Construction method of characteristic chromatogram of sophora flower bud and distinguishing method of sophora flower bud and counterfeit product of sophora flower bud

Abstract

The invention discloses a sophora flower bud characteristic chromatogram construction method and a sophora flower bud and counterfeit product distinguishing method, and the sophora flower bud characteristic chromatogram construction method comprises the following steps: respectively carrying out high performance liquid chromatography detection on a test solution and a reference solution under specific chromatographic conditions to obtain the characteristic chromatogram. According to the method, a sophora flower (sophora flower bud) saponin component characteristic chromatogram is established and used for distinguishing sophora flower bud from counterfeit sophora flower bud and robinia pseudoacacia bud, high performance liquid chromatography is adopted, the sophora flower (sophora flower bud) and counterfeit standard decoction are subjected to comparative research, the internal quality of the sophora flower (sophora flower bud) standard decoction is controlled, the characteristics of the sophora flower (sophora flower bud) standard decoction can be comprehensively reflected, and the method has the advantages that the method is simple and convenient to operate and high in practicability. The method can provide a new analysis means for quality control of standard decoction and formula granules of sophora flower (sophora flower bud), and achieves the purpose of identifying sophora flower (sophora flower bud) and counterfeit standard decoction and formula granules thereof.

Description

Technical Field

[0001] The invention relates to a method for constructing a characteristic spectrum of a sophora bud and a method for distinguishing a sophora bud and its counterfeits, and belongs to the technical field of traditional Chinese medicine identification. Background Art

[0002] Sophora japonica L. is a plant of the Leguminosae family. It is harvested in summer when the flowers bloom or the buds form, and dried in time to remove branches, stems and impurities. The former is commonly known as "sophora flowers" and the latter is commonly known as "sophora rice". It is slightly cold in nature and bitter in taste; it enters the liver and large intestine meridians; it has the effects of cooling blood and stopping bleeding, clearing the liver and purging fire; it is used for blood in stool, tuberculosis, bloody dysentery, metrorrhagia, vomiting blood, epistaxis, liver heat and red eyes, headache and dizziness.

[0003] Modern research shows that Sophora japonica has many kinds of active ingredients, among which flavonoids, triterpenoid saponins, fatty acids, polysaccharides and volatile components are the most studied. The main active ingredients are flavonoids and triterpenoid saponins. Sophora japonica has pharmacological activities such as hemostasis, hypoglycemic, anti-oxidation, gastrointestinal protection, antibacterial, immunity enhancement, antiviral, blood pressure reduction, and anti-tumor.

[0004] Literature reports show that Sophora japonica saponin III has anti-aging, hypoglycemic, and hypolipidemic effects; soybean saponin Bb has the effects of inhibiting adrenaline vasodilation, lowering blood pressure, anti-inflammatory, protecting nerves and regeneration; Sophora japonica saponin I has anticoagulant effects. Saponins are one of the important components of the pharmacological activity of Sophora japonica, and Sophora japonica saponin I, Sophora japonica saponin II, and Sophora japonica saponin III are Sophora japonica-specific components.

[0005] Traditional Chinese medicines are mainly derived from plants and animals in nature, some of which have multiple origins. These traditional Chinese medicines often have confusion in origin and are difficult to distinguish between true and false, which leads to very unstable quality and greatly limits their clinical application effects. Common counterfeit products in the market mainly include the flowers and buds of Robinia pseudoacacia L., a plant of the Leguminosae family, commonly known as Robinia pseudoacacia flowers, and another common counterfeit product is the dried buds of Albizia kalkora (Roxb.) Prain, a plant of the Leguminosae family, commonly known as Albizia kalkora rice.

[0006] At present, there are many methods for comparative research on Sophora japonica and its counterfeits. Research on confusing products mainly focuses on properties and chromatography. Xu Fei et al. compared and identified Sophora japonica and Robinia japonica flowers from the aspects of properties, thin layer chromatography, and ultraviolet spectrum. Fu Fengping et al. used rutin as a control to conduct a qualitative comparison of Sophora japonica and Robinia japonica flowers by thin layer chromatography, colorimetric determination of total flavonoid content, and high performance liquid chromatography determination of rutin content. The chromatograms of Sophora japonica and Robinia japonica flowers are very different. Rutin is the main chemical component in Sophora japonica, while Robinia japonica has a main component different from rutin. The comparison of the properties of medicinal materials in the above studies is not suitable for standard decoctions and formula granules; thin layer chromatography, ultraviolet spectrophotometry, and liquid chromatography are mainly used to analyze flavonoid components commonly present in traditional Chinese medicine, such as rutin, narcissin, quercetin, etc., which leads to poor specificity of the above methods and easy adulteration. Therefore, it is necessary to establish a more specific characteristic spectrum to distinguish the authenticity. Summary of the invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for constructing a characteristic map of Sophora japonica and a method for distinguishing Sophora japonica from its counterfeits.

[0008] Technical solution: To solve the above technical problems, the present invention provides a method for constructing a characteristic map of Sophora japonica, comprising the following steps:

[0009] (1) Preparation of test solution: Take standard decoction of Sophora japonica seed, add extraction solvent to extract, cool, weigh again, make up the lost weight with corresponding reagent, shake well, filter, take the filtrate, evaporate to dryness, re-dissolve the residue with water, shake and extract with saturated n-butanol, combine the n-butanol solution, wash with ammonia test solution, take the n-butanol solution and evaporate to dryness, dissolve the residue with methanol, shake well, filter, and take the filtrate;

[0010] (2) Preparation of reference substance solution: Take soybean saponin Bb reference substance and dissolve it in solvent to obtain;

