A method for constructing characteristic maps of scallion medicinal materials, standard decoctions, and formula granules and its application

Abstract

The present invention belongs to the field of detection technology, and in particular relates to a method for constructing a characteristic spectrum of scallion white medicinal materials, standard decoctions, and formula granules, and its application. The characteristic spectrum construction method provided by the present invention comprises the following steps: preparing a test sample, wherein the test sample is scallion white medicinal materials, scallion white standard decoctions, or scallion white formula granules; dissolving the test sample to obtain a test sample solution; measuring the test sample solution by high performance liquid chromatography to obtain a characteristic spectrum of the corresponding test sample; the chromatographic conditions of the high performance liquid chromatography are: a chromatographic column is a C18 column; mobile phase A is acetonitrile, mobile phase B is a 0.1 vol% formic acid solution, and gradient elution. The characteristic spectrum construction method provided by the present invention can provide a more scientific basis for the identification of scallion white medicinal materials, standard decoctions, and formula granules.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and in particular relates to a method for constructing characteristic maps of scallion medicinal materials, standard decoctions, and formula granules, and applications thereof. Background Art

[0002] Scallion whites are the dried bulbs of the herb, peeled and washed, after removing the outer membrane. Scallion white standard decoctions are freeze-dried powders made from this herb after processing according to a fixed preparation process. Scallion white formula granules are formulated granules made by processing this herb according to the key quality specifications of the standard decoction. To ensure the uniformity and stability of the quality of scallion white slices, standard decoctions, and formula granules, it is essential to establish a new characteristic spectrum method for quality control. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a method for constructing characteristic maps of scallion white medicinal materials, standard decoctions, and formula granules and its application. The construction method provided by the present invention can provide a more scientific basis for the identification of scallion white medicinal materials, standard decoctions, and formula granules.

[0004] The present invention provides a method for constructing characteristic maps of scallion medicinal materials, standard decoctions, and formula granules, comprising the following steps:

[0005] preparing a test sample, wherein the test sample is scallion medicinal material, scallion standard decoction or scallion formula granules;

[0006] Dissolving the test sample to obtain a test sample solution;

[0007] The test solution is measured by high performance liquid chromatography (HPLC) to obtain a characteristic spectrum corresponding to the test sample;

[0008] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is a 0.1 vol% formic acid solution, and the elution is performed in a gradient manner.

[0009] In the construction method provided by the present invention, the dissolving solvent is preferably a 50 vol% methanol solution, which has a high dissolution efficiency, a large amount of chromatographic peak information in the obtained chromatogram, and good separation; the dosage ratio of the test sample to the solvent is preferably (0.5-2) g:50 mL. When the dosage ratio is adjusted, the chromatogram peak shape and separation are good, and the peak size is appropriate.

[0010] In the construction method provided by the present invention, the dissolution is preferably carried out under ultrasonic assistance to improve the efficiency of dissolution; the power of the ultrasonic assistance is preferably 580-620 W, more preferably 600 W; the frequency of the ultrasonic assistance is preferably 35-45 kHz, more preferably 40 kHz; the time of the ultrasonic assistance is preferably 25-35 min, more preferably 30 min.

[0011] In the construction method provided by the present invention, when the test sample is the medicinal material of scallion white, the specific process of dissolving and preparing the test sample solution preferably includes: mixing the test sample with the solvent, ultrasonic-assisted dissolution, cooling, shaking, filtering, and the obtained filtrate is the test sample solution; wherein the dosage ratio of the test sample to the solvent is preferably more preferably 2g:50mL.

[0012] In the construction method provided by the present invention, when the test sample is a standard decoction of scallion whites, the specific process of dissolving and preparing the test sample solution preferably includes: grinding the test sample and mixing it with a solvent, ultrasonic-assisted dissolution, cooling, shaking, filtering, and the resulting filtrate is the test sample solution; wherein the dosage ratio of the test sample to the solvent is preferably 0.5 g:50 mL.

[0013] In the construction method provided by the present invention, when the test sample is scallion formula granules, the specific process of dissolving and preparing the test sample solution preferably includes: grinding the test sample and mixing it with a solvent, ultrasonic-assisted dissolution, cooling, shaking, filtering, and the obtained filtrate is the test sample solution; wherein the dosage ratio of the test sample to the solvent is preferably 0.5g:50mL.

[0014] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the specific process of the gradient elution is preferably:

[0015] 0-5 min, phase A: 1 vol%, phase B: 99 vol%;

[0016] 5-28 min, phase A: 1-5 vol%, phase B: 99-95 vol%;

[0017] 28-44 min, phase A: 5-20 vol%, phase B: 95-80 vol%;

[0018] 44-60 min, phase A: 20-35 vol%, phase B: 80-65 vol%.

[0019] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the flow rate of the mobile phase is preferably 1.0 mL / min. Under this flow rate condition, the chromatogram obtained has a better peak shape, a more stable baseline, and a more complete peak.

[0020] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the detection wavelength is preferably 258 nm. Under this detection wavelength condition, the chromatogram obtained has a larger amount of information and a more stable baseline.

[0021] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the injection volume is preferably 5 μL.

[0022] In the construction method provided by the present invention, when the high performance liquid chromatography determination is performed, the filler in the chromatographic column is octadecylsilane bonded silica gel; the column length of the chromatographic column is preferably 200 to 300 mm, more preferably 250 mm; the inner diameter of the chromatographic column is preferably 4 to 5 mm, more preferably 4.6 mm; the particle size of the filler in the chromatographic column is preferably 3 to 7 μm, more preferably 3.5 μm or 5 μm.

[0023] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the column temperature of the chromatographic column is preferably 30°C. Under this column temperature condition, the peak shape of the chromatogram obtained is more symmetrical, the separation is better, and the peak is more complete.

[0024] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the theoretical plate number calculated based on the adenosine peak should be no less than 5000.

[0025] In the construction method provided by the present invention, it is preferred that the following steps are further included:

[0026] Dissolve the scallion white control medicinal material to obtain a control medicinal material reference solution; dissolve adenosine to obtain a reference substance reference solution;

[0027] The control medicinal material reference solution and the reference substance reference solution are measured by high performance liquid chromatography to obtain a chromatogram of the reference substance; and the components of the characteristic spectrum of the test sample are qualitatively analyzed based on the chromatogram of the reference substance.

[0028] In the construction method provided by the present invention, the specific process of dissolving and preparing the control medicinal material reference solution preferably includes: mixing the scallion white control medicinal material with a solvent, ultrasonically assisted dissolution, shaking, filtering, and the obtained filtrate is the control medicinal material reference solution; wherein, the solvent is preferably a 50 vol% methanol solution; the dosage ratio of the scallion white control medicinal material to the solvent is preferably more preferably 2g:50mL; the power of the ultrasonic assistance is preferably 580~620W, more preferably 600W; the frequency of the ultrasonic assistance is preferably 35~45kHz, more preferably 40kHz; the time of the ultrasonic assistance is preferably 25~35min, more preferably 30min.

