A method for determining HPLC characteristic spectrum of sword bean medicinal materials, standard decoctions, and formula granules and its application

Abstract

The present invention provides a method for determining the HPLC characteristic spectrum of sword bean medicinal materials, standard decoctions, and formula granules and its application. This method uses high performance liquid chromatography for the first time to establish a method for determining the characteristic spectrum of sword bean. The chromatographic conditions of this method have the advantage of a wide selection range, and can achieve the purpose of effective separation in different mobile phases and wavelength ranges. The mobile phase A is methanol, and the mobile phase B can be selected from 0.1 vol% acetic acid, 0.1 vol% phosphoric acid or 0.1 vol% formic acid; the detection wavelength is 320-360 nm. In addition, the preparation method of the test solution also has the advantages of simple preparation and a wide selection range. The HPLC characteristic spectrum of sword bean medicinal materials, standard decoctions, and formula granules obtained according to the above method presents 6 corresponding characteristic peaks, and successfully identifies 1 rutin characteristic peak, wherein rutin is identified for the first time in the characteristic spectrum of sword bean products.

Description

Technical Field

[0001] The invention belongs to the technical field of quality control of traditional Chinese medicine preparations, and specifically relates to a method for determining HPLC characteristic spectra of sword bean medicinal materials, standard decoctions and formula granules and an application thereof. Background Art

[0002] Sword bean is the dried mature seed of the legume plant Canavalia gladiata (Jacq.) DC. The mature fruits are usually harvested in autumn, the seeds are peeled off, and they can be used as medicine after being dried. Sword bean has a sweet and mild taste, is non-toxic, and has the effects of warming the middle and lowering the qi, tonifying the kidney and replenishing the essence, removing phlegm and relieving asthma, and promoting qi and relieving pain. Among them, warming the middle and lowering the qi can be used to treat hiccups, vomiting, abdominal distension, etc.; tonifying the kidney and replenishing the essence can be used to treat kidney deficiency and low back pain; removing phlegm and relieving asthma can be used to treat cold evil damaging the lungs, lung qi stagnation, asthma, cough and gas reversal; promoting qi and relieving pain can be used to treat flank pain and dysmenorrhea.

[0003] Studies have shown that the chemical components of Jack Bean mainly include flavonoids, hemagglutinin, urease, protein, starch, canavanine or fatty acids. At present, the research on Jack Bean is mostly focused on pharmacological effects and clinical applications, and there are no reports on the characteristic spectrum of Jack Bean. In order to effectively compare and analyze the differences and changes of Jack Bean, and to evaluate and control the quality of Jack Bean as a whole, it is urgent to establish a unified method for determining the characteristic spectrum of Jack Bean. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a method for determining the HPLC characteristic spectrum of sword bean medicinal materials, standard decoctions, and formula granules and its application. This method uses high performance liquid chromatography for the first time to establish a method for determining the characteristic spectrum of sword bean medicinal materials, standard decoctions, and formula granules, which can effectively separate the chromatographic components of sword bean and can be used for overall evaluation and control of the quality of sword bean.

[0005] To achieve this object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for determining the HPLC characteristic spectrum of sword bean medicinal materials, standard decoctions, and formula granules, comprising the following steps:

[0007] The test solution and the reference solution are tested and analyzed by liquid chromatography to obtain;

[0008] The reference substance in the reference substance solution is rutin;

[0009] The conditions of the liquid chromatography are: mobile phase A is methanol, mobile phase B is selected from 0.1 vol% acetic acid, 0.1 vol% phosphoric acid or 0.1 vol% formic acid, the detection wavelength is 320-360 nm, and gradient elution is performed. The procedure of gradient elution is:

[0010] 0-3min: 0-15 vol% mobile phase A, 100-85 vol% mobile phase B;

[0011] 3-18 min: 15-35 vol% mobile phase A, 85-65 vol% mobile phase B;

[0012] 18-31min: 35-40vol% mobile phase A, 65-60vol% mobile phase B;

[0013] 31-45 min: 40-55 vol% mobile phase A, 60-45 vol% mobile phase B;

[0014] 45-55min: 55-60vol% mobile phase A, 45-40vol% mobile phase B.

[0015] Preferably, the filler of the chromatographic column in the liquid chromatography is octadecylsilane bonded silica gel; the column temperature is 25 to 35° C.; and the flow rate is 0.8 to 1.2 mL / min.

[0016] Preferably, the detection wavelength is 330 nm.

[0017] Preferably, the column temperature is 30°C and the flow rate is 1.0 mL / min.

[0018] Preferably, the added amount of the test solution and the reference solution is independently 8 to 10 μL.

[0019] Preferably, the test sample solution is obtained by mixing the test sample with a solvent and then performing ultrasonic extraction or reflux extraction.

[0020] Preferably, the ratio of the test sample to the solvent is 1 g: (10-50) mL.

[0021] Preferably, the solvent is selected from 30-80 vol% methanol or 50 vol% ethanol.

[0022] Preferably, the ultrasonic extraction or reflux extraction time is not less than 30 min.

[0023] Preferably, the ultrasonic extraction has a power of 500-600 W and a frequency of 30-40 kHz.

[0024] Preferably, the reference solution is obtained by mixing rutin with 60-80 vol% methanol.

[0025] Preferably, the concentration of the reference substance in the reference substance solution is 15-20 μg / mL.

