Construction method and detection method of characteristic spectrum of Herba Codonopsis pilosulae formula granules

Abstract

The present invention discloses a method for constructing and detecting a characteristic spectrum of Herba Corydalis glutinosae formula granules. The reference solution, the reference medicinal material solution and the test solution are respectively measured by high performance liquid chromatography, and the common peaks are calibrated to obtain the result. Wherein, the chromatographic conditions include: the chromatographic column uses octadecylsilane bonded silica gel as a filler; acetonitrile is used as mobile phase A, and a phosphoric acid aqueous solution with a volume percentage of 0.01 to 0.5% is used as mobile phase B, and gradient elution is performed. The present invention studies the characteristic components shared by Herba Corydalis glutinosae formula granules and Herba Corydalis glutinosae medicinal materials, and finally confirms 7 common characteristic peaks. Therefore, the characteristic spectrum and detection method constructed by the present invention can quickly and comprehensively achieve the purpose of joint control of multiple components in Herba Corydalis glutinosae formula granules, can better characterize the material transfer from medicinal raw materials to formula granules, and ensure the quality of the formula granules.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for constructing and detecting a characteristic spectrum of Herba Codonopsis pilosulae formula particles. Background Art

[0002] The Chinese medicinal material Eriocaulon buergerianum Koern is the dried flower stem capitula of the plant Eriocaulon buergerianum Koern of the Eriocaulonaceae family. It is mainly produced in Jiangsu, Anhui, Zhejiang, Jiangxi, Fujian, Taiwan, Hubei, Hunan, Henan, Guangdong, Guangxi, Hainan, Sichuan, Chongqing, Guizhou, Yunnan and other provinces (municipalities and autonomous regions). It tastes spicy, warm, non-toxic, can dispel wind-heat, improve eyesight and remove cataracts, and is used for wind-heat caused red eyes, swollen and painful photophobia, cataracts in the eyes, and wind-heat headaches.

[0003] Formula granules are made from single-flavor Chinese herbal medicine slices through water extraction, concentration, drying, granulation, etc. Compared with medicinal materials and slices, they have lost their inherent morphology, and the corresponding active ingredients have also changed accordingly. However, the existing technology has only conducted HPLC fingerprint studies on medicinal materials or slices, in which the fingerprint components are mostly fat-soluble components as the research object, and cannot fully reflect the material basis characteristics of the formula granules. At present, there are no relevant reports on the characteristic spectrum of Herba Corydalis formula granules. Therefore, it is difficult for the existing detection methods to effectively compare the differences and changes between the characteristic spectra of Herba Corydalis decoction slices and Herba Corydalis formula granules, and it is difficult to deeply understand the transfer of Herba Corydalis effective substances from raw materials to formula granule preparations.

[0004] Therefore, it is necessary to construct a characteristic spectrum of the Herba Corydalis Glutinosa granules and use the characteristic spectrum for measurement to provide a basis for effectively controlling and comprehensively evaluating the quality of the Herba Corydalis Glutinosa granules. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a method for constructing and detecting a characteristic spectrum of the Herba Corydalis formula granules. The constructed characteristic spectrum of the Herba Corydalis formula granules has 7 characteristic peaks, which is conducive to the comprehensive quality monitoring of multiple characteristic components in the Herba Corydalis formula granules.

[0006] In a first aspect of the present invention, a method for constructing a characteristic spectrum of Herba Codonopsis pilosulae formula particles is provided, comprising the following steps:

[0007] (1) preparing reference substance solution, reference medicinal material solution and test substance solution;

[0008] (2) subjecting the reference substance solution, the reference medicinal material solution and the test solution to high performance liquid chromatography, respectively, and calibrating the common peaks to obtain;

[0009] The conditions of the high performance liquid chromatography include:

[0010] The chromatographic column is filled with octadecylsilane bonded silica gel;

[0011] Acetonitrile was used as mobile phase A, 0.01-0.5% by volume phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed;

[0012] The conditions of the gradient elution are:

[0013]

[0014] The percentage sign in the table means volume percentage.

[0015] According to some embodiments of the present invention, the mobile phase B is a 0.01-0.3% by volume phosphoric acid aqueous solution; preferably a 0.05-0.2% by volume phosphoric acid aqueous solution; more preferably a 0.1% by volume phosphoric acid aqueous solution.

[0016] According to some embodiments of the present invention, the chromatographic column is ACQUITY HSS T3.

[0017] Furthermore, the specifications of the chromatographic column are 100 mm×2.1 mm, 1.8 μm.

[0018] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: the flow rate of the mobile phase is 0.1 to 0.5 mL / min; preferably 0.2 to 0.4 mL / min; more preferably 0.3 mL / min.

[0019] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: the column temperature of the chromatographic column is 25-35°C; preferably 28-32°C; more preferably 30°C.

[0020] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: an injection volume of 0.5 to 2 μL; preferably 0.5 to 1.5 μL; more preferably 1 μL.

[0021] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: a detection wavelength of 250 to 280 nm; preferably 250 to 270 nm; more preferably 260 nm.