[0029] In the construction method provided by the present invention, the specific process of dissolving and preparing the reference substance solution preferably includes: mixing adenosine with a solvent to obtain a reference substance solution; wherein the solvent is preferably a 50 vol% methanol solution; the amount ratio of adenosine to the solvent is preferably (5-20) μg:1 mL, more preferably 10 μg:1 mL.

[0030] In the construction method provided by the present invention, it is preferred that the following steps are further included:

[0031] The invention adopts a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of characteristic spectra of scallion white medicinal material, and obtains a standard characteristic spectrum of scallion white medicinal material consisting of 8 characteristic peaks; in the standard characteristic spectrum, peak 3 corresponding to the peak of an adenosine reference substance is an S peak; the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within the range of ±10% of a specified value, and the specified value is: 0.65 (peak 1), 0.92 (peak 2), 1.00 (peak 3), 1.13 (peak 4), 1.89 (peak 5), 2.43 (peak 6), 2.71 (peak 7), and 3.60 (peak 8).

[0032] In the construction method provided by the present invention, it is preferred that the following steps are further included:

[0033] The similarity of the characteristic spectrum of the scallion white standard decoction was evaluated by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and a standard characteristic spectrum of the scallion white standard decoction consisting of 8 characteristic peaks was obtained; in the standard characteristic spectrum, peak 3 corresponding to the peak of the adenosine reference substance was the S peak, and the relative retention time of each characteristic peak and the S peak was calculated, and the relative retention time was within the range of ±10% of the specified value, and the specified value was: 0.65 (peak 1), 0.92 (peak 2), 1.00 (peak 3), 1.13 (peak 4), 1.89 (peak 5), 2.43 (peak 6), 2.71 (peak 7), and 3.60 (peak 8).

[0034] In the construction method provided by the present invention, it is preferred that the following steps are further included:

[0035] The invention adopts a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to evaluate the similarity of characteristic spectra of scallion white formula granules, and obtains a standard characteristic spectrum of scallion white formula granules consisting of 8 characteristic peaks. In the standard characteristic spectrum, peak 3 corresponding to the peak of a scallion white reference substance is an S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within the range of ±10% of the specified value, and the specified value is: 0.65 (peak 1), 0.92 (peak 2), 1.00 (peak 3), 1.13 (peak 4), 1.89 (peak 5), 2.43 (peak 6), 2.71 (peak 7), and 3.60 (peak 8).

[0036] The present invention also provides a method for identifying scallion white medicinal materials, standard decoctions, and formula granules. The method uses the characteristic spectrum obtained by the construction method described in the above technical solution as the basis for identifying scallion white medicinal materials, standard decoctions, and formula granules.

[0037] Compared with the existing technology, the present invention provides a method for constructing characteristic spectra of scallion white medicinal materials, standard decoctions, and formula granules and its application. The method provided by the present invention can be used to establish HPLC characteristic spectra of scallion white medicinal materials, standard decoctions, and formula granules, has good repeatability, precision and stability, and can provide a more scientific basis for the identification of scallion white medicinal materials, standard decoctions, and formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0039] Figure 1 This is a graph showing the experimental results of investigating the dissolution mode during the preparation of the scallion medicinal material test sample solution provided in Example 1 of the present invention;

[0040] Figure 2 This is a graph showing the experimental results of investigating the dissolving solvent during the preparation of the scallion medicinal material test sample solution provided in Example 1 of the present invention;

[0041] Figure 3 This is a graph showing the experimental results of investigating the amount of solvent added during the preparation of the scallion medicinal material test solution provided in Example 1 of the present invention;

[0042] Figure 4 This is a graph showing the experimental results of investigating the dissolution time during the preparation of a scallion medicinal material test solution provided in Example 1 of the present invention;

[0043] Figure 5 This is a chromatographic peak identification diagram of the characteristic spectrum of the scallion medicinal material provided in Example 1 of the present invention;

[0044] Figure 6 This is a graph showing the experimental results of different chromatographic columns for the scallion herb provided in Example 1 of the present invention;

[0045] Figure 7 This is a characteristic spectrum verification diagram of samples CB01 to CB08 in 16 batches of scallion medicinal materials provided in Example 1 of the present invention;

[0046] Figure 8 This is a characteristic spectrum verification diagram of samples CB09 to CB16 from 16 batches of scallion medicinal materials provided in Example 1 of the present invention;

[0047] Figure 9 This is a comparison of the characteristic spectrum of the medicinal material Scallion White provided in Example 1 of the present invention;

[0048] Figure 10 This is a chromatographic peak identification diagram of the characteristic spectrum of the standard decoction of scallion white provided in Example 2 of the present invention;

[0049] Figure 11 1. This is a graph showing the experimental results of different chromatographic columns for the standard decoction of scallion whites provided in Example 2 of the present invention;

[0050] Figure 12 This is a characteristic spectrum verification diagram of samples CB-BT01 to CB-BT08 in 16 batches of scallion white standard decoction provided in Example 2 of the present invention;

[0051] Figure 13 This is a characteristic spectrum verification diagram of samples CB-BT09 to CB-BT16 in 16 batches of scallion white standard decoction provided in Example 2 of the present invention;

[0052] Figure 14 This is a comparison chart of the characteristic chart of the standard decoction of scallion white provided in Example 2 of the present invention;

[0053] Figure 15 This is a chromatogram of different wavelengths of the scallion formula granules provided in Example 3 of the present invention;

[0054] Figure 16 This is a chromatogram of different mobile phases for the scallion formula granules provided in Example 3 of the present invention;

[0055] Figure 17 This is a column temperature chromatogram of the scallion formula granules provided in Example 3 of the present invention;

[0056] Figure 18 This is a flow rate chromatogram of the scallion formula granules provided in Example 3 of the present invention;

[0057] Figure 19 This is a chromatogram of the delayed investigation of the scallion formula granules provided in Example 3 of the present invention;

[0058] Figure 20 This is a graph showing the experimental results of investigating the dissolution pattern during the preparation of a test solution of the scallion white formula granules provided in Example 3 of the present invention;

[0059] Figure 21 This is a graph showing the experimental results of investigating the dissolving solvent during the preparation of the test solution of the scallion white formula granules provided in Example 3 of the present invention;

[0060] Figure 22 This is a graph showing the experimental results of investigating the amount of solvent added during the preparation of a test solution of the scallion white formula granules provided in Example 3 of the present invention;

[0061] Figure 23 This is a graph showing the experimental results of investigating the dissolution time during the preparation of a test solution of the scallion white formula granules provided in Example 3 of the present invention;

[0062] Figure 24 This is a chromatographic peak identification diagram of the characteristic spectrum of the scallion formula granules provided in Example 3 of the present invention;

[0063] Figure 25 This is a graph showing the experimental results of different chromatographic columns for the scallion white formula granules provided in Example 3 of the present invention;

[0064] Figure 26 This is a characteristic spectrum verification diagram of the scallion formula granules (CB-KL01) provided in Example 3 of the present invention;

[0065] Figure 27 This is a characteristic spectrum verification diagram of the scallion formula granules (CB-KL02) provided in Example 3 of the present invention;

[0066] Figure 28 This is a characteristic spectrum verification diagram of the scallion formula granules (CB-KL03) provided in Example 3 of the present invention;

[0067] Figure 29 This is a comparison graph of the characteristic graph of the scallion formula granules provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0068] The following is a clear and complete description of the technical solutions in 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 making any creative efforts are within the scope of protection of the present invention.