[0011] (3) Ultra-high performance liquid chromatography detection: The test sample solution and the reference substance solution were respectively injected into an ultra-high performance liquid chromatograph, and detected by a charged aerosol detector to obtain a characteristic spectrum; the characteristic spectrum had 10 common peaks, the peak corresponding to the soybean saponin Bb reference substance was the S peak, and the relative retention time of the remaining characteristic peaks and the S peak was calculated. The relative retention time was within the range of ±10% of the specified value, and the specified values ​​of peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, and peak 10 were respectively :0.47, 0.57, 0.84, 0.97, 1.05, 1.15, 1.21, 1.22, 1.28; the chromatographic conditions of the ultra-high performance liquid chromatography are: the chromatographic column is HPH-C18, EC-C18 or SB-C18; acetonitrile is used as mobile phase A, and 0.01 mol / L ammonium formate aqueous solution is used as mobile phase B; the flow rate is 0.28-0.32 ml / min; the column temperature is 38-42°C; the elution gradient is 0-1 min, and the mobile phase A The volume fraction of mobile phase A changes from 21 to 27%, and the volume fraction of mobile phase B changes from 79 to 73%; from 1 to 8 minutes, the volume fraction of mobile phase A changes from 27 to 29%, and the volume fraction of mobile phase B changes from 73 to 71%; from 8 to 11 minutes, the volume fraction of mobile phase A changes from 29 to 31%, and the volume fraction of mobile phase B changes from 71 to 69%; from 11 to 14 minutes, the volume fraction of mobile phase A changes from 31 to 34%, and the volume fraction of mobile phase B changes from 69 to 66%; From 14 to 16 min, the volume fraction of mobile phase A changed from 34 to 36%, and the volume fraction of mobile phase B changed from 66 to 64%; from 16 to 22 min, the volume fraction of mobile phase A was 36%, and the volume fraction of mobile phase B was 64%; from 22 to 25 min, the volume fraction of mobile phase A was 36 to 45%, and the volume fraction of mobile phase B was 64 to 55%; from 25 to 28 min, the volume fraction of mobile phase A was 45%, and the volume fraction of mobile phase B was 55%.

[0012] Wherein, the extraction solvent in step (1) includes water or 30% to 100% methanol.

[0013] Wherein, the extraction method in step (1) includes ultrasonic treatment, heating reflux or shaking extraction.

[0014] Wherein, the extraction time in step (1) is 15 to 60 minutes.

[0015] Wherein, the extraction times in step (1) is 1 to 3 times.

[0016] Among them, the preparation of the standard decoction: take the Sophora japonica (Sophora japonica) slices, weigh them, add water to soak, decoct, filter while hot, concentrate the filtrate under reduced pressure, and freeze-dry to obtain the Sophora japonica (Sophora japonica) standard decoction.

[0017] The present invention also provides a method for establishing a reference map of Sophora japonica, comprising the following steps:

[0018] (1) preparing multiple batches of standard decoction test sample solutions of Sophora japonicae;

[0019] (2) testing the test solution according to the method for constructing the characteristic spectrum of Sophora japonica buds to obtain the characteristic spectrum of Sophora japonica bud standard decoction;

[0020] (3) The obtained characteristic spectrum was imported into the Chinese medicine chromatographic fingerprint similarity evaluation system to establish a reference spectrum of the standard decoction of Sophora japonica seed.

[0021] The present invention also provides a method for distinguishing Sophora japonica seeds and their counterfeits, comprising the following steps:

[0022] (1) Preparation of test solution: Take the test sample, add water or 30% to 100% methanol to extract for 15 to 60 minutes, cool, weigh again, make up the lost weight with the corresponding reagent, shake well, filter, take the filtrate, evaporate to dryness, re-dissolve the residue with water, shake and extract 1 to 3 times with water-saturated n-butanol, combine the n-butanol solutions, wash with ammonia test solution, take the n-butanol solution and evaporate to dryness, dissolve the residue with methanol, shake well, filter, and take the filtrate;

[0023] (2) Preparation of reference solution: Take Sophora japonica reference medicinal material, add water to decoct, take the supernatant, shake and extract with water-saturated n-butanol, take the n-butanol layer, wash with ammonia test solution, evaporate the solvent, fix the volume, shake well, filter, and take the filtrate to obtain the reference medicinal material reference solution; take soybean saponin Bb reference substance, add solvent to dissolve, and obtain the reference substance reference solution;

[0024] (3) Ultra-high performance liquid chromatography detection: Take the test sample solution and the reference substance solution and inject them into the ultra-high performance liquid chromatography respectively, and detect them with a charged aerosol detector to obtain characteristic spectra;

[0025] (4) Discrimination: When the retention times of the 10 characteristic peaks in the chromatogram of the test solution and the reference medicinal material correspond, and the peak corresponding to the soybean saponin Bb reference is the S peak, the relative retention times of peaks 1 to 10 and the S peak are calculated respectively. If the relative retention times are within the range of ±10% of the specified value, and the relative peak area of ​​peak 7 is greater than 1.5, then the sample being tested is Sophora japonica seed; otherwise, the test sample is a counterfeit.

[0026] Wherein, the extraction method in step (1) includes ultrasonic treatment, heating reflux or shaking extraction.

[0027] Wherein, the chromatographic conditions of the ultra-high performance liquid chromatography in step (3) are as follows: the chromatographic column is HPH-C18, EC-C18 or SB-C18; acetonitrile is used as mobile phase A, and 0.01 mol / L ammonium formate aqueous solution is used as mobile phase B; the flow rate is 0.28-0.32 ml / min; the column temperature is 38-42°C; the elution gradient is 0-1 min, the volume fraction of mobile phase A is 21→27%, and the volume fraction of mobile phase B is 79→73%; 1-8 min, the volume fraction of mobile phase A changes from 27→29%, and the volume fraction of mobile phase B changes from 73→71%; 8-11 min, the volume fraction of mobile phase A changes from 29→31 %, the volume fraction of mobile phase B changes from 71→69%; from 11 to 14 min, the volume fraction of mobile phase A changes from 31→34%, and the volume fraction of mobile phase B changes from 69→66%; from 14 to 16 min, the volume fraction of mobile phase A changes from 34→36%, and the volume fraction of mobile phase B changes from 66→64%; from 16 to 22 min, the volume fraction of mobile phase A is 36%, and the volume fraction of mobile phase B is 64%; from 22 to 25 min, the volume fraction of mobile phase A is 36→45%, and the volume fraction of mobile phase B is 64→55%; from 25 to 28 min, the volume fraction of mobile phase A is 45%, and the volume fraction of mobile phase B is 55%.

[0028] Among them, the counterfeit products include Robinia pseudoacacia beans or Sophora japonica beans.