[0022] According to some embodiments of the present invention, the conditions of the high performance liquid chromatography method further include: the detector is a diode array detector.

[0023] According to some embodiments of the present invention, the solvent of the reference solution, the reference medicinal material solution and the test solution is water or a mixed solution of alcohol and water.

[0024] Further, the solvent is a 40-60% by volume methanol aqueous solution, a 40-60% by volume ethanol aqueous solution or water; preferably, the solvent is a 40-60% by volume methanol aqueous solution; more preferably, the solvent is a 50% by volume methanol aqueous solution.

[0025] According to some embodiments of the present invention, the concentration of the test sample in the test solution is 10 to 100 mg / mL; preferably 20 to 80 mg / mL; more preferably 30 to 50 mg / mL.

[0026] According to some embodiments of the present invention, the preparation of the test solution comprises the following steps:

[0027] Mix the test sample with the solvent and extract it.

[0028] Furthermore, the extraction comprises ultrasonic treatment, shaking extraction and / or heating reflux extraction; ultrasonic extraction is preferred.

[0029] When the extraction is ultrasonic extraction, the ultrasonic conditions can be such that the herb is completely dissolved; for example, the ultrasonic power is 200-300 W, preferably 250 W; the ultrasonic frequency is 30-50 kHz, preferably 40 kHz; the ultrasonic time is 15-45 min, preferably 30 min.

[0030] According to some embodiments of the present invention, the reference substance in the reference solution includes protocatechuic acid, vanillic acid, chlorogenic acid and rutin.

[0031] According to some embodiments of the present invention, the concentration of the reference substance in the reference substance solution is 10-100 μg / mL.

[0032] Furthermore, the concentration of protocatechuic acid in the reference solution is 5 to 25 μg / mL, the concentration of vanillic acid in the reference solution is 5 to 25 μg / mL, the concentration of chlorogenic acid in the reference solution is 5 to 25 μg / mL, and the concentration of rutin in the reference solution is 5 to 25 μg / mL.

[0033] According to some embodiments of the present invention, the reference solution can be prepared by conventional methods, such as comprising the following steps:

[0034] Mix the reference substance with the solvent to obtain the product.

[0035] According to some embodiments of the present invention, the control medicinal material solution is prepared by the following steps:

[0036] Add water to the reference medicinal material, decoct or heat under reflux, separate the solid and liquid, take the supernatant, evaporate to dryness, and extract the residue with a solvent to obtain the product.

[0037] Furthermore, the solid-liquid ratio of the control medicinal material to water is 10-100 mg:1 mL; preferably 20-80 mg:1 mL; more preferably 30-50 mg:1 mL.

[0038] Furthermore, the solid-liquid ratio of the reference medicinal material to the solvent is 0.1-0.4 g:1 mL; preferably 0.1-0.3 g:1 mL.

[0039] Furthermore, the heating reflux time is 20 to 40 minutes.

[0040] Furthermore, the extraction comprises ultrasonic treatment, shaking extraction and / or heating reflux extraction; ultrasonic extraction is preferred.

[0041] When the extraction is ultrasonic extraction, the ultrasonic conditions can be such that the herb is completely dissolved; for example, the ultrasonic power is 200-300 W, preferably 250 W; the ultrasonic frequency is 30-50 kHz, preferably 40 kHz; the ultrasonic time is 15-45 min, preferably 30 min.

[0042] The Herba Corydalis Glutinosa granules can be prepared by conventional methods in the art, generally, Herba Corydalis Glutinosa granules are prepared by decocting in water, filtering, concentrating, freeze-drying and granulating.

[0043] The second aspect of the present invention provides the application of the above-mentioned construction method in detecting the quality of Herba Codonopsis pilosulae formula granules.

[0044] Beneficial effects:

[0045] (1) The present invention uses protocatechuic acid, vanillic acid, chlorogenic acid and rutin as reference substances, adopts optimized high performance liquid chromatography conditions, and constructs a characteristic spectrum of the Herba Corydalis formula granules by comparing with the Herba Corydalis control medicinal materials, and finally confirms 7 common characteristic peaks. The characteristic spectrum can be used for qualitative and quantitative analysis of the Herba Corydalis formula granules, and can well characterize the material transfer from the medicinal material raw materials to the formula granules, which can not only well control the quality of the medicinal materials, but also ensure the quality of the formula granules.

[0046] (2) The present invention uses the content determination peak (vanillic acid) as the reference peak, specifies the relative retention time and relative peak area limit range of the other six characteristic peaks, and realizes the quantification of multiple characteristic components, which is in line with the overall action concept of traditional Chinese medicine.

[0047] (3) The characteristic spectrum and detection method constructed by the present invention have good reproducibility, accuracy and reliability, and can quickly and comprehensively realize the quality monitoring of multiple characteristic components of the Herba Corydalis Glutinosa granules, achieving the purpose of multi-component joint control, and providing a reference basis for the quality research and standards of the Herba Corydalis Glutinosa granules.