[0026] Preferably, the standard HPLC characteristic spectrum of the sword bean medicinal material, standard decoction, and formula granules obtained by the determination method presents 6 corresponding characteristic peaks, and 1 rutin characteristic peak is identified.

[0027] In a second aspect, the present invention also provides an application of the HPLC characteristic spectrum obtained by the above-mentioned determination method in detecting the quality of sword bean medicinal materials, standard decoctions, and formula granules.

[0028] In a third aspect, the present invention further provides a quality control method for sword bean medicinal materials, standard decoctions, and formula granules, comprising the following steps:

[0029] Take the samples of sword bean medicinal materials, standard decoctions, and formula granules to be tested, and obtain the HPLC characteristic spectrum of the samples according to the above determination method;

[0030] Compare with the standard HPLC characteristic spectrum obtained by the above-mentioned determination method; if the HPLC fingerprint spectrum of the sample to be tested presents 6 corresponding characteristic peaks, and the RSD value of the relative retention time of each peak in the sample HPLC characteristic spectrum and the relative retention time of each peak in the standard HPLC characteristic spectrum is ≤2.0%, it is judged to be a qualified product.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a method for determining the HPLC characteristic spectrum of sword bean medicinal materials, standard decoctions, and formula granules. The method adopts high performance liquid chromatography for the first time to establish a method for determining the characteristic spectrum of sword bean. The chromatographic conditions of the method are first explored and have the advantage of a wide selection range. The purpose of effective separation can be achieved in different mobile phases and wavelength ranges. The mobile phase A is methanol, and the mobile phase B can be selected from 0.1 vol% acetic acid, 0.1 vol% phosphoric acid or 0.1 vol% formic acid; the detection wavelength is 320-360 nm. In addition, the preparation method of the test solution also has the advantages of simple preparation and a wide selection range. The HPLC characteristic spectrum of sword bean medicinal materials, standard decoctions, and formula granules obtained according to the above method presents 6 corresponding characteristic peaks, and successfully identifies 1 rutin characteristic peak, wherein rutin is identified for the first time in the characteristic spectrum of sword bean products.

[0033] The determination method provided by the present invention has the characteristics of strong operability, convenient analysis and stability, and the quality of sword beans can be effectively controlled by utilizing the obtained high performance liquid fingerprint spectrum. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The liquid chromatograms obtained under different mobile phases using Jack Bean as the test sample;

[0035] Figure 2 The liquid chromatograms obtained at different detection wavelengths using Jack Bean as the test sample;

[0036] Figure 3 The liquid chromatograms obtained at different flow rates using Jack Bean as the test sample;

[0037] Figure 4 The liquid chromatograms obtained at different column temperatures using Jack Bean as the test sample;

[0038] Figure 5 This is the liquid chromatogram obtained after delay investigation using Jack Bean as the test sample;

[0039] Figure 6 This is the liquid chromatogram obtained under different extraction solvents using Jack Bean as the test sample;

[0040] Figure 7 This is the liquid chromatogram obtained at different extraction times using Jack Bean as the test sample;

[0041] Figure 8 This is the liquid chromatogram obtained with different solvent addition amounts using Jack Bean as the test sample;

[0042] Fig. 9 The liquid chromatograms obtained by different extraction methods using Jack Bean as the test sample;

[0043] Fig.10 It is a chromatographic peak identification diagram of the characteristic spectrum of sword bean medicinal material;

[0044] Fig.11 The UV absorption spectra of rutin in the sword bean medicinal material as the test sample and the rutin reference substance;

[0045] Figure a is the UV spectrum of rutin in medicinal materials, and Figure b is the UV spectrum of rutin reference substance;

[0046] Fig.12 This is the liquid chromatogram obtained after different instruments were investigated using Jack Bean as the test sample;

[0047] Fig.13 This is the liquid chromatogram obtained after investigating different chromatographic columns using Jack Bean as the test sample;

[0048] Fig.14 Characteristic spectra of different batches of sword bean medicinal materials;

[0049] Fig.15 It is a reference atlas of sword bean medicinal materials;

[0050] Fig.16 Characteristic spectra of different batches of standard decoction of sword bean;

[0051] Fig.17 It is a reference chart of the standard decoction of sword bean;

[0052] Fig.18 Characteristic spectra of different batches of sword bean formula granules;

[0053] Fig.19 It is a comparison chart of sword bean formula granules;

[0054] Fig. 20 This is a comparison chart of the characteristic spectra of Jack Bean medicinal materials, Jack Bean standard decoction, and Jack Bean formula granules. DETAILED DESCRIPTION

[0055] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] In view of the lack of an evaluation method for overall evaluation and control of the quality of sword bean in the prior art, the present invention provides a method for determining the HPLC characteristic spectrum of sword bean medicinal materials, standard decoctions, and formula granules, comprising the following steps:

[0057] The test solution and the reference solution are tested and analyzed by liquid chromatography to obtain;

[0058] The reference substance in the reference substance solution is rutin.