[0029] The invention also provides application of the differentiation method in quality detection of standard decoction and formula granules of Sophora japonica seed.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The method is simple, reproducible, accurate and reliable, easy to operate, short time, low solvent consumption and little environmental pollution; 2. The present invention adopts ultra-high performance liquid chromatography through a chromatographic method for the difference in components of Sophora japonica, Robinia pseudoacacia and Sophora japonica, and reasonably controls the chromatographic method, using acetonitrile as mobile phase A and 0.01 mol / L ammonium formate solution (adjusting pH value to 4.5 with formic acid) as mobile phase B for gradient elution, and establishing 10 common peaks of Sophora japonica; constructing a characteristic spectrum of Sophora japonica standard decoction, and conducting a comparative study on counterfeit products (Robinia pseudoacacia and Sophora japonica), controlling the intrinsic quality of the entire Sophora japonica standard decoction, being able to more comprehensively reflect the characteristics of Sophora japonica standard decoction and counterfeit products (Robinia pseudoacacia and Sophora japonica), providing a new analytical means for the intrinsic quality control of Sophora japonica standard decoction and formula granules, and achieving the purpose of identifying counterfeit products (Robinia pseudoacacia and Sophora japonica) standard decoction and formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The characteristic spectra of standard decoction of Sophora japonica seed under different chromatographic conditions;

[0032] Figure 2 Characteristic spectra of standard decoction of Sophora japonica seed under different extraction solvents;

[0033] Figure 3 Characteristic spectra of standard decoction of Sophora japonica seed under different extraction methods;

[0034] Figure 4 The characteristic spectrum of standard decoction of Sophora japonica seed at different extraction times;

[0035] Figure 5 The characteristic spectrum of standard decoction of Sophora japonica seed at different extraction times;

[0036] Figure 6 The characteristic spectrum of standard decoction of Sophora japonica seed under different washing conditions;

[0037] Figure 7 The chromatograms are of the standard decoction sample of Sophora japonica (Sophora japonica) and the blank control sample;

[0038] Figure 8 This is a comprehensive investigation of the characteristic spectrum of the standard decoction of Sophora japonica (Sophora japonica) flowers;

[0039] Fig. 9 Characteristic spectra of standard decoction of Sophora japonica seed under different chromatographic columns;

[0040] Fig.10 The characteristic spectra of standard decoction of Sophora japonica seed at different column temperatures;

[0041] Fig.11 The characteristic spectrum of standard decoction of Sophora japonica seed at different flow rates;

[0042] Fig.12 It is an overlay of the characteristic spectra of 24 batches of standard decoctions of Sophora japonica seeds;

[0043] Fig.13 It is the reference spectrum of standard decoction of Sophora japonica seed;

[0044] Fig.14 A control spectrum generated by superimposing the characteristic spectrum of the standard decoction of Robinia pseudoacacia;

[0045] Fig.15 A control spectrum generated by superimposing the characteristic spectrum of the standard decoction of Sophora japonica seed;

[0046] Fig.16 To compare the standard decoction of Sophora japonica and its counterfeit products;

[0047] Fig.17 It is the attribution of peak 5 in the characteristic spectrum of the test product;

[0048] Fig.18 This is the characteristic spectrum of the reference medicinal material Sophora japonica (121270-201403). DETAILED DESCRIPTION

[0049] Unless otherwise specified, the term "test article" as used herein refers to an experimental sample used for detection or identification.

[0050] Unless otherwise specified, the term "reference material" as used herein refers to a standard substance used for identification, inspection, content determination and calibration of instrument performance.

[0051] Unless otherwise specified, the term "precision weighing" used in this article means that the weight should be weighed accurately to one thousandth of the weight taken, the term "weighing" means that the weight should be weighed accurately to one hundredth of the weight taken, the term "precision measuring" means that the volume should be measured accurately to one thousandth of the volume taken, and the term "precision pipetting" refers to the operating method of accurately measuring samples through a microinjector.

[0052] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0053] Instruments, reagents and samples:

[0054] Instruments: Thermo Vanquish ultra-high performance liquid chromatograph; Thermo Vanquish charged aerosol detector (CAD); Chromeleam 7.2SR4 Chameleon workstation; Agilent Technologies 1290 Infinity ultra-high performance liquid chromatograph; 1290 DAD diode array detector; 1290 MCT column oven; 1290 Vialsampler autosampler; 1290 Fiexible pump quaternary pump; OpenLAB CDS2.3 chromatography workstation; ME204E / 02 electronic analytical balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); GKC114 temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); KQ-250E ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); TGL-16C centrifuge (Shanghai Anting Scientific Instrument Factory); acetonitrile (chromatographic grade, Thermo Fisher); Milli-Q pure water system (Millipore);

[0055] Reagents: acetonitrile (chromatographic grade, Thermo Fisher); formic acid (chromatographic grade, Aladdin); ammonium formate (chromatographic grade, Aladdin); ultrapure water; other reagents were of analytical grade.

[0056] Reference material: Soysaponin Bb (batch number: 145102) was purchased from Shanghai Shidande Biotechnology Co., Ltd.

[0057] The reference medicinal material of Sophora japonica seed (batch number: 121270-201403) was purchased from China Food and Drug Inspection Institutes.

[0058] Samples: 24 batches of Sophora japonica (Sophora japonica) medicinal materials were collected; 2 batches of Robinia japonica (Sophora japonica) medicinal materials, and 3 batches of Robinia japonica (Sophora japonica) medicinal materials. The above Sophora japonica (Sophora japonica) medicinal materials all meet the requirements of the "Sophora japonica" item in the 2020 edition of the "Chinese Pharmacopoeia". Impurities and ash were removed from the above medicinal materials to prepare the corresponding decoction pieces.

[0059] Table 1 Origin information

[0060]

[0061] Preparation of standard decoction: take 100g of Sophora japonica (Sophora japonica) slices, weigh them, put them in a casserole, add 1200ml of water and soak them for 30min, use a YMW mechanical split-type decoction pot, first boil over high heat, then simmer over low heat for 30min, select a 200-mesh standard sieve to filter while hot; add 1000ml of water for the second decoction, boil over high heat, then simmer over low heat for 25min, and filter through a 200-mesh standard sieve while hot; combine the two filtrates and concentrate them under reduced pressure at 65°C; concentrate to a relative density of 1.03-1.06 (65°C), divide the concentrate into vials, place them in a freeze dryer, and freeze-dry to obtain the Sophora japonica (Sophora japonica) standard decoction.