[0069] Example 1

[0070] Construction of UPLC characteristic spectrum of Scallion medicinal material:

[0071] 1) Experimental instruments and materials

[0072] High performance liquid chromatograph: Agilent 1260 high performance liquid chromatograph;

[0073] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0074] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0075] Ultrasonic cleaner: KQ600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0076] Chromatographic columns: chromatographic column 1, chromatographic column 2, and chromatographic column 3 (all filled with octadecylsilane bonded silica gel, column length 250 mm, inner diameter 4.6 mm, and filler particle size 3.5 μm);

[0077] Acetonitrile and formic acid were of chromatographic grade, water was ultrapure water, and the remaining reagents were of analytical grade;

[0078] Adenosine (China Food and Drug Control Institutes, batch number: 110879-202204, purity: 99.7%);

[0079] Guanosine (China Food and Drug Inspection Institute, batch number: 111977-202202, purity: 88.6%);

[0080] Scallion white control medicinal material (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 230149-202303);

[0081] Scallion white medicinal material (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: CB01, CB02, CB03, CB04, CB05, CB06, CB07, CB08, CB09, CB10, CB11, CB12, CB13, CB14, CB15, CB16).

[0082] 2) Feature spectrum detection method

[0083] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 3.5 μm); acetonitrile was used as mobile phase A, and 0.1 vol% formic acid solution was used as mobile phase B, with gradient elution performed as specified in Table 1; the flow rate was 1.0 mL / min; the column temperature was 30°C; the detection wavelength was 258 nm; and the number of theoretical plates calculated based on the adenosine peak should be no less than 5000.

[0084] Table 1 Gradient elution program

[0085] Time (minutes) Mobile phase A (vol%) Mobile phase B (vol%) 0～5 1 99 5～28 1→5 99→95 28～44 5→20 95→80 44～60 20→35 80→65

[0086] Preparation of reference solution: Take 2 g of scallion white reference medicinal material, place it in a stoppered conical flask, add 50 mL of 50 vol% methanol solution, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference medicinal material solution; take an appropriate amount of adenosine reference substance, accurately weigh it, and add 50 vol% methanol solution to make a solution containing 10 μg of adenosine per 1 mL, which is used as the reference substance solution.

[0087] Preparation of test solution: Take about 2 g of the product powder (passed through No. 2 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 50 vol% methanol solution, stopper it tightly, weigh it, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50 vol% methanol solution, shake it well, filter it, and take the filtrate to obtain the product.

[0088] Determination method: Accurately aspirate 5 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0089] 3) Preparation of test solution

[0090] 3.1) Investigation of dissolution mode

[0091] Take 2 g of the powder of this product (batch number: CB01), place it in a stoppered conical flask, add 25 mL of 50 vol% methanol solution, stopper it, heat reflux and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes respectively, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0092] According to the experimental conditions proposed above, the reflux treatment and ultrasonic treatment of the test solution were chromatographically determined, and the results are shown in Figure 1 , Figure 1 This is an experimental result diagram of the dissolution mode of the scallion medicinal material provided in Example 1 of the present invention. Figure 1 It can be seen that there is little difference in the effects of reflux and ultrasonic treatment of the test sample. The ultrasonic method is fast and simple, so the dissolution method of the test sample is determined to be ultrasonic-assisted dissolution.

[0093] 3.2) Investigation of dissolving solvent

[0094] Take 2 g of the powder of this product (batch number: CB01) and place it in a stoppered conical flask. Add 25 mL each of methanol, 50 vol% methanol, 70 vol% methanol, and water. Ultrasonicate (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate to obtain the test solution.

[0095] According to the experimental conditions proposed above, the test solutions prepared with different solvents were chromatographically determined. The results are shown in Figure 2 , Figure 2 This is a graph showing the experimental results of the investigation of the dissolving solvent in the preparation process of the scallion medicinal material test solution provided in Example 1 of the present invention. Figure 2 It can be seen that when the solvent is 50 vol% methanol, the extraction efficiency is higher, and the extraction solvent is determined to be 50 vol% methanol.

[0096] 3.3) Investigation of solvent addition amount

[0097] Take 2 g of the powder of this product (batch number: CB01) and place it in a stoppered conical flask. Add 25 mL, 50 mL, and 100 mL of 50 vol% methanol respectively. Ultrasonicate (power 600 W, frequency 40 kHz) for 30 minutes. Shake well, filter, and take the filtrate to obtain the test solution.

[0098] According to the experimental conditions proposed above, the test solution with different solvent addition amounts was chromatographically determined. The results are shown in Figure 3 , Figure 3 This is a graph showing the experimental results of investigating the amount of solvent added during the preparation of the scallion medicinal material test solution provided in Example 1 of the present invention. Figure 3 It can be seen that when the amount of solvent added is 50 mL, the peak shape and separation of each chromatographic peak are better, and the peak size is appropriate, so the solvent amount is selected as 50 mL.

[0099] 3.4) Investigation of dissolution time

[0100] Take 2 g of the powder of this product (batch number: CB01), place it in a stoppered conical flask, add 25 mL of 50 vol% methanol, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 15 minutes, 30 minutes, and 60 minutes respectively. Shake well, filter, and take the filtrate to obtain the test solution.

[0101] According to the experimental conditions proposed above, the test solution treated with ultrasound for different times was subjected to chromatographic determination. The results are shown in Figure 4 , Figure 4 This is a graph showing the experimental results of the dissolution time during the preparation of the scallion medicinal material test solution provided in Example 1 of the present invention. Figure 4 It can be seen that when the ultrasonic-assisted dissolution time is 30 minutes, the medicinal ingredients have been completely extracted, so the dissolution time is determined to be 30 minutes.

[0102] 4) Methodological investigation

[0103] 4.1) Chromatographic peak identification

[0104] According to the experimental conditions proposed above, prepare the scallion medicinal material test solution, scallion medicinal material reference solution, and adenosine reference solution;

[0105] Preparation of guanosine reference substance solution: Take an appropriate amount of guanosine reference substance, accurately weigh it, and add 50 vol% methanol solution to make a solution containing 50 μg of guanosine per 1 mL, which is used as the guanosine reference substance solution.

[0106] Preparation of negative control solution: Referring to the experimental conditions proposed above, a negative control solution lacking the scallion herb (ie, blank solution) was prepared.