[0048] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0050] Figure 1 is an ultraviolet absorption spectrum of the substances corresponding to each chromatographic peak in Example 1 of the present invention in the wavelength range of 190 to 400 nm, wherein A is a spectrum of the substance corresponding to chromatographic peak 1 (protocatechuic acid), B is a spectrum of the substance corresponding to chromatographic peak 2, C is a spectrum of the substance corresponding to chromatographic peak 3 (vanillic acid), D is a spectrum of the substance corresponding to chromatographic peak 4 (chlorogenic acid), E is a spectrum of the substance corresponding to chromatographic peak 5, F is a spectrum of the substance corresponding to chromatographic peak 6, and G is a spectrum of the substance corresponding to chromatographic peak 7 (rutin);

[0051] Figure 2 : are characteristic spectra of the Herba Codonopsis pilosulae formula granules under different detection wavelength conditions of Example 1 of the present invention, wherein A is the spectrum corresponding to the ultraviolet absorption wavelength of 210 nm, B is the spectrum corresponding to the ultraviolet absorption wavelength of 300 nm, and C is the spectrum corresponding to the ultraviolet absorption wavelength of 260 nm;

[0052] Figure 3 are characteristic spectra of the Herba Codonopsis pilosulae granules under different mobile phase B conditions in Example 1 of the present invention, wherein A is the spectrum corresponding to a 0.1% phosphoric acid aqueous solution, B is the spectrum corresponding to a 0.1% acetic acid aqueous solution, and C is the spectrum corresponding to a 0.1% formic acid aqueous solution;

[0053] Figure 4 1 is a characteristic spectrum of the Herba Codonopsis pilosulae formula granules under different mobile phase gradients in Example 1 of the present invention, wherein A is the spectrum corresponding to gradient 1, and B is the spectrum corresponding to gradient 2;

[0054] Figure 5 : are characteristic spectra of the Herba Codonopsis pilosulae formula granules under different extraction conditions in Example 1 of the present invention, wherein A is the spectrum corresponding to ultrasonic extraction, and B is the spectrum corresponding to reflux extraction;

[0055] Figure 6 are characteristic spectra of the Herba Codonopsis pilosulae formula granules under different extraction solvent conditions in Example 1 of the present invention, wherein A is the spectrum corresponding to methanol, B is the spectrum corresponding to anhydrous ethanol, C is the spectrum corresponding to water, and D is the spectrum corresponding to 50% methanol;

[0056] Figure 7are characteristic spectra of the Herba Codonopsis pilosulae formula granules at different extraction times in Example 1 of the present invention, wherein A is the spectrum corresponding to extraction for 15 minutes, B is the spectrum corresponding to extraction for 30 minutes, and C is the spectrum corresponding to extraction for 45 minutes;

[0057] Figure 8 are characteristic spectra of the Herba Codonopsis pilosulae formula granules at different extraction amounts in Example 1 of the present invention, wherein A is the spectrum corresponding to a sampling amount of 0.5 g, B is the spectrum corresponding to a sampling amount of 1 g, and C is the spectrum corresponding to a sampling amount of 2 g;

[0058] Fig. 9 It is the specific experimental chromatogram of the Herba Codonopsis pilosulae granules, wherein A is the spectrum corresponding to the blank solution (50% methanol in water), B is the spectrum corresponding to the mixed reference solution, C is the spectrum corresponding to the negative sample solution, D is the spectrum corresponding to the test solution, and E is the spectrum corresponding to the reference medicinal material solution;

[0059] Fig.10 It is a characteristic spectrum with a retention time twice that of the Herba Codonopsis pilosulae formula granules;

[0060] Fig.11 It is an overlay of the characteristic spectra of the Herba Corydalis Glutinosa granules of the present invention, wherein A is the characteristic spectrum of the mixed reference substance, B is the characteristic spectrum of the reference medicinal material, C is an overlay of the characteristic spectra of three batches of Herba Corydalis Glutinosa granules, and D is the control characteristic spectrum of the Herba Corydalis Glutinosa granules. DETAILED DESCRIPTION

[0061] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0062] If not otherwise specified, the reagents, equipment and methods used in the examples are all conventional in the art. In addition, if not otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0063] Example 1 Construction of characteristic spectrum of Herba Codonopsis pilosulae formula granules

[0064] 1. Preparation of reference solution

[0065] Take appropriate amount of protocatechuic acid, vanillic acid, chlorogenic acid, and rutin reference substances, add 50% methanol solution to make a solution containing 20 μg of each per 1 mL.

[0066] 2. Preparation of reference medicinal material solution

[0067] Take 2 g of the control medicinal material, add 50 mL of water, heat and reflux for 30 minutes, filter once, evaporate the filtrate to dryness, add 10 mL of 50% methanol solution to the residue and ultrasonically treat for 30 minutes, and take the filtrate after secondary filtration to obtain the product.

[0068] 3. Preparation of test solution

[0069] Take an appropriate amount of Herba Codonopsis pilosulae granules and grind them into powder. Accurately weigh 1g of the granules. Add 25mL of 50% methanol solution and ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes. Take it out, let it cool, weigh it again, make up the lost weight with 50% methanol solution, shake well, filter, and you have it.