[0062] Preparation of reference solution: Take 0.5g of Sophora japonica reference medicinal material, place it in a stoppered conical flask, add 25ml of water, stopper it, heat and reflux for 60 minutes, cool it, weigh it again, make up the lost weight with water, shake it well, filter it, take 20ml of the filtrate, shake and extract it twice with water-saturated n-butanol, 20ml each time, combine the n-butanol solutions, wash it with ammonia test solution until it is colorless, take the n-butanol solution and evaporate it to dryness, dissolve the residue with methanol, transfer it to a 2ml volumetric flask, add methanol to the scale, shake it well, filter it, and take the filtrate as the reference solution of the reference medicinal material.

[0063] Take an appropriate amount of soybean saponin Bb reference substance, weigh it accurately, and add methanol to prepare a solution containing 20 μg of soybean saponin Bb per 1 ml, which serves as the reference substance solution.

[0064] Example 1 Determination of chromatographic conditions

[0065] Chromatographic conditions 1: HPH-C18 (150 mm × 2.1 mm, 1.9 μm); acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution according to the regulations in Table 2; flow rate 0.35 ml per minute; column temperature 40°C; detection by charged aerosol detector, the results are shown in Figure 1 .

[0066] Table 2 Gradient elution table

[0067]

[0068] Chromatographic conditions 2: HPH-C18 (150 mm × 2.1 mm, 1.9 μm); acetonitrile as mobile phase A, 10 mM ammonium formate aqueous solution (pH = 4.8) as mobile phase B, gradient elution according to the provisions in Table 3; flow rate 0.30 ml per minute; column temperature 40 ° C; evaporation tube temperature 70 ° C, the results are shown in Figure 1 .

[0069] Table 3 Gradient elution table

[0070]

[0071] Chromatographic conditions 3: HPH-C18 (150 mm × 2.1 mm, 1.9 μm); acetonitrile as mobile phase A, 10 mM ammonium formate aqueous solution (pH = 4.5) as mobile phase B, gradient elution according to the provisions in Table 4; flow rate 0.30 ml per minute; column temperature 40 ° C; evaporation tube temperature 70 ° C, the results are shown in Figure 1 .

[0072] Table 4 Gradient elution table

[0073]

[0074] After optimization, acetonitrile-0.01mol / L ammonium formate aqueous solution (adjusted to pH 4.5 with formic acid) was finally selected as the mobile phase. The chromatographic conditions were finally determined as follows: HPH-C18 (150mm×2.1mm, 1.9μm); acetonitrile as mobile phase A, 0.01mol / L ammonium formate aqueous solution (adjusted to pH 4.5 with formic acid) as mobile phase B, gradient elution according to the provisions in Table 5; flow rate of 0.30ml per minute; column temperature of 40℃; detection by charged aerosol detector. The theoretical plate number calculated based on the soybean saponin Bb peak should be no less than 5000.

[0075] Table 5 Gradient elution table

[0076]

[0077] Example 2: Preparation of saponin characteristic spectrum test solution in Sophora japonica flower (Sophora japonica seed)

[0078] 1. Investigation of different extraction solvents

[0079] Take an appropriate amount of this product (batch number: HM-DG-1, a standard decoction prepared from Sophora japonica seeds with batch number HM-YC-1), grind it into powder, take 0.5g, weigh it accurately, put it in a stoppered conical flask, accurately add 25ml of water, 30% methanol, 50% methanol, 70% methanol, and methanol respectively, stopper it, and treat it with ultrasound (power 250W, frequency 40kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with the corresponding reagent, shake it well, filter it, take 20ml of the filtrate, evaporate it to dryness, dissolve the residue in 20ml of water, extract it by shaking with saturated n-butanol with water twice, 20ml each time, combine the n-butanol solutions, wash it 3 times with ammonia test solution, take the n-butanol solution and evaporate it to dryness, dissolve the residue in methanol, transfer it to a 2ml volumetric flask, add methanol to the scale, shake it well, filter it, take the filtrate, and you have it. 1 μl of each test solution was accurately drawn and injected into the ultra-high performance liquid chromatograph, and the results were measured according to the chromatographic conditions determined in Example 1. Figure 2 .

[0080] Table 6 Comparison of different extraction solvents (peak area / sample weight)

[0081]

[0082] Conclusion: Water, 30% methanol, 50% methanol, 70% methanol and methanol extraction solvents were investigated respectively. It was found that 50% methanol had a higher extraction efficiency and better peak separation when used as the extraction solvent. Therefore, 50% methanol was determined to be the extraction solvent.

[0083] 2. Investigation of different extraction methods

[0084] Take an appropriate amount of this product (batch number: HM-DG-1), grind it into powder, take 0.5g, weigh it accurately, put it in a stoppered conical flask, accurately add 25ml of 50% methanol, seal it, ultrasonically treat it (power 250W, frequency 40kHz), heat it under reflux, shake it for 30 minutes, let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, take 20ml of the filtrate, evaporate it to dryness, dissolve the residue with 20ml of water, shake it and extract it twice with saturated n-butanol with 20ml each time, combine the n-butanol solution, wash it 3 times with ammonia test solution, take the n-butanol solution and evaporate it to dryness, dissolve the residue with methanol, transfer it to a 2ml volumetric flask, add methanol to the scale, shake it well, filter it, take the filtrate, and you're done. Accurately take 1μl of each test solution, inject it into the ultra-high performance liquid chromatograph, and measure it according to the chromatographic conditions determined in Example 1. The results are shown in Table 7. Figure 3 .

[0085] Table 7 Comparison of different extraction methods (peak area / sample weight)

[0086]

[0087] Conclusion: Ultrasonic treatment has high extraction efficiency and is easy to operate, so ultrasonic treatment was finally selected as the extraction method.