[0107] The above solution was subjected to chromatographic detection, and the characteristic spectrum peaks of the scallion medicinal material were located according to the detection results. The results are shown in Figure 5 , Figure 5 This is the peak identification diagram of the characteristic spectrum of the medicinal material Scallion White provided in Example 1 of the present invention. The results show that Peak 3 (S) is the adenosine peak and Peak 4 is the guanosine peak. In the following methodological investigation, eight characteristic peaks in the sample were investigated.

[0108] 4.2) Precision test

[0109] Take the test solution of the medicinal material scallion (CB01) and inject it continuously 6 times according to the proposed experimental method, 5 μL each time, and calculate the retention time of each characteristic peak. The results are shown in Table 2.

[0110] Table 2 Precision investigation-retention time

[0111]

[0112]

[0113] As shown in Table 2, the RSD values ​​of the retention times of the peaks were 0.11% to 1.82%, indicating that the instrument had good precision.

[0114] 4.3) Repeatability study

[0115] Six portions of scallion white medicinal material (CB01) were accurately weighed, and the test solution was prepared and chromatographically determined according to the proposed experimental method. The results are shown in Table 3.

[0116] Table 3 Repeatability study - relative retention time of characteristic peaks

[0117]

[0118] As shown in Table 3, the RSD range of the relative retention times of the six samples was 0.00% to 0.16%, indicating that the method had good repeatability.

[0119] 4.4) Intermediate precision study

[0120] Based on the experimental conditions proposed above, different personnel (A, B) accurately weighed 1 portion of the medicinal material (CB01) of scallion white at different times (T1, T2) to prepare the test sample and conduct the determination. The results are shown in Table 4.

[0121] Table 4 Relative retention time of characteristic peaks under investigation by different personnel and time

[0122]

[0123] As can be seen from Table 4, when different personnel measured the same sample at different times, the RSDs of the relative retention times of the characteristic peaks ranged from 0.51% to 6.88%, indicating good method stability.

[0124] 4.5) Column durability assessment

[0125] Based on the experimental conditions proposed above, the analysis was carried out using chromatographic columns 1, 2 and 3 respectively. The results are shown in Figure 6 , Figure 6 The graph shows the experimental results of different chromatographic columns for testing the medicinal material Scallion White provided in Example 1 of the present invention. The results show that the RSDs of the relative retention times of the characteristic peaks when tested on the samples using the above three chromatographic columns ranged from 0.51% to 3.39%, indicating that the chromatographic columns of this method have good durability.

[0126] 4.6) Stability study

[0127] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 3h, 6h, 12h, 16h, and 24h respectively. The results are shown in Table 5.

[0128] Table 5 Stability Study - Retention Time

[0129]

[0130]

[0131] It can be seen from Table 5 that the RSD of the corresponding characteristic peak retention time is between 0.06% and 0.20%, and the sample solution is relatively stable within 24 hours.

[0132] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above eight characteristic peaks were included in the subsequent investigation.

[0133] 4.7) Determination of characteristic peaks and establishment of reference maps

[0134] The proposed method was used to analyze the characteristic patterns of 16 batches of scallion medicinal materials and calculate the relative retention time. The results are shown in Figures 7-8 , Table 6, Figure 7 This is a characteristic spectrum verification diagram of samples CB01 to CB08 in 16 batches of scallion medicinal materials provided in Example 1 of the present invention. Figure 7 Among them, S1 to S8 are: CB01, CB02, CB03, CB04, CB05, CB06, CB07, CB08; Figure 8 This is a characteristic spectrum verification diagram of samples CB09 to CB16 from 16 batches of scallion medicinal materials provided in Example 1 of the present invention. Figure 8 Among them, S1~S8 are: CB09, CB10, CB11, CB12, CB13, CB14, CB15, and CB16 respectively.

[0135] Table 6 Relative retention time of 16 batches of scallion medicinal materials

[0136]

[0137] Eight robust peaks were selected as characteristic peaks based on their stability in relative retention time, detectability across all batches, and relatively high peak height. Based on the methodological review and validation results from 16 batches of medicinal materials, the theoretical plate number (TPN) calculated based on the adenosine peak was determined to be no less than 5,000.

[0138] Final regulations: There should be 8 characteristic peaks in the chromatogram of the test sample, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the control medicinal material reference substance, among which Peak 3 should correspond to the retention time of the adenosine reference substance peak; the peak corresponding to the adenosine reference substance peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.65 (peak 1), 0.92 (peak 2), 1.00 (peak 3), 1.13 (peak 4), 1.89 (peak 5), 2.43 (peak 6), 2.71 (peak 7), and 3.60 (peak 8).

[0139] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 16 batches of scallion white medicinal materials, and a comparison map of the characteristic maps of scallion white medicinal materials was established, such as Figure 9 As shown, Figure 9 This is a comparison of the characteristic atlas of the medicinal material Scallion White provided in Example 1 of the present invention.

[0140] 5) Determination of the characteristic spectrum of scallion medicinal material

[0141] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 3.5 μm); acetonitrile was used as mobile phase A, and 0.1 vol% formic acid solution was used as mobile phase B, with gradient elution performed as specified in Table 1; the flow rate was 1.0 mL / min; the column temperature was 30°C; the detection wavelength was 258 nm; and the number of theoretical plates calculated based on the adenosine peak should be no less than 5000.

[0142] Preparation of reference solution: Take 2 g of scallion white reference medicinal material, place it in a stoppered conical flask, add 50 mL of 50 vol% methanol solution, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference medicinal material solution; take an appropriate amount of adenosine reference substance, accurately weigh it, and add 50 vol% methanol solution to make a solution containing 10 μg of adenosine per 1 mL, which is used as the reference substance solution.

[0143] Preparation of test solution: Take about 2 g of the product powder (passed through No. 2 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 50 vol% methanol solution, stopper it tightly, weigh it, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50 vol% methanol solution, shake it well, filter it, and take the filtrate to obtain the product.

[0144] Determination method: Accurately aspirate 5 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0145] There should be 8 characteristic peaks in the chromatogram of the test sample, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the control medicinal material reference substance, among which Peak 3 should correspond to the retention time of the adenosine reference substance peak; the peak corresponding to the adenosine reference substance peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is: 0.65 (peak 1), 0.92 (peak 2), 1.00 (peak 3), 1.13 (peak 4), 1.89 (peak 5), 2.43 (peak 6), 2.71 (peak 7), and 3.60 (peak 8).

[0146] Example 2

[0147] Construction of UPLC characteristic spectrum of scallion white standard decoction:

[0148] 1) Experimental instruments and materials

[0149] Scallion white standard decoction (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: CB-BT01, CB-BT02, CB-BT03, CB-BT04, CB-BT05, CB-BT06, CB-BT07, CB-BT08, CB-BT09, CB-BT10, CB-BT11, CB-BT12, CB-BT13, CB-BT14, CB-BT15, CB-BT16);

[0150] Other experimental instruments and materials are the same as those in Example 1 and will not be described in detail.