[0070] 4. Chromatographic conditions and system suitability parameters

[0071] ACQUITY HSS T3, 100 mm × 2.1 mm, 1.8 μm for the chromatographic column;

[0072] Flow rate: 0.3 mL / min;

[0073] Column temperature: 30°C;

[0074] Detection wavelength: 260nm;

[0075] Injection volume: 1 μL;

[0076] Use acetonitrile as mobile phase A and 0.1% by volume phosphoric acid aqueous solution as mobile phase B, and perform gradient elution under the conditions shown in the following table:

[0077]

[0078] The percentage sign in the table means volume percentage.

[0079] According to the chromatographic conditions under the aforementioned “4. Chromatographic conditions and system suitability parameters”, the reference solution prepared under “1. Preparation of reference solution”, the reference medicinal material solution prepared under “2. Preparation of reference medicinal material solution” and the test sample solution prepared under “3. Preparation of test sample solution” were sampled and tested.

[0080] 5. Investigation of chromatographic conditions and preparation methods

[0081] 5.1. Investigation of detection wavelength

[0082] The test solution was injected into the liquid chromatograph and detected online in the ultraviolet region of 190-400 nm by a diode array detector. The ultraviolet absorption spectra of each proposed characteristic peak were shown in Figure 1 .

[0083] From the UV absorption spectra of the proposed chromatographic peaks of the characteristic spectrum of the herbaceous granules, it can be seen that the 7 chromatographic peaks proposed in the characteristic spectrum of the herbaceous granules show maximum absorption near 260nm. Therefore, the characteristic spectrum of the herbaceous granules at 210nm, 260nm, and 300nm UV absorption wavelengths was compared, and the results are shown in Figure 2 .

[0084] The results showed that when the detection wavelength was 260nm, the response values ​​of the chromatographic peaks were large, the peak shapes were excellent, and the peak areas were similar. Therefore, the detection wavelength of 260nm was selected as the detection wavelength of the characteristic spectrum of the Herba Corydalis Glutinosa granules.

[0085] 5.2. Investigation of mobile phase types

[0086] In the preliminary condition exploration process, the present invention adopts the program of "Gradient 1" in Table 2 for gradient elution, and on this basis, further investigates the influence of three different mobile phases B on the chromatographic peak when the mobile phase A is acetonitrile, the mobile phase B is 0.1% formic acid aqueous solution, 0.1% acetic acid aqueous solution, and 0.1% phosphoric acid aqueous solution, respectively, to determine the chromatographic conditions of the characteristic spectrum of the Herba Corydalis glutinosae formula granules. The results are shown in Table 1. Figure 3 .

[0087] Table 1 Comparison of different mobile phase types

[0088]

[0089] The results show that when acetonitrile-0.1% phosphoric acid aqueous solution is used as the mobile phase, the peak shape of the chromatographic peak is better, the separation degree is higher, and the baseline is more stable. Therefore, acetonitrile-0.1% phosphoric acid aqueous solution is selected as the mobile phase B component of HPLC detection.

[0090] 5.3. Investigation of mobile phase gradient

[0091] ACQUITY HSS T3, 100mm×2.1mm, 1.8μm was used as the chromatographic column, acetonitrile was used as the mobile phase A, 0.1% phosphoric acid aqueous solution was used as the mobile phase B, and gradient elution was performed according to the elution programs in Tables 2 and 3 respectively; the flow rate was 0.3mL per minute; and the column temperature was 30°C.

[0092] Table 2 Gradient 1

[0093]

[0094] Table 3 Gradient 2

[0095]

[0096] The changes in the chromatographic peaks were investigated, and the results are shown in Table 4 and Figure 4 .

[0097] Table 4 Comparison of different mobile phase gradients

[0098]

[0099] The results show that when the gradient in "Table 3 Gradient 2" is used for gradient elution, the peak shape of each proposed chromatographic peak is good, the symmetry is good, and the baseline is relatively stable. Therefore, it is finally determined to use the gradient "Table 3 Gradient 2" as the chromatographic condition for the determination of the characteristic spectrum of the Herba Corydalis Root Granules.

[0100] In summary, the chromatographic conditions for the determination of the content of the herbaceous sorghum formula granules are as follows: octadecylsilane bonded silica gel as the filler (chromatographic column: ACQUITY HSS T3, column length 100mm, inner diameter 2.1mm, particle size 1.8μm); flow rate 0.3mL per minute, column temperature 30℃; detection wavelength 260nm; injection volume 1μL; acetonitrile as mobile phase A, 0.1% by volume phosphoric acid aqueous solution as mobile phase B, gradient elution according to the elution program recorded in "Table 3 Gradient 2", when the gradient time is increased to about 35min, no target chromatographic peak appears, therefore, the gradient time is set to 35min, the specific gradient elution program is as shown in the following table, the theoretical plate number calculated based on the vanillic acid peak should be not less than 5000.