[0088] 3. Investigation of different extraction times

[0089] Take an appropriate amount of this product (batch number: HM-DG-1), grind it into powder, take 0.5g, weigh it accurately, put it in a stoppered conical flask, accurately add 25ml of 50% methanol, seal it, and ultrasonically treat it (power 250W, frequency 40kHz) for 15 minutes, 30 minutes, 45 minutes, and 60 minutes respectively. Let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, take 20ml of the filtrate, evaporate it to dryness, dissolve the residue with 20ml of water, shake and extract it twice with saturated n-butanol with water, 20ml each time, combine the n-butanol solution, wash it 3 times with ammonia test solution, take the n-butanol solution and evaporate it to dryness, dissolve the residue with methanol, transfer it to a 2ml volumetric flask, add methanol to the scale, shake it well, filter it, take the filtrate, and you're done. Accurately take 1μl of each test solution, inject it into the ultra-high performance liquid chromatograph, and measure it according to the chromatographic conditions determined in Example 1. The results are shown in Table 8. Figure 4 .

[0090] Table 8 Comparison of different extraction times

[0091]

[0092] Conclusion: From the results, it can be seen that the extraction efficiency is higher after 30 minutes of ultrasonic treatment, indicating that the extraction is basically complete, and the extraction time is confirmed to be 30 minutes.

[0093] 4. Investigation of different extraction times

[0094] Take an appropriate amount of this product (batch number: HM-DG-1), grind it into powder, take 0.5g, weigh it accurately, put it in a stoppered conical flask, accurately add 25ml of 50% methanol, seal it, and treat it ultrasonically (power 250W, frequency 40kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, take 20ml of the filtrate, evaporate it to dryness, dissolve the residue with 20ml of water, shake it with water-saturated n-butanol for 0 times, 1 time, 2 times, and 3 times, 20ml each time, combine the n-butanol solution, wash it 3 times with ammonia test solution, take the n-butanol solution and evaporate it to dryness, dissolve the residue with methanol, transfer it to a 2ml volumetric flask, add methanol to the scale, shake it well, filter it, take the filtrate, and you're done. Accurately take 1μl of each test solution, inject it into the ultra-high performance liquid chromatograph, and measure it according to the chromatographic conditions determined in Example 1. The results are shown in Table 9. Figure 5 .

[0095] Table 9 Comparison of different extraction times

[0096]

[0097]

[0098] Conclusion: From the results, it can be seen that there are more impurity peaks when there is no extraction, and the peak shape is good after extraction, with fewer impurity peaks and better separation. In addition, the extraction efficiency does not increase much after two extractions, indicating that the extraction is twice and is relatively sufficient. Taking comprehensive considerations, the number of extractions is determined to be 2 times.

[0099] 5. Investigation of different washing conditions

[0100] Take an appropriate amount of this product (batch number: HM-DG-1), grind it into powder, take 0.5g, weigh it accurately, put it in a stoppered conical flask, accurately add 25ml of 50% methanol, seal it, and treat it ultrasonically (power 250W, frequency 40kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, take 20ml of the filtrate, evaporate it to dryness, dissolve the residue with 20ml of water, shake and extract it twice with saturated n-butanol with water, 20ml each time, combine the n-butanol solution, wash it with water 3 times, wash it with ammonia test solution 3 times, and do not add any reagents, 20ml each time, take the n-butanol solution and evaporate it to dryness, dissolve the residue with methanol, transfer it to a 2ml volumetric flask, add methanol to the scale, shake it well, filter it, take the filtrate, and you are done. Accurately take 1μl of each test solution, inject it into the ultra-high performance liquid chromatograph, and measure it according to the chromatographic conditions determined in Example 1. The results are shown in Figure 6 .

[0101] Conclusion: After ammonia washing, there are fewer impurities and the peak shape is good, so the final washing condition is determined to be ammonia washing.

[0102] Based on the above results, the preparation method of the test solution in the following examples of the present invention is:

[0103] Take an appropriate amount of standard decoction of Sophora japonica (Sophora japonica seed), grind it into powder, take 0.5g, put it in a stoppered conical flask, add 25ml of 50% methanol, stopper it, ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes, shake well, filter, take 20ml of the filtrate, evaporate to dryness, dissolve the residue in 20ml of water, shake and extract twice with water-saturated n-butanol, 20ml each time, combine the n-butanol solutions, wash three times with ammonia test solution, 20ml each time, discard the ammonia test solution, evaporate the n-butanol solution, dissolve the residue in methanol, transfer it to a 2ml volumetric flask, add methanol to the scale, shake well, filter, take the filtrate, and obtain.

[0104] Example 3: Methodological verification of the characteristic spectrum detection method of saponins in the standard decoction of Sophora japonica (Sophora japonica)

[0105] 1. Exclusive inspection

[0106] Take the standard decoction sample of Sophora japonica (Sophora japonica) (batch number: HM-DG-1) and the blank control sample and test them according to the method under Example 2. The results are shown in Figure 7 .

[0107] Conclusion: The extraction solvent has no interference on the determination of characteristic peaks. The method is specific for the determination of saponin components in the standard decoction of Sophora japonica (Sophora japonica) flowers.

[0108] 2. Overall inspection

[0109] Take a sample of Sophora japonica (Sophora japonica) standard decoction (batch number: HM-DG-1), and change the elution gradient under the chromatographic conditions determined in Example 1: keep the elution gradient at the highest acetonitrile ratio, and double the elution time. The preparation conditions of the test solution are the same as those in Example 2, and record the chromatogram. The results are shown in Figure 8 .

[0110] Conclusion: No obvious chromatographic peaks were eluted after the original gradient elution, indicating that the chromatographic conditions basically met the requirements of information maximization.

[0111] 3. Precision inspection

[0112] Take the same batch number sample (batch number: HM-DG-1), prepare the test solution according to the test solution preparation method of Example 2, inject 6 times continuously, 1 μl each time, detect according to the chromatographic conditions determined in the example, and record the chromatogram. Take the peak corresponding to soybean saponin as the reference peak (peak S), calculate the relative retention time and relative peak area of ​​each peak, and the results are shown in Table 10 and Table 11.

[0113] Table 10 Precision test results - relative retention time

[0114]

[0115] Table 11 Precision test results - relative peak area

[0116]

[0117] The results showed that the relative retention time RSD of each characteristic peak was less than 1%, and the relative peak area RSD was less than 2%, indicating good precision.