[0151] 2) Feature spectrum detection method

[0152] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 3.5 μm); acetonitrile was used as mobile phase A, and 0.1 vol% formic acid solution was used as mobile phase B, with gradient elution performed as specified in Table 1; the flow rate was 1.0 mL / min; the column temperature was 30°C; the detection wavelength was 258 nm; and the number of theoretical plates calculated based on the adenosine peak should be no less than 5000.

[0153] Preparation of reference solution: Take 2 g of scallion white reference medicinal material, place it in a stoppered conical flask, add 50 mL of 50 vol% methanol solution, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference medicinal material solution; take an appropriate amount of adenosine reference substance, accurately weigh it, and add 50 vol% methanol solution to make a solution containing 10 μg of adenosine per 1 mL, which is used as the reference substance solution.

[0154] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 50 vol% methanol solution, stopper it tightly, weigh it, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50 vol% methanol solution, shake it well, filter it, and take the filtrate to obtain it.

[0155] Determination method: Accurately aspirate 5 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0156] 3) Methodological investigation

[0157] 3.1) Chromatographic peak identification

[0158] According to the experimental conditions proposed above, prepare the test solution of scallion white standard decoction, the reference solution of scallion white reference medicinal material, and the reference solution of adenosine reference substance;

[0159] Preparation of guanosine reference substance solution: Take an appropriate amount of guanosine reference substance, accurately weigh it, and add 50 vol% methanol solution to make a solution containing 50 μg of guanosine per 1 mL, which is used as the guanosine reference substance solution.

[0160] Preparation of negative control solution: Referring to the experimental conditions proposed above, prepare a negative control solution (ie, blank solution) lacking the scallion white standard decoction.

[0161] The above solution was subjected to chromatographic detection, and the characteristic peaks of the scallion white standard decoction were located according to the detection results. Figure 10 , Figure 10 This is a chromatographic peak identification diagram of the characteristic spectrum of the standard decoction of scallion white provided in Example 2 of the present invention. The results show that Peak 3 (S) is the adenosine peak and Peak 4 is the guanosine peak. In the following methodological investigation, 8 characteristic peaks in the sample were investigated.

[0162] 3.2) Precision test

[0163] Take the test solution of scallion white standard decoction (batch number: CB-BT01) and inject it continuously 6 times according to the proposed experimental method, 5 μL each time, and calculate the retention time of each characteristic peak. The results are shown in Table 7.

[0164] Table 7 Precision Investigation-Retention Time

[0165]

[0166]

[0167] As shown in Table 7, the RSD values ​​of the retention times of the peaks ranged from 0.06% to 2.64%, indicating that the instrument had good precision.

[0168] 3.3) Repeatability study

[0169] Accurately weigh 6 portions of scallion white standard decoction (batch number: CB-BT01), and prepare the test solution and perform chromatographic determination according to the proposed experimental method. The results are shown in Table 8.

[0170] Table 8 Repeatability study - relative retention time of characteristic peaks

[0171]

[0172] As shown in Table 8, the RSD range of the relative retention times of the six samples was 0.00% to 0.17%, indicating that the method had good repeatability.

[0173] 3.4) Intermediate precision study

[0174] Based on the experimental conditions proposed above, different personnel (A, B) accurately weighed one portion of the scallion white standard decoction (batch number: CB-BT01) at different times (T1, T2) to prepare the test samples for determination. The results are shown in Table 9.

[0175] Table 9 Relative retention time of characteristic peaks under investigation by different personnel and time

[0176]

[0177] As can be seen from Table 9, when different personnel measured the same sample at different times, the RSDs of the relative retention times of the characteristic peaks ranged from 0.72% to 9.98%, indicating good method stability.

[0178] 3.5) Column durability assessment

[0179] Based on the experimental conditions proposed above, the analysis was carried out using chromatographic columns 1, 2 and 3 respectively. The results are shown in Figure 11 , Figure 11 This is a graph showing the experimental results of different chromatographic columns for the standard decoction of scallion white provided in Example 2 of the present invention. The results show that the RSDs of the relative retention times of the characteristic peaks when tested on the samples using the above three chromatographic columns ranged from 0.51% to 4.10%, indicating that the chromatographic columns used in this method have good durability.

[0180] 3.6) Stability study

[0181] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 3h, 6h, 12h, 16h, and 24h respectively. The results are shown in Table 10.

[0182] Table 10 Stability Study - Retention Time

[0183]

[0184]

[0185] It can be seen from Table 10 that the RSD of the corresponding characteristic peak retention time is between 0.06% and 0.27%, and the sample solution is relatively stable within 24 hours.

[0186] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above eight characteristic peaks were included in the subsequent investigation.

[0187] 3.7) Determination of characteristic peaks and establishment of reference spectrum

[0188] The proposed method was used to analyze the characteristic patterns of 16 batches of scallion white standard decoction samples and calculate the relative retention time. The results are shown in Figures 12-13 , Table 11, Figure 12 This is a characteristic spectrum verification diagram of samples CB-BT01 to CB-BT08 in 16 batches of scallion white standard decoction provided in Example 2 of the present invention. Figure 12 Among them, S1 to S8 are: CB-BT01, CB-BT02, CB-BT03, CB-BT04, CB-BT05, CB-BT06, CB-BT07, CB-BT08; Figure 13 This is a characteristic spectrum verification diagram of samples CB-BT09 to CB-BT16 in 16 batches of scallion white standard decoction provided in Example 2 of the present invention. Figure 13 Among them, S1~S8 are: CB-BT09, CB-BT10, CB-BT11, CB-BT12, CB-BT13, CB-BT14, CB-BT15, and CB-BT16.

[0189] Table 1 Relative retention time of 1116 batches of scallion standard decoction

[0190]

[0191]

[0192] Eight robust peaks were selected as characteristic peaks based on their stability in relative retention time, detectability across all sample batches, and relatively high peak height. Based on the methodological review and validation results from 16 batches of standard decoctions, the theoretical plate number (TPN) calculated based on the adenosine peak was determined to be no less than 5,000.

[0193] Final regulations: The test sample chromatogram should show eight characteristic peaks, and their retention times should correspond to the eight characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the adenosine reference peak. The peak corresponding to the adenosine reference peak is the S peak. The relative retention time of each characteristic peak to the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.65 (peak 1), 0.92 (peak 2), 1.00 (peak 3), 1.13 (peak 4), 1.89 (peak 5), 2.43 (peak 6), 2.71 (peak 7), and 3.60 (peak 8).

[0194] Sixteen batches of standard decoctions of scallion white were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison spectrum of the characteristic spectrum of standard decoctions of scallion white was established. Figure 14 As shown, Figure 14 This is a comparison graph of the characteristic graph of the standard decoction of scallion white provided in Example 2 of the present invention.