[0101]

[0102]

[0103] 5.4. Investigation of test sample preparation methods

[0104] (1) Investigation of extraction methods

[0105] Considering that the Herba Codonopsis pilosulae granules are obtained by water extraction and are easier to extract and dissolve than medicinal materials, the two extraction methods of heating reflux and ultrasonic extraction were compared.

[0106] The specific experimental steps are as follows: take about 1g of the formula granules of Codonopsis pilosula, weigh two portions in parallel, weigh accurately, place in a stoppered conical bottle, accurately add 25mL of 50% methanol solution to each, weigh the weight, reflux extract for 30 minutes for one portion, and ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes for another portion, take out, cool, weigh, make up the lost weight with 50% methanol solution, mix well, filter, and obtain. Take 1μL of each of the two extracts and inject them into the liquid chromatograph, and measure according to the chromatographic conditions under "4. Chromatographic conditions and system suitability parameters". Select the appropriate extraction method according to the parameters of each characteristic peak (peak shape, separation degree, symmetry, total peak area and other parameters).

[0107] The results showed that there was no significant difference between the total peak area of ​​reflux extraction and ultrasonic extraction. Figure 5 ,After comprehensive consideration, ultrasonic extraction was selected as the extraction method in the preparation of the test solution.

[0108] (2) Investigation of extraction solvent

[0109] The extraction solvents were investigated for the preparation of the test solution of the Herba Corydalis Glutinosa granules, and the extraction efficiency of the Herba Corydalis Glutinosa granules was investigated when methanol, ethanol, water, 50% methanol solution, and 50% ethanol solution were used as the extraction solvents.

[0110] The specific experimental steps are as follows: take about 1g of this product, weigh five portions in parallel, weigh accurately, place in a stoppered conical bottle, accurately add 25mL of the extraction solvent to be investigated, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, take out, cool, weigh the weight, make up the lost weight with the extraction solvent to be investigated, mix well, filter, and obtain. According to the chromatographic conditions under "4. Chromatographic conditions and system suitability parameters", the determination is carried out, and the appropriate type of extraction solvent is selected according to the parameters of each characteristic peak (peak shape, separation degree, symmetry, total peak area and other parameters).

[0111] The results showed that when the extraction solvent was ethanol, the proposed characteristic peak substances were difficult to extract. The extraction efficiency of methanol was lower than that of water. The extraction efficiency of 50% methanol solution, 50% ethanol solution and water was basically the same. Figure 6 In order to ensure sufficient extraction and easy preservation of organic solvents, and taking into account factors such as peak shape and separation, 50% methanol was selected as the extraction solvent for the preparation of the test sample after comprehensive consideration.

[0112] (3) Investigation of extraction time

[0113] The specific experimental steps are as follows: take about 1g of this product, weigh three portions in parallel, weigh accurately, place in a stoppered conical bottle, accurately add 25mL of 50% methanol solution, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz) for 15, 30, and 45 minutes, take out, cool, weigh the weight, make up the lost weight with 50% methanol, mix well, filter, and obtain. Determine according to the chromatographic conditions under "4. Chromatographic conditions and system suitability parameters". Compare the parameter changes of each characteristic peak in the results of each extraction time (based on parameters such as peak shape, separation effect, symmetry, total peak area, etc.) to select the appropriate extraction time.

[0114] The results showed that there was no significant difference in the peak area of ​​each characteristic peak and the total peak area after conversion of the sampling volume at the three extraction times of 15 minutes, 30 minutes and 45 minutes. The results showed that there was no difference in the peak area of ​​each characteristic peak of the test sample at each ultrasonic extraction time, and no other impurity peaks appeared as time went on; the results are shown in Figure 7 After comprehensive investigation, 30 minutes was proposed as the extraction time for subsequent research.

[0115] (4) Investigation of sampling quantity

[0116] The specific experimental steps are as follows: take about 0.5g, 1g, and 2g of the product, weigh accurately, place in a stoppered conical bottle, accurately add 25mL of 50% methanol solution, weigh the weight, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, take out, cool, weigh, make up the lost weight with 50% methanol, mix, filter, and obtain. Determine according to the chromatographic conditions under "4. Chromatographic conditions and system suitability parameters". Compare the parameter changes of each characteristic peak in the results of each sampling amount (peak shape, resolution, symmetry factor, total peak area and other parameters) to select the appropriate sampling amount.

[0117] The results showed that the total peak area of ​​the test samples with a sampling volume of 0.5g, 1g and 2g was compared and found that there was no significant difference after the sampling volume conversion, indicating that the extraction efficiency of the proposed characteristic peaks in the test samples with a sampling volume of 0.5g, 1g and 2g was basically the same, indicating that the above-mentioned extraction parameters can stably extract 1g of each characteristic peak substance; the results are shown in Figure 8 After comprehensive consideration, 1g was determined as the sampling amount for the characteristic spectrum of Glechoma longituba particles.

[0118] In summary: the main parameters of the test solution preparation method are as follows: take about 1.0 g of the product, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 50% methanol solution, weigh the weight, ultrasonically treat (250 W, frequency 40 kHz) for 30 minutes, take it out, cool, weigh it again, make up the lost weight with 50% methanol solution, shake well, filter, and obtain.