[0118] 4. Repeatability Investigation

[0119] Take an appropriate amount of Sophora japonica (Sophora japonica) standard decoction (batch number: HM-DG-1) and make 6 parallel portions. Prepare test solutions according to the test solution preparation method determined in Example 2, inject 1 μl of each, and determine the peak area value according to the chromatographic conditions determined in the example. The results are shown in Tables 12 and 13 below.

[0120] Table 12 Repeatability test results (relative retention time)

[0121]

[0122] Table 13 Repeatability test results (relative peak area)

[0123]

[0124] The results showed that the relative retention time RSD of each characteristic peak was less than 1%, and the relative peak area RSD was less than 2%, indicating that the method had good repeatability.

[0125] 5. Stability test

[0126] Take the same batch number sample (batch number: HM-DG-1), prepare the test solution according to the test solution preparation method determined in Example 2, inject once every 0, 4, 8, 12, 16, 20, and 24 hours, inject 1 μl respectively, detect according to the chromatographic conditions determined in the example, and record the chromatogram. Take the peak corresponding to soybean saponin as the reference peak, calculate the relative retention time and relative peak area of ​​each common peak. The results are shown in Table 14 and Table 15.

[0127] Table 14 Stability test results - relative retention time

[0128]

[0129] Table 15 Stability test results - relative peak area

[0130]

[0131] The results showed that the relative retention time RSD of each characteristic peak was less than 1%, and the relative peak area RSD was less than 3% except for peak 2 and peak 8. The sample had good stability within 24 hours.

[0132] 6. Intermediate precision inspection

[0133] Take 3 portions of Sophora japonica (Sophora japonica) standard decoction (batch number: HM-DG-1), and prepare sample test solutions by two experimenters according to the test solution preparation method determined in Example 2, and inject 1 μl of each sample on different instruments at different times, and detect according to the chromatographic conditions determined in the example, and record the chromatogram. Take the peak corresponding to soybean saponin as the reference peak, and calculate the relative retention time of each common peak. The results are shown in Table 16.

[0134] Table 16 Intermediate precision test results (relative retention time)

[0135]

[0136] The results showed that the RSD of the relative retention time of each characteristic peak was less than 1%, and the intermediate precision of the samples was good.

[0137] 7. Durability inspection

[0138] 7.1 Investigation of different chromatographic columns

[0139] This experiment investigated the effects of three different types of chromatographic columns, with other chromatographic conditions being the same as those in Example 1 and the preparation method of the test sample being the same as that in Example 2 on the characteristic spectrum of the standard decoction of Sophora japonica (Sophora japonica) flower. The results are shown in Fig. 9 .

[0140] The results showed that the sample of Sophora japonica (Sophora japonica) could be separated well on the HPH C18 column, but the separation effect was poor on the EC-C18 and SB-C18 columns. Therefore, the Infinity Poroshell HPH-C18 (Angilent, 2.1mm×150mm, 1.9μm) column was used in the subsequent study.

[0141] 7.2 Investigation of different column temperatures

[0142] An Infinity Poroshell HPH-C18 (Angilent, 2.1 mm × 150 mm, 1.9 μm) chromatographic column was used with a flow rate of 0.30 ml / min. The sample separation effects at column temperatures of 38°C, 40°C, and 42°C were investigated. Other chromatographic conditions were the same as in Example 1. The preparation method of the test sample was the same as in Example 2. The results are shown in Table 1. Fig.10 and Table 17.

[0143] Table 17 Effect of column temperature on separation effect (relative retention time)

[0144]

[0145] The results showed that small column temperature changes within the range of 38°C to 42°C could meet the system suitability requirements, and a column temperature of 40°C was used in subsequent studies.

[0146] 7.3 Investigation of different flow rates

[0147] An Infinity Poroshell HPH-C18 (Angilent, 2.1 mm × 150 mm, 1.9 μm) chromatographic column was used, and the column temperature was 40°C. The sample separation effects at flow rates of 0.28 ml / min, 0.30 ml / min, and 0.32 ml / min were investigated. Other chromatographic conditions were the same as in Example 1. The preparation method of the test sample was the same as in Example 2. The results are shown in Table 1. Fig.11 , Table 18.

[0148] Table 18 Effect of flow rate on separation effect (relative retention time)

[0149]

[0150] The results showed that small flow rate changes in the range of 0.28ml / min to 0.32ml / min could meet the system suitability requirements, and a flow rate of 0.30ml / min was used in subsequent studies.

[0151] Example 4: Determination of characteristic spectrum of saponin components in standard decoction of Sophora japonica flower (Sophora japonica seed)

[0152] 1. Chromatographic conditions: Use HPH-C18 (150 mm × 2.1 mm, 1.9 μm) as the chromatographic column; use acetonitrile as the mobile phase A, 0.01 mol / L ammonium formate aqueous solution (adjusted to pH 4.5 with formic acid) as the mobile phase B, and perform gradient elution according to the provisions in Table 5; flow rate is 0.30 ml per minute; column temperature is 40°C; use a charged aerosol detector for detection. The theoretical plate number calculated based on the soybean saponin Bb peak should not be less than 5000.

[0153] 2. Preparation of reference solution: Take 0.5g of Sophora japonica reference medicinal material, place it in a stoppered conical flask, add 25ml of water, stopper it, heat and reflux for 60 minutes, cool it, weigh it again, make up the lost weight with water, shake it well, filter it, take 20ml of the filtrate, shake and extract it twice with water-saturated n-butanol, 20ml each time, combine the n-butanol solution, wash it with ammonia test solution until it is colorless, take the n-butanol solution and evaporate it to dryness, dissolve the residue in methanol, transfer it to a 2ml volumetric flask, add methanol to the scale, shake it well, filter it, take the filtrate as the reference solution of the reference medicinal material. Take an appropriate amount of soybean saponin Bb reference substance, accurately weigh it, add methanol to make a solution containing 20μg of soybean saponin Bb per 1ml, as the reference solution of the reference substance.