[0195] 5) Determination of characteristic spectrum of standard decoction of scallion white

[0196] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 3.5 μm); acetonitrile was used as mobile phase A, and 0.1 vol% formic acid solution was used as mobile phase B, with gradient elution performed as specified in Table 1; the flow rate was 1.0 mL / min; the column temperature was 30°C; the detection wavelength was 258 nm; and the number of theoretical plates calculated based on the adenosine peak should be no less than 5000.

[0197] Preparation of reference solution: Take 2 g of scallion white reference medicinal material, place it in a stoppered conical flask, add 50 mL of 50 vol% methanol solution, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference medicinal material solution; take an appropriate amount of adenosine reference substance, accurately weigh it, and add 50 vol% methanol to make a solution containing 10 μg of adenosine per 1 mL, which is used as the reference substance solution.

[0198] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 50 vol% methanol solution, stopper it tightly, weigh it, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50 vol% methanol solution, shake it well, filter it, and take the filtrate to obtain it.

[0199] Determination method: Accurately aspirate 5 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0200] The test sample chromatogram should show eight characteristic peaks, and their retention times should correspond to the eight characteristic peaks in the control medicinal material reference chromatogram. Peak 3 should correspond to the retention time of the adenosine reference peak. The peak corresponding to the adenosine reference peak is the S peak. Calculate the relative retention time of each characteristic peak to the S peak, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.65 (peak 1), 0.92 (peak 2), 1.00 (peak 3), 1.13 (peak 4), 1.89 (peak 5), 2.43 (peak 6), 2.71 (peak 7), and 3.60 (peak 8).

[0201] Example 3

[0202] Construction of UPLC characteristic spectrum of scallion formula granules:

[0203] 1) Experimental instruments and materials

[0204] Scallion formula granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers CB-KL01, CB-KL02, CB-KL03);

[0205] Other experimental instruments and materials are the same as those in Example 1 and will not be described in detail.

[0206] 2) Feature spectrum detection method

[0207] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 3.5 μm); acetonitrile was used as mobile phase A, and 0.1 vol% formic acid solution was used as mobile phase B, with gradient elution performed as specified in Table 1; the flow rate was 1.0 mL / min; the column temperature was 30°C; the detection wavelength was 258 nm; and the number of theoretical plates calculated based on the adenosine peak should be no less than 5000.

[0208] Preparation of reference solution: Take 2 g of scallion white reference medicinal material, place it in a stoppered conical flask, add 50 mL of 50 vol% methanol solution, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference medicinal material solution; take an appropriate amount of adenosine reference substance, accurately weigh it, and add 50 vol% methanol solution to make a solution containing 10 μg of adenosine per 1 mL, which is used as the reference substance solution.

[0209] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 50 vol% methanol solution, stopper it tightly, weigh it, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50 vol% methanol solution, shake it well, filter it, and take the filtrate to obtain it.

[0210] Determination method: Accurately aspirate 5 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0211] 3) Investigation of chromatographic conditions

[0212] 3.1) Wavelength selection

[0213] Based on the experimental conditions proposed above, a diode array detector was used to perform full-band scanning of resveratrol and the test solution, and the chromatograms of the test solution at wavelengths of 220nm, 258nm, 290nm, 310nm, and 330nm were extracted respectively. The results are shown in Figure 15 , Figure 15 This is a chromatogram of different wavelengths of the scallion formula granules provided in Example 3 of the present invention. Figure 15 It can be seen that when the detection wavelength is 258 nm, the chromatographic peak information volume is larger and the chromatogram baseline is more stable, so the detection wavelength is determined to be 258 nm.

[0214] 3.2) Mobile phase selection

[0215] Based on the experimental conditions proposed above, the separation effects of three different mobile phases were investigated: acetonitrile (phase A)-0.1 vol% formic acid (phase B), acetonitrile (phase A)-0.1 vol% phosphoric acid (phase B), and methanol (phase A)-0.1 vol% formic acid (phase B). The results are shown in Figure 16 , Figure 16 This is a chromatogram of different mobile phases of the scallion formula granules provided in Example 3 of the present invention. Figure 16 It can be seen that under the conditions of acetonitrile-0.1 vol% formic acid solution gradient elution, the chromatogram baseline is relatively stable and there are many chromatographic peaks. Therefore, acetonitrile-0.1 vol% formic acid solution gradient elution is used as the mobile phase for the characteristic spectrum determination method of scallion formula granules.

[0216] 3.3) Column temperature investigation

[0217] Based on the experimental conditions proposed above, the test solution was chromatographed at column temperatures of 25°C, 30°C, and 35°C. The results are shown in Figure 17 and Table 12, Figure 17 This is a column temperature chromatogram of the scallion formula granules provided in Example 3 of the present invention.

[0218] Table 12 Column temperature investigation - relative retention time

[0219]

[0220]

[0221] pass Figure 17 As can be seen from Table 12, when the column temperature is 25℃~35℃, the relative retention time RSD is 1.76%~3.08%, and the durability is good; when the column temperature is 30℃, the chromatogram peak shape is more symmetrical, the separation is better, and the peak is more complete, so the column temperature is determined to be 30℃.

[0222] 3.4) Flow rate investigation

[0223] Based on the experimental conditions proposed above, the test solution was chromatographed at flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min, respectively. The results are shown in Figure 18 and Table 13, Figure 18 This is a chromatogram of the flow rate of the scallion formula granules provided in Example 3 of the present invention.

[0224] Table 13 Flow rate investigation - relative retention time

[0225]

[0226] pass Figure 18 As can be seen from Table 13, when the flow rate is 0.8mL / min~1.2mL / min, the relative retention time RSD value is 1.34%~11.57%; when the flow rate is 0.8mlL / min, peak 8 does not appear. When the flow rate is 1.0mL / min, the chromatogram peak shape is better, the baseline is more stable, and the peak is more complete, so the flow rate is determined to be 1.0mL / min.

[0227] 3.5) Latency Investigation

[0228] Based on the experimental conditions proposed above, a delay test was conducted and the results are shown in Figure 19 , Figure 19 This is a chromatogram of the delayed investigation of the scallion formula granules provided in Example 3 of the present invention. Figure 19It can be seen that the sample has basically no useful chromatographic peak after 60 minutes, so the sample detection time is set to 60 minutes.

[0229] 4) Preparation of test solution

[0230] 4.1) Investigation of dissolution mode

[0231] Take an appropriate amount of this product (batch number: CB-KL01), grind it into powder, take 0.5 g, place it in a stoppered conical flask, add 25 mL of 50 vol% methanol solution, stopper it, heat it under reflux and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes respectively, cool it, shake it well, filter it, and take the filtrate to obtain the test solution.

[0232] According to the experimental conditions proposed above, the reflux treatment and ultrasonic treatment of the test solution were chromatographically determined, and the results are shown in Figure 20 , Figure 20 This is the experimental result diagram of the dissolution mode during the preparation of the test solution of the scallion formula granules provided in Example 3 of the present invention. Figure 20 It can be seen that there is little difference in the effects of reflux and ultrasonic treatment of the test sample. The ultrasonic method is fast and simple, so the dissolution method of the test sample is determined to be ultrasonic-assisted dissolution.