[0119] 6 Methodological Validation

[0120] 6.1 Exclusivity

[0121] In this verification, reagent blank solution (take an appropriate amount of 50% methanol solution as the reagent blank solution), reference substance solution, reference medicinal material solution, negative sample solution (take an appropriate amount of dextrin and prepare the negative sample solution according to the test sample preparation method), and test sample solution were selected for specificity experiments.

[0122] The results show that the retention time of the test solution is consistent with that of the reference solution and the reference medicinal material solution, and the reagent blank sample and the negative sample have no interference near the retention time of the target substance. Fig. 9 , indicating that the specificity meets the method validation requirements.

[0123] 6.2 Double retention time investigation

[0124] The specific experimental steps are as follows: take a portion of the test solution and detect the characteristic spectrum at twice the retention time.

[0125] The experimental results are shown in Fig.10 The results showed that there were no other chromatographic peaks in the test solution after 60 minutes, indicating that the method had good overall performance.

[0126] 6.3 Instrument precision test

[0127] The test solution was injected 6 times, and the determination was carried out according to the method. Peak 3 was used as the reference peak, and the RSD of the relative retention time and relative peak area of ​​each characteristic peak was calculated. The results are shown in Tables 5 and 6. The RSD of the relative retention time of each characteristic peak was less than 1%, and the RSD of the relative peak area was less than 2%. The test results showed that the instrument had good precision.

[0128] Table 5 Relative retention time results of instrument precision test

[0129]

[0130] Table 6 Relative peak area results of instrument precision test

[0131]

[0132]

[0133] 6.4 Intermediate precision of different instruments

[0134] The same batch of Herba Codonopsis pilosulae granules was used to prepare the test solution according to the method under “3. Preparation of test solution”. According to the chromatographic conditions under “4. Chromatographic conditions and system suitability parameters”, the samples were tested on Agilent HPLC and Waters HPLC, and the chromatograms were recorded. The relative retention time and relative peak area RSD (%) of each characteristic peak were calculated. The experimental results are shown in Tables 7 and 8.

[0135] Table 7 Intermediate precision relative retention time results of different instruments

[0136]

[0137] Table 8 Results of intermediate precision relative peak area tests for different instruments

[0138]

[0139]

[0140] The results showed that the characteristic spectra of the Herba Codonopsis pilosulae formula granules were determined using Agilent high performance liquid chromatograph and Waters high performance liquid chromatograph respectively. The relative retention time RSD range of the 7 characteristic peaks in the characteristic spectrum was 0.31% to 4.53%, and the relative peak area RSD range was 6.57% to 94.13%, which were quite different. However, the relative retention times were all within the standard value range, and no relative peak area standard was established. Comprehensively judging, the intermediate precision of the method (different instruments) met the requirements.

[0141] 6.5 Intermediate precision among different personnel

[0142] Three different experimenters took the same batch of Herba Codonopsis pilosulae granules, prepared the test solution according to the method under “3. Preparation of test solution”, injected and tested according to the chromatographic conditions under “4. Chromatographic conditions and system suitability parameters”, recorded the chromatograms, calculated the relative retention time of each characteristic peak and the RSD (%) of the relative peak area. The experimental results are shown in Tables 9 and 10.

[0143] Table 9 Results of the intermediate precision relative retention time test of different personnel

[0144]

[0145] Table 10 Results of the relative peak area test of intermediate precision of different personnel

[0146]

[0147]

[0148] The results showed that the characteristic spectrum of Coptis chinensis granules was determined by three different personnel. The RSD range of relative retention time of each chromatographic peak was 0.19% to 0.73%, which was less than 2%. The RSD range of relative peak area was 1.57% to 17.75%, which was quite different. However, no relative peak area standard was established. Comprehensively judging, the intermediate precision of this method (by different personnel) met the requirements.

[0149] 6.6 Method reproducibility

[0150] Take 6 portions of the same batch of Herba Codonopsis pilosulae granules, prepare 6 portions of test solutions in parallel according to the method under “3. Preparation of test solution”, perform sampling and detection according to the chromatographic conditions under “4. Chromatographic conditions and system suitability parameters”, record the chromatogram, calculate the relative retention time and relative peak area RSD (%) of each characteristic peak, and the experimental results are shown in Tables 11 and 12.

[0151] Table 11 Method repeatability relative retention time test results

[0152]

[0153]

[0154] Table 12 Method repeatability relative peak area test results

[0155]

[0156] The results showed that the RSD range of the relative retention time of each chromatographic peak was 0.06% to 0.77%, and the RSD range of the relative peak area was 0.45% to 2.96%, both less than 3%, and the relative retention time was in compliance with the regulations, indicating that the method had good repeatability.

[0157] 6.7 Durability

[0158] Durability refers to the degree to which the test results are not affected by small changes in the test conditions. By changing the sample weight, mobile phase flow rate, column temperature and other conditions, the influence on the test results was investigated to provide a basis for the established method to be used for the production inspection of Herba Corydalis glutinosae formula granules.