[0154] 3. Preparation of test solution: Take an appropriate amount of Sophora japonica (Sophora japonica) standard decoction sample, grind it into powder, take 0.5g, put it in a stoppered conical flask, add 25ml of 50% methanol, stopper it, ultrasonically treat it (power 250W, frequency 40kHz) for 30 minutes, shake well, filter, take 20ml of the filtrate, evaporate to dryness, dissolve the residue in 20ml of water, shake and extract twice with water-saturated n-butanol, 20ml each time, combine the n-butanol solution, wash three times with ammonia test solution, 20ml each time, discard the ammonia test solution, evaporate the n-butanol solution, dissolve the residue in methanol, transfer it to a 2ml volumetric flask, add methanol to the scale, shake well, filter, take the filtrate, and obtain.

[0155] 4. Determination method: Accurately pipette 1 μl of reference solution and test solution respectively and inject into ultra-high performance liquid chromatography.

[0156] 5. Establishment of feature maps

[0157] The characteristic spectra of 24 batches of standard decoction samples of Sophora japonica (prepared from Sophora japonica HM-YC-1 to 24) were superimposed using the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012.0 Version)" issued by the Chinese Pharmacopoeia Committee, and the common peaks with relatively stable relative retention time and relative peak area RSD of Sophora japonica (Sophora japonica) were selected as characteristic peaks. Fig.12 , generate the control map see Fig.13 The relative retention times and relative peak areas are shown in Tables 19 and 20.

[0158] Table 19 Sample determination results (relative retention time)

[0159]

[0160]

[0161] Table 19 Sample measurement results (relative peak area)

[0162]

[0163] There are 10 characteristic peaks in the chromatogram of the test sample, and the retention times of the 10 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other, among which Peak 5 should correspond to the retention time of the reference peak of soybean saponin Bb reference substance; the peak corresponding to the peak of soybean saponin Bb reference substance is peak S, and the relative retention times of the remaining characteristic peaks and peak S are calculated. The relative retention times should be within ±10% of the specified values, and the specified values ​​are: 0.47 (peak 1), 0.57 (peak 2), 0.84 (peak 3), 0.97 (peak 4), 1.05 (peak 6), 1.15 (peak 7), 1.21 (peak 8), 1.22 (peak 9), and 1.28 (peak 10).

[0164] 7. Comparison between Sophora japonica seeds and counterfeit products

[0165] The characteristic spectra of four batches of Robinia pseudoacacia decoction samples (HM-DG-1 to 4) were superimposed to generate a reference spectrum using the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System (2012.0 Version)" issued by the Chinese Pharmacopoeia Committee. Fig.14 , the characteristic spectra of 4 batches of Sophora japonica standard decoction samples were superimposed to generate a reference spectrum. Fig.15 . Comparison of standard decoction of Sophora japonica seed and counterfeit products Fig.16 , indicating that there are obvious differences in the spectra of the standard decoctions of Sophora japonica and its counterfeits (Robinia pseudoacacia and Robinia pseudoacacia).

[0166] 8. Research on the material basis of Sophora japonica and identification of characteristic peaks

[0167] Liquid chromatography-mass spectrometry (LC-MS) was used to collect high-resolution mass spectrometry data according to the liquid chromatography conditions of the characteristic spectrum of the standard decoction of Sophora japonica, and the molecular formula of the compounds in the standard decoction of Sophora japonica was inferred by combining isotope analysis. The common peaks were attributed by comparing with references and related reference substances. The retention time of peak 5 was consistent with that of the reference substance, so peak 5 in the characteristic spectrum of the test sample was identified as soybean saponin Bb, see Fig.17 The retention times of the 10 characteristic peaks in the standard decoction of Sophora japonica and the 10 characteristic peaks in the reference medicinal material of Sophora japonica are consistent, see Fig.18 .

[0168] 9. Determination of ingredients to distinguish Sophora japonica from counterfeit products (Robinia japonica, Robinia japonica)

[0169] Through mass spectrometry and reference substances, the difference components were determined, and the components were screened to distinguish the standard decoctions of Sophora japonica, Robinia pseudoacacia, and Sophora japonica. Sophora japonica and counterfeit products (Robinia pseudoacacia, Sophora japonica) can be distinguished by Peak 7. Due to the phenomenon of partial adulteration in actual production, the relative peak area of ​​Peak 7 was further used to distinguish the adulterated samples, and the minimum value of 1.80 was lowered by 15%, that is, 1.53 was used as the specified value. Finally, it was stipulated that soybean saponin Bb was used as the reference peak. If the relative peak area of ​​Peak 7 was greater than 1.50, the sample under test was Sophora japonica.

[0170] Table 2 Relative peak area range of Sophora japonica seed and counterfeit peak 7

[0171]

[0172] The review states:

[0173] a. If the retention times of the 10 characteristic peaks in the chromatogram of the tested sample and the reference medicinal material correspond, and the corresponding peak of the soybean saponin Bb reference substance is taken as the S peak, the relative retention time of peaks 1 to 10 and the S peak is calculated, and the relative retention time is within the range of ±10% of the specified value, and the relative peak area of ​​peak 7 is greater than 1.5, then the tested sample is Sophora japonica;

[0174] b. If the retention times of the 10 characteristic peaks in the chromatogram of the tested sample and the reference medicinal material correspond, and the corresponding peak of the soybean saponin Bb reference material is taken as the S peak, the relative retention time of peaks 1 to 10 and S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value, and the relative peak area of ​​peak 7 is less than 1.5, then the tested sample is a counterfeit;

[0175] c. If the retention times of the 10 characteristic peaks in the chromatogram of the tested sample and the reference medicinal material do not correspond, and the corresponding peak of the soybean saponin Bb reference material is taken as the S peak, and the relative retention times of peaks 1 to 10 and S peak are calculated, and their relative retention times are not within the range of ±10% of the specified value, and the relative peak area of ​​peak 7 is less than 1.5, then the tested sample is a counterfeit.