[0059] In the present invention, the test solution is the corresponding solution of sword bean medicinal material, standard decoction, and formula granules. In some embodiments of the present invention, the test solution is obtained by mixing the test sample with a solvent, and then ultrasonically extracting or refluxing. Wherein, the solvent is selected from 30-80 vol% methanol or 50 vol% ethanol, preferably 30-80 vol% methanol, and more preferably 80 vol% methanol. In the present invention, based on the mass of the test sample 1g, the amount of solvent added is 10-50 mL, which can be 10 mL, 25 mL, 50 mL, and preferably 10 mL. According to the present invention, after the test sample is mixed with the solvent, the obtained mixed solution is obtained by ultrasonic extraction or reflux extraction. To ensure complete extraction, the time of the ultrasonic extraction or reflux extraction is preferably not less than 30 min, preferably 30 min. Since there is little difference between the two when reflux and ultrasonic extraction are performed on the test sample, the preferred extraction method of the present invention is ultrasonic extraction, and the power of the ultrasonic extraction is 500-600 W and the frequency is 30-40 kHz.

[0060] In some specific embodiments of the present invention, the method for preparing the test solution of the characteristic spectrum of the sword bean medicinal material is preferably: take an appropriate amount of the product (i.e., the sword bean medicinal material), take 1.0 g, place it in a stoppered conical flask, add 10 mL of 80% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, filter, and take the filtrate to obtain.

[0061] The method for preparing the test solution of the characteristic spectrum of the standard decoction of sword bean is preferably as follows: take 1 g of the powder of the product (i.e., the standard decoction of sword bean), place it in a stoppered conical flask, add 25 mL of 80% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain.

[0062] The method for preparing the test solution of the characteristic spectrum of the sword bean formula granules is preferably as follows: take an appropriate amount of the product (i.e., the sword bean formula granules), grind it into powder, take 1.0 g, put it in a stoppered conical flask, add 25 mL of 80% methanol, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, cool it, filter it, and take the filtrate to obtain it.

[0063] In the present invention, the reference substance in the reference solution is rutin. The reference solution is preferably prepared according to the following method: take an appropriate amount of rutin reference substance, accurately weigh it, add 60-80 vol% methanol to make a solution containing 15-20 μg per 1 mL, preferably add 80 vol% methanol to make a solution containing 20 μg per 1 mL, as a reference solution. In the present invention, the reference substance rutin is a flavonoid compound, which is mainly found in leguminous plants and has anti-inflammatory, antioxidant, antibacterial and other effects. The present invention successfully identified rutin and used it as a reference substance reference peak for the characteristic spectra of sword bean medicinal materials, decoction pieces, standard decoctions and formula granules.

[0064] In the present invention, the determination method further comprises the step of subjecting the sword bean control medicinal material reference solution to liquid chromatography detection and analysis. The sword bean control medicinal material reference solution is preferably prepared according to the following method: take 1 g of sword bean control medicinal material, place it in a stoppered conical flask, add 50 mL of water, boil for 30 minutes, cool, filter, take the supernatant, evaporate to dryness, add 2 to 4 mL of 60 to 80 vol% methanol to the residue to dissolve it, preferably add 2 mL of 80 vol% methanol to the residue to dissolve it, filter, and take the filtrate as the sword bean control medicinal material reference solution.

[0065] In the present invention, the determination method further comprises the step of subjecting the negative solution to liquid chromatography detection and analysis, wherein the negative solution is 60-80 vol% methanol.

[0066] In the present invention, the filler of the chromatographic column in the liquid chromatography is octadecylsilane bonded silica gel; the column temperature is 25-35°C, preferably 30°C; the flow rate is 0.8-1.2mL / min, preferably 1.0mL / min; the mobile phase A is methanol, and the mobile phase B is selected from 0.1vol% acetic acid, 0.1vol% phosphoric acid or 0.1vol% formic acid, preferably 0.1vol% phosphoric acid. The detection wavelength is 320-360nm, preferably 330nm. The present invention preferably performs gradient elution, and the procedure of gradient elution is as follows:

[0067] 0-3min: 0-15 vol% mobile phase A, 100-85 vol% mobile phase B;

[0068] 3-18 min: 15-35 vol% mobile phase A, 85-65 vol% mobile phase B;

[0069] 18-31min: 35-40vol% mobile phase A, 65-60vol% mobile phase B;

[0070] 31-45 min: 40-55 vol% mobile phase A, 60-45 vol% mobile phase B;

[0071] 45-55min: 55-60vol% mobile phase A, 45-40vol% mobile phase B.

[0072] In some specific embodiments of the present invention, the preferred liquid chromatography conditions are: octadecylsilane bonded silica gel as filler; methanol as mobile phase A, 0.1 vol% phosphoric acid solution as mobile phase B, gradient elution as specified in the following table; flow rate of 1.0 mL per minute; column temperature of 30°C; detection wavelength of 330 nm. The theoretical plate number calculated based on the rutin peak shall not be less than 5000. The mobile phase gradient elution procedure is as follows:

[0073] 0-3min: 0-15 vol% mobile phase A, 100-85 vol% mobile phase B;

[0074] 3-18 min: 15-35 vol% mobile phase A, 85-65 vol% mobile phase B;

[0075] 18-31min: 35-40vol% mobile phase A, 65-60vol% mobile phase B;

[0076] 31-45 min: 40-55 vol% mobile phase A, 60-45 vol% mobile phase B;

[0077] 45-55min: 55-60vol% mobile phase A, 45-40vol% mobile phase B.

[0078] In the present invention, the test solution and the reference solution are preferably added in an amount of 8 to 10 μL, preferably 10 μL, respectively, and added to a high performance liquid chromatograph, and detected and analyzed according to the above-mentioned chromatographic conditions. As a result, it was found that the standard HPLC characteristic spectrum of the sword bean medicinal material, standard decoction, and formula granules obtained by the determination method showed 6 corresponding characteristic peaks, and a rutin characteristic peak was successfully identified for the first time.

