSD detection: Preparation of HPLC-ELSD test solution: Take Astragalus granules, grind them into powder, take 3 g, weigh accurately, add 40-60 mL of water, ultrasonicate for 20-40 minutes, shake well, filter, measure the filtrate, extract with water-saturated n-butanol, evaporate the n-butanol extract, dissolve the residue with methanol, transfer to a volumetric flask, add methanol to dilute to the mark, shake well, and use as HPLC-ELSD test solution; each 1 mL of HPLC-ELSD test solution contains 0.2-0.4 g Astragalus granules; Preparation of HPLC-ELSD reference solution: astragaloside IV reference substance, astragaloside I reference substance, astragaloside II reference substance, astragaloside III reference substance, isoastragaloside I reference substance, formononetin reference substance, and isoastragaloside II reference substance, place them in the same container, and add methanol to dissolve them to prepare a mixed reference solution; each 1 mL of HPLC-ELSD reference solution contains 25-100 μg of astragaloside IV, 25-100 μg of astragaloside I, 25-100 μg of astragaloside II, 25-100 μg of astragaloside III, 25-100 μg of isoastragaloside I, 25-100 μg of formononetin, and 50-200 μg of isoastragaloside II; Determination method: Accurately pipette 5-25 μL of HPLC-ELSD reference solution and HPLC-ELSD test solution respectively, inject into liquid chromatograph, measure according to the chromatographic conditions of HPLC-ELSD detection, record the chromatogram, and compare the HPLC-ELSD test solution chromatogram with the HPLC-ELSD reference solution chromatogram.

5. The characteristic spectrum detection method of Astragalus granules according to claim 1 or 2, characterized in that: The HPLC-VWD test includes the following steps: Preparation of HPLC-VWD test solution: Grind Astragalus granules into powder, take 1 g, weigh accurately, add 5-15 mL of 60-80% methanol aqueous solution, sonicate for 20-40 minutes, shake well, filter, and take the filtrate as the HPLC-VWD test solution; Preparation of HPLC-VWD reference solution: take the reference substance of calycosin glucoside, calycosin isoflavone, formononetin and formononetin, put them in the same container, and add methanol to dissolve them to prepare a mixed reference solution; each 1 mL of HPLC-VWD reference solution contains 25-90 μg of calycosin glucoside, 7-30 μg of calycosin, 25-90 μg of formononetin and 7-30 μg of formononetin; Determination method: Take 5-25 μL of HPLC-VWD reference solution and HPLC-VWD test solution, respectively, inject them into the liquid chromatograph, determine according to the chromatographic conditions of HPLC-VWD detection, record the chromatogram, and compare the chromatogram of HPLC-VWD test solution with the chromatogram of HPLC-VWD reference solution.

6. The use of a characteristic spectrum detection method for Astragalus granules according to any one of claims 1 to 5, characterized in that: Application in quality control of Huangqi granules, where 13 characteristic peaks should appear in the HPLC-ELSD test sample chromatogram, of which peaks 3, 6, 7, 8, 9, 11 and 12 should correspond to the retention times of the chromatographic peaks of formononetin, astragaloside IV, astragaloside III, astragaloside II, isoastragaloside II, astragaloside I and isoastragaloside I in the HPLC-ELSD reference chromatogram, respectively; the peak corresponding to the astragaloside IV reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value, which is: peak 1: 0.099, peak 2: 0.615, peak 4: 0.801, peak 5: 0.931, peak 10: 1.136, peak 13: 1.273; Among them, 7 characteristic peaks should be presented in the HPLC-VWD test sample chromatogram, among which peak 1, peak 2, peak 4, and peak 5 should correspond to the retention time of the chromatographic peaks of calycosin glucoside, formononetin, calycosin, and formononetin in the HPLC-VWD reference chromatogram, respectively. The peak corresponding to the calycosin reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: Peak 3: 0.865, Peak 6: 1.648, Peak 7: 1.

711.

7. A quality control method for Astragalus granules, characterized in that: Its quality control indicators include the characteristic spectrum of Huangqi granules and the content of calycosin isoflavone glucoside; The characteristic spectrum of the Astragalus granules is detected by the characteristic spectrum detection method described in any one of claims 1 to 5. There should be 13 characteristic peaks in the HPLC-ELSD test sample chromatogram, among which peak 3, peak 6, peak 7, peak 8, peak 9, peak 11, and peak 12 should correspond to the chromatographic peak retention times of formononetin, astragaloside IV, astragaloside III, astragaloside II, isoastragaloside II, astragaloside I, and isoastragaloside I in the HPLC-ELSD reference chromatogram, respectively; the peak corresponding to the astragaloside IV reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value, and the specified values ​​are: peak 1: 0.099, peak 2: 0.615, peak 4: 0.801, peak 5: 0.931, peak 10: 1.136, peak 13: 1.273; The HPLC-VWD test sample chromatogram should show 7 characteristic peaks, of which peak 1, peak 2, peak 4, and peak 5 should correspond to the retention time of the chromatographic peaks of calycosin glucoside, formononetin, calycosin, and formononetin in the HPLC-VWD reference chromatogram, respectively. The peak corresponding to the calycosin reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value, and the specified values ​​are: peak 3: 0.865, peak 6: 1.648, peak 7: 1.711; The content of calycosin isoflavone glucoside was detected by liquid chromatography; the chromatographic conditions were: octadecylsilane bonded silica gel as filler; acetonitrile as mobile phase A, 0.1-0.4% formic acid solution as mobile phase B, and gradient elution according to the following program: 0 min, 13~17% A, 83~87% B; 20 min, 28-32%A, 68-72%B; 30 min, 28-32%A, 68-72%B; 34 min, 13~17% A, 83~87% B; Each bag of Astragalus granules contains Astragalus isoflavone glucoside C 22 H 22 O 10 It shall not be less than 1.0mg.

8. The quality control method of Astragalus granules according to claim 7, characterized in that: The gradient elution procedure for the determination of the content of calycosin isoflavone glucoside is as follows: 0 min, 15% A, 85% B; 20 min, 30% A, 70% B; 30 min, 30% A, 70% B; 34 min, 15% A, 85% B.

9. The quality control method of Astragalus granules according to claim 7 or 8, characterized in that: The detection wavelength was 240-280 nm, acetonitrile was used as mobile phase A, 0.1-0.4% formic acid solution was used as mobile phase B for gradient elution, and the flow rate of the mobile phase was 0.8-1.2 mL / min.

10. The quality control method of Astragalus granules according to claim 7 or 8, characterized in that: The detection of the content of calycosin isoflavone glucoside includes the following steps: Prepare the test solution: Take an appropriate amount of Astragalus granules, grind them into powder, take 1g, use 25-100mL of 30-50% methanol solution as solvent, ultrasonically extract for 10-30min, take out, cool, make up the lost weight with solvent, shake well, filter, and take the filtrate as the test solution; Preparation of reference substance solution: Take an appropriate amount of calycosin glucoside reference substance, accurately weigh it, and add methanol to make a solution containing 10-40 μg per 1 mL as the reference substance solution; Determination method: Take 5-20 μl of the test solution and the reference solution, respectively, and inject them into the liquid chromatograph. Determine according to the chromatographic conditions for the determination of the content of calycosin isoflavone glucoside, record the chromatogram, and calculate the content of calycosin isoflavone glucoside in the test solution by the peak area according to the external standard method.

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