[0233] 4.2) Investigation of dissolving solvent

[0234] Take an appropriate amount of this product (batch number: CB-KL01), grind it into powder, take 0.5 g, place it in a stoppered conical flask, add 25 mL each of methanol, 50 vol% methanol, 70 vol% methanol, and water, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes. Shake well, filter, and take the filtrate to obtain the test solution.

[0235] According to the experimental conditions proposed above, the test solutions prepared with different solvents were chromatographically determined. The results are shown in Figure 21 , Figure 21 This is a graph showing the experimental results of the investigation of the dissolving solvent during the preparation of the test solution of the scallion white formula granules provided in Example 3 of the present invention. Figure 21 It can be seen that the chromatographic peaks obtained by using 50 vol% methanol as the extraction solvent have a large amount of information and good separation, and the extraction solvent is determined to be 50 vol% methanol.

[0236] 4.3) Investigation of solvent addition amount

[0237] Take an appropriate amount of this product (batch number: CB-KL01), grind it into powder, take 0.5 g, place it in a stoppered conical flask, add 10 mL, 25 mL, and 50 mL of 50 vol% methanol respectively, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes. Shake well, filter, and take the filtrate to obtain the test solution.

[0238] According to the experimental conditions proposed above, the test solution with different solvent addition amounts was chromatographically determined. The results are shown in Figure 22 , Figure 22 This is the experimental result of investigating the amount of solvent added during the preparation of the test solution of the scallion formula granules provided in Example 3 of the present invention. Figure 22 It can be seen that when the amount of solvent added is 50 mL, the size of the chromatographic peak is more appropriate, so the solvent amount is selected as 50 mL.

[0239] 4.4) Investigation of dissolution time

[0240] Take an appropriate amount of this product (batch number: CB-KL01), grind it into powder, take 0.5 g, place it in a stoppered conical flask, add 50 mL of 50 vol% methanol, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 15 minutes, 30 minutes, and 60 minutes respectively. Shake well, filter, and take the filtrate to obtain the test solution.

[0241] According to the experimental conditions proposed above, the test solution treated with ultrasound for different times was subjected to chromatographic determination. The results are shown in Figure 23 , Figure 23 This is a graph showing the experimental results of the dissolution time during the preparation of the test solution of the scallion white formula granules provided in Example 3 of the present invention. Figure 23 It can be seen that when the ultrasonic-assisted dissolution time is 30 minutes, the medicinal ingredients have been completely extracted, so the dissolution time is determined to be 30 minutes.

[0242] 5) Methodological investigation

[0243] 5.1) Chromatographic peak identification

[0244] According to the experimental conditions proposed above, the scallion formula granule test solution, scallion reference medicinal material reference solution, and adenosine reference solution were prepared;

[0245] Preparation of guanosine reference substance solution: Take an appropriate amount of guanosine reference substance, accurately weigh it, and add 50 vol% methanol solution to make a solution containing 50 μg of guanosine per 1 mL, which is used as the guanosine reference substance solution.

[0246] Preparation of negative control solution: Referring to the experimental conditions proposed above, a negative control solution (ie, blank solution) lacking scallion white formula granules was prepared.

[0247] The above solution was subjected to chromatographic detection, and the characteristic spectrum peaks of the scallion white formula particles were located according to the detection results. The results are shown in Figure 24 , Figure 24 This is a chromatographic peak identification diagram of the characteristic spectrum of the scallion white formula granules provided in Example 3 of the present invention. The results show that Peak 3 (S) is the adenosine peak and Peak 4 is the guanosine peak. In the following methodological investigation, eight characteristic peaks in the sample were examined.

[0248] 5.2) Precision test

[0249] Take the test solution of scallion formula granules (batch number: CB-KL01) and inject 5 μL of the sample 6 times continuously according to the proposed experimental method. The retention time of each characteristic peak is calculated. The results are shown in Table 14.

[0250] Table 14 Precision Investigation-Retention Time

[0251]

[0252]

[0253] As shown in Table 14, the RSD values ​​of the retention times of the peaks were 0.01% to 0.31%, indicating that the instrument had good precision.

[0254] 5.3) Repeatability study

[0255] Accurately weigh 6 portions of scallion formula granules (batch number: CB-KL01), and prepare the test solution and perform chromatographic determination according to the proposed experimental method. The results are shown in Table 15.

[0256] Table 15 Repeatability study - relative retention time of characteristic peaks

[0257]

[0258] As shown in Table 15, the RSD range of the relative retention times of the six samples was 0.00% to 0.16%, indicating that the method had good repeatability.

[0259] 5.4) Intermediate precision study

[0260] Based on the experimental conditions proposed above, different personnel (A, B) accurately weighed 1 portion of scallion formula granules (batch number: CB-KL01) at different times (T1, T2) to prepare test samples for determination. The results are shown in Table 4.

[0261] Table 16 Relative retention time of characteristic peaks in different personnel and time periods

[0262]

[0263] As can be seen from Table 16, when different people measured the same sample at different times, the RSDs of the relative retention times of the characteristic peaks were 0.72% to 3.89%, indicating good method stability.

[0264] 5.5) Column durability assessment

[0265] Based on the experimental conditions proposed above, the analysis was carried out using chromatographic columns 1, 2 and 3 respectively. The results are shown in Figure 25 , Figure 25 The following is a graph showing the experimental results of different chromatographic columns for the scallion granules provided in Example 3 of the present invention. The results show that the RSDs of the relative retention times of the characteristic peaks for the samples tested using the three chromatographic columns ranged from 0.51% to 4.10%, indicating that the chromatographic columns used in this method have good durability.

[0266] 5.6) Stability study

[0267] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 3h, 6h, 12h, 16h, and 24h respectively. The results are shown in Table 17.

[0268] Table 17 Stability Study - Retention Time

[0269]

[0270] It can be seen from Table 17 that the RSD of the corresponding characteristic peak retention time is between 0.06% and 0.27%, and the sample solution is relatively stable within 24 hours.

[0271] In summary, the RSDs of the relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above eight characteristic peaks were included in the subsequent investigation.

[0272] 5.7) Determination of characteristic peaks and establishment of reference maps

[0273] The proposed method was used to analyze the characteristic spectrum of three batches of scallion granule samples and calculate the relative retention time. The results are shown in Figures 26-28 , Table 18, Figure 26 This is a characteristic spectrum verification diagram of the scallion formula granules (CB-KL01) provided in Example 3 of the present invention. Figure 27 This is a characteristic spectrum verification diagram of the scallion formula granules (CB-KL02) provided in Example 3 of the present invention. Figure 28 This is a characteristic spectrum verification diagram of the scallion formula granules (CB-KL03) provided in Example 3 of the present invention.