[0079] The present invention conducts methodological investigation on the determination method, including precision test, repeatability investigation, intermediate precision investigation (investigation on different instruments, investigation on different personnel and time), durability investigation (investigation on durability of chromatographic column, investigation on stability), and the results show that the determination method has good precision, repeatability and durability.

[0080] The present invention also adopts the proposed determination method to measure the characteristic spectra of different batches of sword bean medicinal materials, standard decoctions, and formula granules, and calculates the relative retention time and relative peak area. The results show that the characteristic peaks can be detected and the peaks are relatively high. Therefore, a total of 6 peaks with good repeatability are selected as characteristic peaks.

[0081] In the present invention, when the test sample is a medicinal material, it is stipulated that: 6 characteristic peaks should be present in the chromatogram of the test sample, and the retention time should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material reference, the peak corresponding to the peak of the rutin reference reference is the S peak, and the relative retention time of peak 1, peak 2, peak 3, peak 5, peak 6 and S peak is calculated; the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.67 (peak 1), 0.70 (peak 2), 0.88 (peak 3), 1.32 (peak 5), 1.35 (peak 6).

[0082] When the test sample is a standard decoction, it is stipulated that: 6 characteristic peaks should be present in the chromatogram of the test sample, and the retention time should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the peak of the rutin reference substance is the S1 peak. The relative retention time of peak 1, peak 2, peak 3, peak 5, peak 6 and S peak is calculated; the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.67 (peak 1), 0.70 (peak 2), 0.88 (peak 3), 1.32 (peak 5), 1.35 (peak 6).

[0083] When the test sample is a formula granule, it is stipulated that: 6 characteristic peaks should be present in the chromatogram of the test sample, and the retention time should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the peak of the rutin reference substance is the S peak. The relative retention time of peak 1, peak 2, peak 3, peak 5, peak 6 and S peak is calculated; the relative retention time is within the range of ±10% of the specified value. The specified values ​​are: 0.67 (peak 1), 0.71 (peak 2), 0.88 (peak 3), 1.33 (peak 5), 1.36 (peak 6).

[0084] The present invention also adopts the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) to synthesize 21 batches of characteristic spectra of sword bean medicinal materials, 21 batches of characteristic spectra of sword bean standard decoctions and 3 batches of sword bean formula granules, and establishes a comparison spectrum of the characteristic spectra of sword bean medicinal materials, sword bean standard decoctions and sword bean formula granules.

[0085] The present invention also provides an application of a comparison map of the characteristic maps of the sword bean medicinal material, the standard sword bean decoction and the sword bean formula granules in detecting the quality of the sword bean medicinal material, the standard sword bean decoction and the formula granules.

[0086] The present invention also provides a quality control method for sword bean medicinal materials, standard decoctions, and formula granules, comprising the following steps:

[0087] Take the samples of sword bean medicinal materials, standard decoctions, and formula granules to be tested, and obtain the HPLC characteristic spectrum of the samples according to the above determination method;

[0088] Compare with the standard HPLC characteristic spectrum obtained by the above-mentioned determination method; if the HPLC fingerprint spectrum of the sample to be tested presents 6 corresponding characteristic peaks, and the RSD value of the relative retention time of each peak in the sample HPLC characteristic spectrum and the relative retention time of each peak in the standard HPLC characteristic spectrum is ≤2.0%, it is judged to be a qualified product.

[0089] In order to further illustrate the present invention, the following examples are used to explain it in detail. The experimental raw materials used in the following examples of the present invention are all common commercial products. The "%" involved below, unless otherwise specified, all represent volume percentages.

[0090] Example 1

[0091] 1 sword bean medicinal material

[0092] An HPLC characteristic spectrum of Jack Bean medicinal materials was established, which can be used as an overall quality control method for Jack Bean medicinal materials.

[0093] 1.1 Materials, reagents, and instruments

[0094] High performance liquid chromatograph: Agilent 1260 high performance liquid chromatograph, Shimadzu LC-20AD high performance liquid chromatograph, Waters 2695 high performance liquid chromatograph;

[0095] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instrument Co., Ltd.);

[0096] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0097] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0098] Chromatographic columns: Agilent ZORBAX SB-Aq250×4.6mm, 5μm; Ecosil120-5-AQ PLUS 4.6*250mm, 5μm; Waters HSS T3 4.6*250mm, 5μm.

[0099] Methanol (Sigma-Aldrich, chromatographic grade); phosphoric acid, acetic acid, formic acid (chromatographic grade); water was ultrapure water, and other reagents were of analytical grade;

[0100] Rutin reference substance (China Food and Drug Control Institutes, batch number: 100080-202012, purity: 91.6%);

[0101] Jack bean control medicinal material (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTYD001301).

[0102] 1.2 Chromatographic conditions and system suitability test

[0103] Octadecylsilane bonded silica gel is used as filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5.0 μm); methanol is used as mobile phase A, 0.1% phosphoric acid solution is used as mobile phase B, and gradient elution is performed according to the provisions in the following table; flow rate is 1.0 mL per minute; column temperature is 30°C; detection wavelength is 330 nm. The number of theoretical plates calculated based on the rutin peak shall not be less than 5000.