[0159] (1) Flow rate durability

[0160] Take the same batch of Herba Codonopsis pilosulae granules, prepare the test solution according to the method under “3. Preparation of test solution”, and perform injection and detection according to the chromatographic conditions under “4. Chromatographic conditions and system suitability parameters”. Set the mobile phase flow rates to 0.28 mL / min, 0.3 mL / min, and 0.32 mL / min, respectively, record the chromatograms, calculate the relative retention time and relative peak area RSD (%) of each characteristic peak, and examine the effects of different flow rates of the same mobile phase on the test results. The experimental results are shown in Tables 13 and 14.

[0161] Table 13 Flow rate durability relative retention time results

[0162]

[0163] Table 14 Flow rate durability relative peak area results

[0164]

[0165]

[0166] The above results show that when the mobile phase flow rate changes at (0.3±0.02) mL / min, the relative retention time RSD of the seven characteristic peaks ranges from 0.24% to 1.90%, which is less than 2%, and the relative peak area RSD of the seven characteristic peaks ranges from 2.36% to 80.08%, which has a large difference. However, no relative peak area standard has been established. The durability results at different flow rates meet the method validation requirements.

[0167] (2) Column temperature durability

[0168] The same batch of Herba Codonopsis pilosulae granules were used to prepare the test solution according to the method under “3. Preparation of test solution”, and the sample was injected and tested according to the chromatographic conditions under “4. Chromatographic conditions and system suitability parameters”. The difference was that the column temperature was set to 28°C, 30°C and 32°C respectively. The chromatogram was recorded, and the relative retention time and RSD of the relative peak area of ​​each characteristic peak were calculated. The effect of different column temperatures of the same mobile phase on the test results was investigated. The experimental results are shown in Tables 15 and 16.

[0169] Table 15 Column temperature durability relative retention time results

[0170]

[0171] Table 16 Column temperature durability relative peak area results

[0172]

[0173] The above results show that when the column temperature is (30±2)℃, the relative retention time RSD of the four characteristic peaks ranges from 0.26% to 2.71%, which is less than 3%, and the relative peak area RSD ranges from 4.96% to 61.73%, which shows a large difference. However, no relative peak area standard has been established, and the column temperature durability results meet the method validation requirements.

[0174] (3) Acid concentration durability

[0175] The same batch of Herba Codonopsis pilosulae granules was used to prepare the test solution according to the method under “3. Preparation of test solution” and the chromatographic conditions under “4. Chromatographic conditions and system suitability parameters”, with the difference that the concentration of phosphoric acid solution in the mobile phase was 0.08%, 0.10% and 0.12%. The samples were injected for detection, the chromatogram was recorded, the relative retention time and RSD of the relative peak area of ​​each characteristic peak were calculated, and the effect of different acid concentrations on the test results was investigated. The experimental results are shown in Tables 17 and 18.

[0176] Table 17 Acid concentration durability test relative retention time results

[0177]

[0178] Table 18 Acid concentration durability test relative peak area results

[0179]

[0180] The above results show that when the acid concentration is between (0.08% and 0.12%), the relative retention time RSD of the seven characteristic peaks ranges from 0.13% to 1.96%, which is less than 2%. The acid concentration durability results meet the method validation requirements.

[0181] (4) Column durability

[0182] Take the same batch of Herba Codonopsis pilosulae granules, prepare the test solution according to the method under "3. Preparation of test solution", and follow the chromatographic conditions under "4. Chromatographic conditions and system suitability parameters", except that (ACQUITY Three different types of chromatographic columns (HSS T3, 100mm×2.1mm, 1.8μm; ZORBAX SB-C18, 100mm×2.1mm, 1.8μm; AcclaimTMRSLC120C18, 100mm×2.1mm, 2.2μm) were used for sample injection and detection, and the chromatograms were recorded. The relative retention time and relative peak area RSD of each characteristic peak were calculated. The experimental results are shown in Tables 19 and 20.

[0183] Table 19 Column durability test relative retention time test results

[0184]

[0185]

[0186] Table 20 Column durability test relative peak area test results

[0187]

[0188] The results showed that ACQUITY HSS T3, 100mm×2.1mm, 1.8μm; ZORBAX SB-C18, 100mm×2.1mm, 1.8μm; Acclaim TM RSLC 120C18, 100mm×2.1mm, 2.2μm three different types of chromatographic columns were used to determine the characteristic spectrum of the formula granules of Gujingcao. The RSD range of the relative retention time of each chromatographic peak was 0.60% to 7.65%, and the RSD range of the relative peak area was 6.43% to 20.92%, both of which were quite different. Taking into account the relative retention time requirements of the characteristic spectrum of Gujingcao granules and the separation effect of each characteristic peak, ACQUITY Determination by HSS T3 column.

[0189] (5) Sample solution stability

[0190] Take the same batch of Herba Codonopsis pilosulae granules, prepare the test solution according to the method under "3. Preparation of test solution", and inject and test at 0, 2, 4, 8, 12, 18, and 24 hours according to the chromatographic conditions under "4. Chromatographic conditions and system suitability parameters". Record the chromatogram, calculate the relative retention time and relative peak area RSD of each characteristic peak, and examine the stability of the test solution. The experimental results are shown in Tables 21 and 22.