Claims

1. A method for constructing a characteristic spectrum of Sophora japonica, characterized in that: The following steps are involved: (1) Preparation of test solution: Take standard decoction of Sophora japonica seed, add extraction solvent to extract, cool, weigh again, make up the lost weight with corresponding reagent, shake well, filter, take the filtrate, evaporate to dryness, re-dissolve the residue with water, shake and extract with saturated n-butanol, combine the n-butanol solution, wash with ammonia test solution, take the n-butanol solution and evaporate to dryness, dissolve the residue with methanol, shake well, filter, and take the filtrate; (2) Preparation of reference substance solution: Take soybean saponin Bb reference substance and dissolve it in solvent to obtain; (3) Ultra-high performance liquid chromatography detection: The test sample solution and the reference substance solution were respectively injected into an ultra-high performance liquid chromatograph, and detected by a charged aerosol detector to obtain a characteristic spectrum; the characteristic spectrum had 10 common peaks, the peak corresponding to the soybean saponin Bb reference substance was the S peak, and the relative retention time of the remaining characteristic peaks and the S peak was calculated. The relative retention time was within the range of ±10% of the specified value, and the specified values ​​of peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, and peak 10 were respectively :0.47, 0.57, 0.84, 0.97, 1.05, 1.15, 1.21, 1.22, 1.28; the chromatographic conditions of the ultra-high performance liquid chromatography are: the chromatographic column is HPH-C18, EC-C18 or SB-C18; acetonitrile is used as mobile phase A, and 0.01 mol / L ammonium formate aqueous solution is used as mobile phase B; the flow rate is 0.28-0.32 ml / min; the column temperature is 38-42°C; the elution gradient is 0-1 min, and the mobile phase A The volume fraction of mobile phase A changes from 21 to 27%, and the volume fraction of mobile phase B changes from 79 to 73%; from 1 to 8 minutes, the volume fraction of mobile phase A changes from 27 to 29%, and the volume fraction of mobile phase B changes from 73 to 71%; from 8 to 11 minutes, the volume fraction of mobile phase A changes from 29 to 31%, and the volume fraction of mobile phase B changes from 71 to 69%; from 11 to 14 minutes, the volume fraction of mobile phase A changes from 31 to 34%, and the volume fraction of mobile phase B changes from 69 to 66%; From 14 to 16 min, the volume fraction of mobile phase A changed from 34 to 36%, and the volume fraction of mobile phase B changed from 66 to 64%; from 16 to 22 min, the volume fraction of mobile phase A was 36%, and the volume fraction of mobile phase B was 64%; from 22 to 25 min, the volume fraction of mobile phase A was 36 to 45%, and the volume fraction of mobile phase B was 64 to 55%; from 25 to 28 min, the volume fraction of mobile phase A was 45%, and the volume fraction of mobile phase B was 55%.

2. The method according to claim 1, characterized in that: The extraction solvent in step (1) includes water or 30% to 100% methanol.

3. The method according to claim 1, characterized in that: The extraction method in step (1) includes ultrasonic treatment, heating reflux or shaking extraction.

4. The method according to claim 1, characterized in that: The extraction time in step (1) is 15 to 60 minutes.

5. The method according to claim 1, characterized in that: The extraction is performed 1 to 3 times in step (1).

6. A method for distinguishing Sophora japonica seeds from their counterfeits, characterized in that: The following steps are involved: (1) Preparation of test solution: Take the test sample, add water or 30% to 100% methanol to extract for 15 to 60 minutes, cool, weigh again, make up the lost weight with the corresponding reagent, shake well, filter, take the filtrate, evaporate to dryness, re-dissolve the residue with water, shake and extract 1 to 3 times with water-saturated n-butanol, combine the n-butanol solutions, wash with ammonia test solution, take the n-butanol solution and evaporate to dryness, dissolve the residue with methanol, shake well, filter, and take the filtrate; (2) Preparation of reference solution: Take Sophora japonica reference medicinal material, add water to decoct, take the supernatant, shake and extract with water-saturated n-butanol, take the n-butanol layer, wash with ammonia test solution, evaporate the solvent, fix the volume, shake well, filter, and take the filtrate to obtain the reference medicinal material reference solution; take soybean saponin Bb reference substance, add solvent to dissolve, and obtain the reference substance reference solution; (3) Ultra-high performance liquid chromatography detection: Take the test sample solution and the reference substance solution and inject them into the ultra-high performance liquid chromatography respectively, and detect them with a charged aerosol detector to obtain characteristic spectra; (4) Discrimination: When the retention times of the 10 characteristic peaks in the chromatogram of the test solution and the reference medicinal material correspond, and the peak corresponding to the soybean saponin Bb reference is the S peak, the relative retention times of peaks 1 to 10 and the S peak are calculated respectively. If the relative retention times are within the range of ±10% of the specified value, and the relative peak area of ​​peak 7 is greater than 1.5, then the sample being tested is Sophora japonica seed; otherwise, the test sample is a counterfeit.

7. The method according to claim 6, characterized in that: The extraction method in step (1) includes ultrasonic treatment, heating reflux or shaking extraction.

8. The method according to claim 6, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography in step (3) are as follows: the chromatographic column is HPH-C18, EC-C18 or SB-C18; acetonitrile is used as mobile phase A, and 0.01 mol / L ammonium formate aqueous solution is used as mobile phase B; the flow rate is 0.28-0.32 ml / min; the column temperature is 38-42° C.; the elution gradient is 0-1 min, the volume fraction of mobile phase A is 21→27%, and the volume fraction of mobile phase B is 79→73%; 1-8 min, the volume fraction of mobile phase A changes from 27→29%, and the volume fraction of mobile phase B changes from 73→71%; 8-11 min, the volume fraction of mobile phase A changes from 29→31%, The volume fraction of mobile phase B changes from 71 to 69%; from 11 to 14 min, the volume fraction of mobile phase A changes from 31 to 34%, and the volume fraction of mobile phase B changes from 69 to 66%; from 14 to 16 min, the volume fraction of mobile phase A changes from 34 to 36%, and the volume fraction of mobile phase B changes from 66 to 64%; from 16 to 22 min, the volume fraction of mobile phase A is 36%, and the volume fraction of mobile phase B is 64%; from 22 to 25 min, the volume fraction of mobile phase A is 36 to 45%, and the volume fraction of mobile phase B is 64 to 55%; from 25 to 28 min, the volume fraction of mobile phase A is 45%, and the volume fraction of mobile phase B is 55%.

9. The method according to claim 6, characterized in that: The counterfeits include Robinia pseudoacacia beans or Robinia pseudoacacia beans.

10. Use of the method according to any one of claims 6 to 9 in quality testing of standard decoction and formula granules of Sophora japonica.

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