[0104] Table 1 Proposed mobile phase gradient

[0105] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～3 0→15 100→85 3～18 15→35 85→65 18～31 35→40 65→60 31～45 40→55 60→45 45～55 55→60 45→40

[0106] 1.3 Preparation of reference solution

[0107] Take 1g of sword bean reference medicinal material, place it in a stoppered conical flask, add 50mL of water, boil for 30 minutes, cool, filter, take the supernatant, evaporate to dryness, add 2mL of 80% methanol to the residue to dissolve, filter, take the filtrate as the reference solution of the reference medicinal material. Take an appropriate amount of rutin reference substance, accurately weigh it, add 80% methanol to make a solution containing 20μg per 1mL, as the reference solution of the reference substance.

[0108] 1.4 Preparation of test solution

[0109] Take 1.0 g of the powder of this product, place it in a stoppered conical flask, add 10 mL of 80% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, filter, and take the filtrate to obtain the product.

[0110] 1.5 Determination method

[0111] The determination method is to accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0112] 1.6 Chromatographic conditions and system suitability test

[0113] 1.6.1 Mobile phase selection

[0114] Based on the experimental conditions proposed above, the separation effects of three different mobile phases were investigated: methanol-0.1% phosphoric acid, methanol-1% phosphoric acid, and methanol-1% formic acid. Figure 1 .

[0115] The results showed that the separation purpose could be achieved under the three mobile phases. Among them, the chromatographic peak baseline was relatively stable and there were more chromatographic peaks under the gradient elution condition of methanol-0.1% phosphoric acid solution. Therefore, it is more preferred to use the gradient elution of methanol-0.1% phosphoric acid solution as the mobile phase for the characteristic spectrum determination method of the standard decoction of sword bean.

[0116] 1.6.2 Wavelength Selection

[0117] Based on the experimental conditions proposed above, the diode array detector was used to scan the whole band of the test solution, and the chromatograms of the test solution at wavelengths of 320nm, 330nm, 340nm, 350m, and 360nm were extracted respectively. Figure 2 .

[0118] The results show that the separation purpose can be achieved at a detection wavelength of 320-360 nm. At 330 nm, the chromatographic peak has a larger amount of information and the chromatogram baseline is more stable, so the detection wavelength is more preferably determined to be 330 nm.

[0119] 1.6.3 Flow rate investigation

[0120] Based on the experimental conditions proposed above, the flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min were investigated. Fig.15 .See Figure 3 .

[0121] The results showed that when the flow rate was 1.0 mL / min, the chromatogram peak shape was better and the separation was moderate. Therefore, the flow rate was determined to be 1.0 mL / min.

[0122] 1.6.4 Column temperature investigation

[0123] Based on the experimental conditions proposed above, the column temperatures of 25℃, 30℃ and 35℃ were investigated respectively. Figure 4 .

[0124] The results show that the separation purpose can be achieved under the above three column temperatures, but when the column temperature is 30℃, the chromatogram peak shape is better and the separation degree is moderate. Therefore, the column temperature is more preferably determined to be 30℃.

[0125] 1.6.5 Delay test

[0126] Based on the experimental conditions proposed above, a delay test was conducted. The results are shown in Figure 5 .

[0127] The results showed that the sample had almost no chromatographic peak after 55 minutes, so the sample detection time was set at 55 minutes.

[0128] 1.6.6 System suitability test results

[0129] Octadecylsilane bonded silica gel was used as filler; methanol was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 1.0 mL per minute; the column temperature was 30°C; the detection wavelength was 330 nm. The theoretical plate number calculated based on the rutin peak should not be less than 5000. The mobile phase gradient is shown in Table 2.

[0130] Table 2 Mobile phase gradient

[0131] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～3 0→15 100→85 3～18 15→35 85→65 18～31 35→40 65→60 31～45 40→55 60→45 45～55 55→60 45→40

[0132] 1.7 Preparation of test solution

[0133] 1.7.1 Investigation of extraction solvent

[0134] Take 1.0g of the powder (passed through No. 4 sieve) and place it in a stoppered conical flask. Use 10mL of each of ethanol, water, methanol, 80% methanol, 50% ethanol, and 30% methanol as the extraction solvents for the test product. Seal the flask tightly and treat with ultrasound (power 600W, frequency 40kHz) for 30 minutes. Cool, shake well, filter, and take the filtrate. The results are shown in the table. Figure 6 .

[0135] The results show that when the extraction solvent is 80% methanol, 50% ethanol, and 30% methanol, the separation purpose can be achieved. However, when the extraction solvent is 80% methanol, the peak shape of each characteristic peak is good and the separation degree is moderate. Therefore, it is more preferred to temporarily set the extraction solvent to 80% methanol.

[0136] 2.4.2 Extraction time investigation

[0137] Take 1.0g of the powder (passed through a No. 4 sieve) and place it in a stoppered conical flask. Add 10mL of 80% methanol, seal it tightly, and treat it with ultrasound. The extraction time of the test product is 15 minutes, 30 minutes, and 45 minutes respectively. Cool it, shake it well, filter it, and take the filtrate to obtain the Figure 7 .

[0138] The results showed that when the extraction time was 30 minutes, the peak shape and separation of the chromatogram were better. Therefore, the extraction time was determined to be 30 minutes.

[0139] 2.4.3 Investigation of solvent addition amount

[0140] Take 1.0g of the powder (passed through a No. 4 sieve) of this product, place it in a stoppered conical flask, add 10mL, 25mL, and 50mL of 80% methanol respectively, seal it tightly, treat it with ultrasound for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain the product. Figure 8 .