[0191] Table 21 Results of relative retention time test of sample solution stability test

[0192]

[0193]

[0194] Table 22 Sample solution stability test relative peak area test results

[0195]

[0196] The results showed that when the same test solution was sampled and tested within 24 hours, the relative retention time RSD of the seven characteristic peaks ranged from 0.02% to 0.23%, less than 2%, and the relative peak area RSD ranged from 1.09% to 3.39%, less than 5%, indicating that the test solution was stable within 24 hours.

[0197] Through the above series of experimental optimization, under the chromatographic conditions of the optimal characteristic spectrum, 7 characteristic peaks should be present in the chromatogram of the test solution after positioning and identifying each peak with the reference substance, peak 1 is protocatechuic acid, peak 2 is unknown, peak 3 is vanillic acid, peak 4 is chlorogenic acid, peak 5 is unknown, peak 6 is unknown, and peak 7 is rutin; taking peak 3 as the S peak, the relative retention time of peaks 2, 5, 6 and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value, and the specified values ​​are: 0.74 (peak 2), 1.24 (peak 5), 1.79 (peak 6).

[0198] Example 2 Detection of characteristic spectrum of Herba Codonopsis pilosulae formula granules

[0199] Three batches of samples of Herba Corydalis glutinosae formula granules (batch numbers: ZS211018, ZS211019, and ZS211020, all from Hunan Chunguang Jiuhui Modern Chinese Medicine Co., Ltd.) were taken and their characteristic spectra were tested according to the characteristic spectrum detection method of Herba Corydalis glutinosae formula granules. The results showed that there were 7 common peaks in the characteristic chromatograms of the three batches of pilot samples, and the retention times of the 7 characteristic peaks in the chromatograms of the reference medicinal material reference materials were corresponding. The characteristic spectrum detection results are shown in Fig.11 , Table 23.

[0200] Table 23 Detection of characteristic spectra of different batches of Herba Codonopsis pilosulae formula granules

[0201]

[0202] Conclusion: From the comprehensive analysis of the above experimental contents, it can be seen that the method used to determine the characteristic spectrum characteristic peaks (7 characteristic peaks) in the Herba Corydalis formula granules has good precision, durability, stability and specificity through methodological verification, indicating that the established method is suitable for the determination of the characteristic spectrum of Herba Corydalis formula granules.

[0203] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for constructing a characteristic spectrum of Herba Codonopsis pilosulae formula granules, characterized in that: The Herba Corydalis glutinosae formula granules are prepared by boiling Herba Corydalis glutinosae decoction pieces in water, filtering, concentrating, freeze-drying and granulating; The construction method comprises the following steps: (1) preparing a reference solution, a reference medicinal material solution and a test solution, wherein the solvents of the reference solution, the reference medicinal material solution and the test solution are water or a mixed solution of alcohol and water; the reference substance in the reference solution comprises protocatechuic acid, vanillic acid, chlorogenic acid and rutin; (2) The reference substance solution, the reference medicinal material solution and the test solution are respectively measured by high performance liquid chromatography, and the common peaks are calibrated to obtain; The conditions of the high performance liquid chromatography include: The chromatographic column was ACQUITY HSS T3; The flow rate of the mobile phase is 0.1~0.5mL / min; The column temperature of the chromatographic column is 25~35℃; The injection volume is 0.5~2μL; The detection wavelength is 250~280nm; Acetonitrile was used as mobile phase A, 0.01-0.5% by volume phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed; The conditions of the gradient elution are:

2. The construction method according to claim 1, characterized in that: The solvent is a methanol aqueous solution with a volume percentage of 40-60%, an ethanol aqueous solution with a volume percentage of 40-60%, or water.

3. The construction method according to claim 1, characterized in that: The concentration of the test sample in the test solution is 10-100 mg / mL.

4. The construction method according to claim 1, characterized in that: The concentration of the reference substance in the reference substance solution is 10-100 μg / mL.

5. The construction method according to claim 1, characterized in that: The control medicinal material solution is prepared by the following steps: adding water to the control medicinal material, decocting or heating under reflux, separating the solid and the liquid, taking the supernatant, evaporating to dryness, and extracting the residue with a solvent to obtain the control medicinal material solution.

6. The construction method according to claim 5, characterized in that: The solid-to-liquid ratio of the control medicinal material to water is 10-100 mg:1 mL.

7. The construction method according to claim 5, characterized in that: The solid-to-liquid ratio of the reference medicinal material to the solvent is 0.1-0.4 g:1 mL.

8. The construction method according to claim 5, characterized in that: The extraction includes ultrasonic extraction, shaking extraction and / or heating reflux extraction.

9. The construction method according to claim 8, characterized in that: The extraction is ultrasonic extraction.

10. Application of the construction method according to any one of claims 1 to 9 in detecting the quality of Herba Codonopsis pilosulae formula granules.

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