[0274] Table 183 Relative retention time of batch scallion formula granules

[0275]

[0276] Eight peaks were selected as characteristic peaks based on their stable relative retention times, detectability across all sample batches, and relatively high peak heights. Based on the methodological review results and validation results from three batches of particles, the theoretical plate number (TPN) calculated based on the adenosine peak was determined to be no less than 5,000.

[0277] Final regulations: There should be 8 characteristic peaks in the chromatogram of the test sample, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the control medicinal material reference substance, among which Peak 3 should correspond to the retention time of the adenosine reference substance peak; the peak corresponding to the adenosine reference substance peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.65 (peak 1), 0.92 (peak 2), 1.00 (peak 3), 1.13 (peak 4), 1.89 (peak 5), 2.43 (peak 6), 2.71 (peak 7), and 3.60 (peak 8).

[0278] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize three batches of scallion white formula granules, and a comparison map of the characteristic spectrum of scallion white formula granules was established. Figure 29 As shown, Figure 29 This is a comparison graph of the characteristic graph of the scallion formula granules provided in Example 3 of the present invention.

[0279] 5) Determination of characteristic spectrum of scallion formula particles

[0280] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 3.5 μm); acetonitrile was used as mobile phase A, and 0.1 vol% formic acid solution was used as mobile phase B, with gradient elution performed as specified in Table 1; the flow rate was 1.0 mL / min; the column temperature was 30°C; the detection wavelength was 258 nm; and the number of theoretical plates calculated based on the adenosine peak should be no less than 5000.

[0281] Preparation of reference solution: Take 2 g of scallion white reference medicinal material, place it in a stoppered conical flask, add 50 mL of 50 vol% methanol solution, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the filtrate as the reference medicinal material solution; take an appropriate amount of adenosine reference substance, accurately weigh it, and add 50 vol% methanol solution to make a solution containing 10 μg of adenosine per 1 mL, which is used as the reference substance solution.

[0282] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 50 vol% methanol solution, stopper it tightly, weigh it, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50 vol% methanol solution, shake it well, filter it, and take the filtrate to obtain it.

[0283] Determination method: Accurately aspirate 5 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0284] There should be 8 characteristic peaks in the chromatogram of the test sample, and the retention times should correspond to the 8 characteristic peaks in the chromatogram of the control medicinal material reference substance, among which Peak 3 should correspond to the retention time of the adenosine reference substance peak; the peak corresponding to the adenosine reference substance peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is: 0.65 (peak 1), 0.92 (peak 2), 1.00 (peak 3), 1.13 (peak 4), 1.89 (peak 5), 2.43 (peak 6), 2.71 (peak 7), and 3.60 (peak 8).

[0285] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing characteristic maps of scallion medicinal materials, standard decoctions, and formula granules, characterized in that: The following steps are involved: preparing a test sample, wherein the test sample is scallion medicinal material, scallion standard decoction or scallion formula granules; Dissolve the test sample to obtain a test sample solution; the dissolving solvent is 50 vol% methanol solution; The test solution is measured by high performance liquid chromatography to obtain a characteristic spectrum corresponding to the test sample; The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is a 0.1 vol% formic acid solution, and the gradient elution is performed; the detection wavelength is 258 nm; The gradient elution is specifically as follows: 0~5min, phase A: 1vol%, phase B: 99vol%; 5~28min, phase A: 1~5vol%, phase B: 99~95vol%; 28~44min, phase A: 5~20vol%, phase B: 95~80vol%; 44~60min, phase A: 20~35vol%, phase B: 80~65vol%.

2. The construction method according to claim 1, characterized in that The dosage ratio of the test sample to the solvent is (0.5~2) g:50 mL.

3. The construction method according to claim 1, characterized in that The dissolution is carried out under the assistance of ultrasound, wherein the power of the ultrasound assistance is 580-620 W, the frequency is 35-45 kHz, and the time is 25-35 min.

4. The construction method according to claim 1, wherein The chromatographic conditions of the high performance liquid chromatography method also include: a mobile phase flow rate of 1.0 mL / min; an injection volume of 5 μL; a column temperature of 30° C.; and a theoretical plate number of not less than 5000 calculated based on the adenosine peak.

5. The construction method according to claim 1, characterized in that The following steps are also included: Dissolve the scallion white control medicinal material to obtain a control medicinal material reference solution; dissolve adenosine to obtain a reference substance reference solution; The control medicinal material reference solution and the reference substance reference solution are measured by high performance liquid chromatography to obtain a chromatogram of the reference substance; and the components of the characteristic spectrum of the test sample are qualitatively analyzed based on the chromatogram of the reference substance.

6. The construction method according to claim 5, characterized in that: The following steps are also included: The similarity of the characteristic spectrum of the scallion white medicinal material was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and a standard characteristic spectrum of the scallion white medicinal material consisting of 8 characteristic peaks was obtained; in the standard characteristic spectrum, peak 3 corresponding to the peak of the adenosine reference substance was the S peak, and the relative retention time of each characteristic peak and the S peak was calculated. The relative retention time was within the range of ±10% of the specified value, and the specified value was: 0.65 (peak 1), 0.92 (peak 2), 1.00 (peak 3), 1.13 (peak 4), 1.89 (peak 5), 2.43 (peak 6), 2.71 (peak 7), 3.60 (peak 8).

7. The construction method according to claim 6, characterized in that: The following steps are also included: The similarity of the characteristic spectrum of the scallion white standard decoction was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and a standard characteristic spectrum of the scallion white standard decoction consisting of 8 characteristic peaks was obtained; in the standard characteristic spectrum, peak 3 corresponding to the adenosine reference peak was the S peak, and the relative retention time of each characteristic peak and the S peak was calculated. The relative retention time was within the range of ±10% of the specified value, and the specified value was: 0.65 (peak 1), 0.92 (peak 2), 1.00 (peak 3), 1.13 (peak 4), 1.89 (peak 5), 2.43 (peak 6), 2.71 (peak 7), 3.60 (peak 8).

8. The construction method according to claim 6, characterized in that: The following steps are also included: The similarity of the characteristic spectrum of the scallion white formula granules was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and a standard characteristic spectrum of the scallion white formula granules consisting of 8 characteristic peaks was obtained; in the standard characteristic spectrum, peak 3 corresponding to the peak of the scallion white reference substance was the S peak, and the relative retention time of each characteristic peak and the S peak was calculated. The relative retention time was within the range of ±10% of the specified value, and the specified value was: 0.65 (peak 1), 0.92 (peak 2), 1.00 (peak 3), 1.13 (peak 4), 1.89 (peak 5), 2.43 (peak 6), 2.71 (peak 7), 3.60 (peak 8).

9. A method for identifying scallion medicinal materials, standard decoctions, and formula granules, characterized in that: The characteristic spectrum obtained by the construction method according to any one of claims 1 to 8 is used as the basis for identifying scallion medicinal materials, standard decoctions, and formula granules.

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