[0141] The results showed that when the amount of extraction solvent added was 10 mL, the peak shape and separation of each chromatographic peak were better, so the solvent amount was selected to be 10 mL.

[0142] 2.4.4 Investigation of extraction methods

[0143] Take 1.0g of the powder of this product (passed through a No. 4 sieve), place it in a stoppered conical flask, add 10mL of 80% methanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, filter, and take the filtrate to obtain; take another 1.0g of the powder of this product, place it in a stoppered conical flask, add 10mL of 80% methanol, seal it, weigh it, reflux it for 30 minutes, cool, shake it well, weigh it, make up the lost weight with 80% methanol, filter it, and take the filtrate to obtain. Fig. 9 .

[0144] The results showed that there was little difference between reflux extraction and ultrasonic extraction of the test sample. After comprehensive consideration, ultrasonic extraction was determined as the extraction method of the test sample.

[0145] In summary, the preparation method of the test solution of the characteristic spectrum of sword bean medicinal material is determined as follows: take an appropriate amount of the product, take 1.0 g, put it in a stoppered conical flask, add 10 mL of 80% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, filter, and take the filtrate to obtain.

[0146] 1.8 Methodological Review

[0147] 1.8.1 Chromatographic peak identification

[0148] Preparation of test solution: Take 1.0 g of the product, place it in a stoppered conical flask, add 10 mL of 80% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, filter, and take the filtrate to obtain

[0149] Preparation of reference solution: Take 1g of sword bean reference medicinal material, place it in a conical flask, add 50mL of water, boil for 30 minutes, cool, filter, take the supernatant, evaporate to dryness, add 2mL of 80% methanol to the residue to dissolve, filter, take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of rutin reference substance, accurately weigh it, add 80% methanol to make a solution containing 20μg per 1mL, as the reference substance solution.

[0150] Preparation of negative control solution: Take 400mL of methanol in a 500mL volumetric flask, add ultrapure water to the scale, shake well, filter, and take the filtrate to obtain the negative control solution.

[0151] Locate the characteristic peaks of the standard decoction of sword bean. Fig.10 .

[0152] The results showed that peak 4 was rutin. In the following methodological investigation, six characteristic peaks in the sample were investigated.

[0153] 1.8.2 Precision test

[0154] Take the test solution of sword bean medicinal material, inject it 6 times continuously according to the proposed experimental method, 10 μL each time, and calculate the relative retention time and relative peak area of ​​each characteristic peak. See Table 3.

[0155] Table 3 Precision investigation-retention time

[0156]

[0157] The results showed that the RSD values ​​of the retention times of the peaks were 0.04% to 0.11%. The instrument had good precision.

[0158] 1.8.3 Repeatability study

[0159] Accurately weigh 6 portions of Jack Bean medicinal materials, prepare and measure them according to the proposed experimental method (see Table 4).

[0160] Table 4 Repeatability study - relative retention time ratio

[0161]

[0162]

[0163] The results showed that the RSD of the relative retention time of each characteristic peak was between 0.06% and 0.08%, indicating that the method had good repeatability.

[0164] 1.8.4 Intermediate precision study

[0165] 1.8.4.1 Investigation of different instruments

[0166] Based on the experimental conditions proposed above, three portions of Jack Bean were weighed to prepare test solutions, which were then measured on Waters e2695, Shimadzu LC-20, and Agilent 1260 HPLC. Fig.12 , Table 5.

[0167] Table 5 Different instruments investigated - relative retention time ratios

[0168]

[0169] The results showed that when the three instruments were used to detect the test samples, the RSD of the relative retention time of each characteristic peak was 0.35% to 0.65%, indicating that the instruments had good durability.

[0170] 1.8.4.2 Inspection by different personnel and time

[0171] Based on the experimental conditions proposed above, different personnel (A, B) accurately weighed two portions of sword bean medicinal materials at different times (T1, T2) to prepare test samples for determination. See Tables 6 and 7.

[0172] Table 6 Different personnel and time inspection - relative retention time ratio

[0173]

[0174]

[0175] Table 7 Relative peak area ratio of characteristic peaks for different personnel and time periods

[0176]

[0177] The results showed that under different sample preparation personnel and different sample preparation time conditions, the RSD of the relative retention time of each characteristic peak was between 0.03% and 0.06%, indicating that the method had good stability.

[0178] 1.8.5 Durability test

[0179] 1.8.5.1 Column durability test

[0180] Based on the above experimental conditions, the chromatographic columns Agilent ZORBAX SB-Aq250×4.6mm, 5μm; Ecosil120-5-AQ PLUS 4.6*250mm, 5μm; Waters HSS T3 4.6*250mm, 5μm for analysis and investigation, see Fig.13 , Table 8.

[0181] Table 8 Chromatographic column durability investigation - relative retention time ratio

[0182]

[0183]

[0184] The results showed that the RSD of the relative retention time of each characteristic peak of different brands of chromatographic columns was between 1.13 and 2.19%.

[0185] 1.8.5.2 Stability test

[0186] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 4h, 8h, 12h, 16h and 24h respectively. See Table 9-10.

[0187] Table 9 Stability Study - Retention Time

[0188]

[0189] Table 10 Stability Study - Peak Area

[0190]

[0191] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.04 and 0.16. The sample solution was relatively stable within 24 hours.

[0192] 2.6 Determination of characteristic peaks and establishment of reference spectrum

[0193] The proposed method was used to measure the characteristic spectra of 21 batches of samples of this product, and the relative retention time and relative peak area were calculated. Fig.14 , Table 11.

[0194] Table 11 Relative retention time ratios of 21 batches of sword bean medicinal materials

[0195]

[0196]

[0197] According to the principle that the relative retention time is stable and each batch of samples can be detected and the peak is relatively high, a total of 6 peaks with good repeatability were selected as characteristic peaks. The results showed that the RSD of the relative retention time of the 6 characteristic peaks of 21 batches of sword bean medicinal materials was less than 1.0%.

[0198] Final regulations: The test sample should show 6 characteristic peaks in the chromatogram, and the retention time should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the peak of the rutin reference material is the S peak. The relative retention time of peak 1, peak 2, peak 3, peak 5, peak 6 and S peak is calculated; the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.67 (peak 1), 0.70 (peak 2), 0.88 (peak 3), 1.32 (peak 5), 1.35 (peak 6).

[0199] The characteristic spectra of 21 batches of sword bean medicinal materials were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a reference spectrum of the characteristic spectrum of sword bean standard decoction was established, such as Fig.15 .

[0200] Example 2

[0201] 2. Standard decoction of sword beans

[0202] The HPLC characteristic spectrum of the standard decoction of Jack Bean was established, which can be used as an overall quality control method for the standard decoction of Jack Bean.

[0203] 2.1 Materials, reagents, and instruments

[0204] 2.1.1 Experimental instruments and materials

[0205] High performance liquid chromatograph: Agilent inifity1260 high performance liquid chromatograph, Shimadzu LC20 high performance liquid chromatograph, Waters2695 high performance liquid chromatograph;

[0206] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instrument Co., Ltd.);

[0207] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0208] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0209] Chromatographic columns: Agilent ZORBAX SB-Aq 250×4.6mm, 5μm, Ecosil120-5-AQ PLUS 250×4.6mm, 5μm, Waters HSS T3 250×4.6mm, 5μm.

[0210] 2.1.2 Reagents and test drugs

[0211] Methanol (Sigma-Aldrich, chromatographic grade); phosphoric acid, acetic acid, formic acid (chromatographic grade); water was ultrapure water, and other reagents were of analytical grade;

[0212] Rutin reference substance (China Food and Drug Control Institutes, batch number: 100080-202012, purity: 91.6%);

[0213] Jack bean control medicinal material (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTYD001301);

[0214] Jack bean standard decoction freeze-dried powder (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd.).

[0215] 2.2 Chromatographic conditions and system suitability test

[0216] Same as 1.2 Chromatographic conditions and system suitability test.

[0217] 2.3 Preparation of reference solution

[0218] Prepare the reference solution in the same way as in 1.3.

[0219] 2.4 Preparation of test solution

[0220] Take 1 g of the powder of this product, place it in a stoppered conical flask, add 25 mL of 80% methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0221] 2.5 Determination method

[0222] Same as 1.5 Chromatographic conditions and system suitability test.

[0223] 2.6 Determination of characteristic peaks and establishment of reference spectrum

[0224] The proposed method was used to measure the characteristic spectra of 21 batches of samples of this product, and the relative retention time and relative peak area were calculated. Fig.16 , Table 12.

[0225] Table 12 Relative retention time ratios of 21 batches of standard decoction of sword bean

[0226]

[0227]

[0228] According to the principle that the relative retention time is stable and each batch of samples can be detected and the peak is relatively high, a total of 6 peaks with good repeatability were selected as characteristic peaks. The results showed that the relative retention time of each characteristic peak was stable and within the range of ±10% of the average value, so the relative retention time of each peak was temporarily set at ±10%.

[0229] Final regulations: The test sample should show 6 characteristic peaks in the chromatogram, and the retention time should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the rutin reference peak is the S1 peak. The relative retention time of peak 1, peak 2, peak 3, peak 5, peak 6 and S peak is calculated; the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.67 (peak 1), 0.70 (peak 2), 0.88 (peak 3), 1.32 (peak 5), 1.35 (peak 6).

[0230] The characteristic spectra of 21 batches of standard decoctions of sword bean were synthesized by using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison spectrum of the characteristic spectra of standard decoctions of sword bean was established, such as Fig.17 shown.

[0231] Example 3

[0232] 3. Sword Bean Formula Granules

[0233] An HPLC characteristic spectrum of sword bean formula granules was established, which can be used as an overall quality control method for sword bean formula granules.

[0234] 3.1 Materials, reagents and instruments

[0235] Same as 1.1. Materials, reagents and instruments.

[0236] Sword bean formula granules (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 19120071, 2305037, 2305038, 2305039)

[0237] 3.2 Chromatographic conditions and system suitability test

[0238] Same as 1.2 Chromatographic conditions and system suitability test.

[0239] 3.3 Preparation of reference solution

[0240] Same as 1.3 for preparation of reference solution.

[0241] 3.4 Preparation of test solution

[0242] Take an appropriate amount of this product, grind it into powder, take 1.0g, put it in a stoppered conical flask, add 25mL of 80% methanol, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, cool, filter, and take the filtrate to obtain the product.

[0243] 3.5 Determination method

[0244] Same as 1.5 Chromatographic conditions and system suitability test.

[0245] 3.6 Determination of characteristic peaks and establishment of reference spectrum

[0246] The proposed method was used to measure the characteristic spectra of three batches of samples of this product, and the relative retention time and relative peak area were calculated. Fig.18 , Table 13.

[0247] Table 13 Relative retention time ratios of three batches of sword bean formula granules

[0248]

[0249] According to the principle that the relative retention time is stable and each batch of samples can be detected and the peak is relatively high, a total of 6 peaks with good durability were selected as characteristic peaks. According to the results of the methodological investigation and the verification results of 3 batches of particles, the theoretical plate number calculated based on the rutin peak is tentatively set to be no less than 5000. The relative retention time of each characteristic peak is stable and within the range of ±10% of the average value, so the relative retention time of each peak is temporarily set at ±10%.

[0250] Final regulations: The test sample should show 6 characteristic peaks in the chromatogram, and the retention time should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the peak of the rutin reference material is the S peak. The relative retention time of peak 1, peak 2, peak 3, peak 5, peak 6 and S peak is calculated; the relative retention time is within the range of ±10% of the specified value. The specified values ​​are: 0.67 (peak 1), 0.71 (peak 2), 0.88 (peak 3), 1.33 (peak 5), 1.36 (peak 6).

[0251] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize three batches of sword bean formula granules, and a comparison spectrum of the characteristic spectrum of sword bean formula granules was established. Fig.19 shown.

[0252] The present invention adopts high performance liquid chromatography for the first time to establish a method for simultaneously determining the characteristic spectrum of sword bean medicinal materials, sword bean standard decoction, and sword bean formula granules, and adopts the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) to combine the reference spectrum of sword bean medicinal materials, sword bean standard decoction, and sword bean formula granules on one graph for comparison. Fig. 20 After research, the present invention can analyze the differences and changes of sword beans, which is conducive to the overall evaluation of the scientificity and rationality of the sword bean-related process, and can also control the intrinsic quality of sword beans as a whole, ensuring the clinical efficacy of sword bean formula granules.

[0253] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for determining the HPLC characteristic spectrum of sword bean medicinal materials, standard decoctions, and formula granules, characterized in that: The following steps are involved: The test solution and the reference solution are tested and analyzed by liquid chromatography to obtain; The reference substance in the reference substance solution is rutin; The conditions of the liquid chromatography are as follows: mobile phase A is methanol, mobile phase B is selected from 0.1 vol% acetic acid, 0.1 vol% phosphoric acid or 0.1 vol% formic acid, the detection wavelength is 320-360 nm, and gradient elution is performed. The procedure of gradient elution is: 0-3 min: 0-15 vol% mobile phase A, 100-85 vol% mobile phase B; 3-18 min: 15-35 vol% mobile phase A, 85-65 vol% mobile phase B; 18-31 min: 35-40 vol% mobile phase A, 65-60 vol% mobile phase B; 31-45 min: 40-55 vol% mobile phase A, 60-45 vol% mobile phase B; 45-55 min: 55-60 vol% mobile phase A, 45-40 vol% mobile phase B; The test sample solution is obtained by mixing the test sample with a solvent and then ultrasonically extracting or refluxing the mixture; The filler of the chromatographic column in the liquid chromatography is octadecylsilane bonded silica gel; The standard HPLC characteristic spectrum of the sword bean medicinal material, standard decoction and formula granules obtained by the determination method presents 6 corresponding characteristic peaks, and 1 rutin characteristic peak is identified.

2. The measuring method according to claim 1, characterized in that The column temperature of the chromatographic column in the liquid chromatography is 25-35° C.; the flow rate is 0.8-1.2 mL / min.

3. The measuring method according to claim 2, characterized in that The detection wavelength is 330 nm; The column temperature was 30°C and the flow rate was 1.0 mL / min.

4. The measuring method according to any one of claims 1 to 3, characterized in that The amount of the test solution and the reference solution added was 8-10 μL each independently.

5. The measuring method according to claim 1, characterized in that The ratio of the test sample to the solvent is 1 g: (10-50) mL; The solvent is selected from 30-80 vol% methanol or 50 vol% ethanol; The ultrasonic extraction or reflux extraction time is not less than 30 min; The ultrasonic extraction has a power of 500-600 W and a frequency of 30-40 kHz.

6. The measuring method according to claim 1, characterized in that The reference solution is obtained by mixing rutin with 60-80 vol% methanol; The concentration of the reference substance in the reference substance solution is 15-20 μg / mL.

7. Application of the HPLC characteristic spectrum obtained by the determination method according to any one of claims 1 to 6 in detecting the quality of sword bean medicinal materials, standard decoctions, and formula granules.

8. A quality control method for sword bean medicinal materials, standard decoctions, and formula granules, characterized in that: The following steps are involved: Take the samples of sword bean medicinal materials, standard decoctions, and formula granules to be tested, and obtain the sample HPLC characteristic spectrum according to the determination method described in any one of claims 1 to 6; Compare with the standard HPLC characteristic spectrum obtained by the determination method according to any one of claims 1 to 6; if the HPLC fingerprint of the sample to be tested presents 6 corresponding characteristic peaks, and the RSD value of the relative retention time of each peak in the sample HPLC characteristic spectrum and the relative retention time of each peak in the standard HPLC characteristic spectrum is ≤2.0%, it is judged to be a qualified product.

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