Method for constructing characteristic chromatograms of antifebrile dichroa and preparation thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a construction method of a specific chromatogram of antifebrile dichroa and a preparation thereof, and the construction method comprises the following steps: (1) preparing a test solution; (2) the test solution is taken and detected by adopting a high performance liquid chromatography, octadecylsilane chemically bonded silica is taken as a filler, a mobile phase comprises an aqueous solution containing ammonium acetate and acetonitrile, the gradient elution procedure comprises 0-1-16 min-35-40 min, and the volume percentage of acetonitrile in the mobile phase is 5%-6%-5%-11%-28%-30%. According to the method, effective separation of six common characteristic peaks including all characteristic peaks including dichroa alkaloid can be realized, the obtained characteristic spectrum is stable in baseline, good in peak shape of the characteristic peaks and high in separation degree, and the integrity and characteristics of antifebrile dichroa and a preparation thereof can be fully reflected.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for constructing a characteristic chromatogram of Dichroa febrifuga and its preparations. Background Art

[0002] Dichroa febrifuga is the dried root of the plant Dichroa febrifuga Lour. of the Saxifragaceae family. It can be dug in autumn, the fibrous roots are removed, washed, and dried in the sun. It is bitter, pungent, and cold in nature; it belongs to the lung, liver, and heart meridians; it has a strong effect of expelling phlegm and inducing vomiting, and is mostly used for phlegm retention, malaria, etc. The "Dichroa febrifuga" item in the Chinese Pharmacopoeia (2020 Edition) only includes its character, microscopic identification, thin-layer identification, and inspection items such as moisture and total ash. Summary of the Invention

[0003] Therefore, the purpose of the present invention is to provide a method for constructing a characteristic chromatogram of Dichroa febrifuga and its preparations. According to the characteristics of Dichroa febrifuga and its preparations, this method establishes the characteristic chromatogram of this variety, with a large number of characteristic peaks, high resolution, obvious characteristics, and can comprehensively control the quality of Dichroa febrifuga and its preparations.

[0004] For this purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a method for constructing a characteristic chromatogram of Dichroa febrifuga and its preparations, including the following steps: (1) Preparation of the test solution; (2) Taking the test solution and detecting it by high performance liquid chromatography, using octadecylsilane chemically bonded silica gel as the filler, the mobile phase includes an aqueous solution containing ammonium acetate and acetonitrile, and the gradient elution program includes: 0 → 1 - 16 min → 35 - 40 min, and the volume percentage of acetonitrile in the mobile phase is: 5% - 6% → 5% - 11% → 28% - 30%.

[0006] Optionally, step (2) also satisfies at least one of the following 1) - 4):

[0007] 1) The detection wavelength is 226 - 304 nm, the flow rate is 0.20 - 0.35 mL / min, and the column temperature is 20 - 35 °C; optionally, the detection wavelength is 235 nm, the flow rate is 0.25 - 0.30 mL / min, and the column temperature is 25 - 30 °C;

[0008] 2) During the detection process, a chromatographic column with a specification of 2.1 mm × 100 mm, 1.7 μm is used; optionally, it is an ACQUITY UPLC CSH C18 or ACQUITY UPLC BEH C18 chromatographic column;

[0009] 3) The injection volume is 1 - 5 μL; optionally, it is 1 - 3 μL;

[0010] 4) The concentration of ammonium acetate in the aqueous solution containing ammonium acetate is 0.025 - 0.050 mol / L; optionally, it is 0.050 mol / L.

[0011] Optionally, the gradient elution program includes: 0 → 2 min → 11 min → 16 min → 40 min, and the volume percentage of acetonitrile in the mobile phase is: 5% → 5% → 10% → 11% → 28%; or, it includes: 0 → 1 min → 13 min → 35 min, and the volume percentage of acetonitrile in the mobile phase is: 6% → 6% → 10% → 30%.

[0012] Optionally, in step (1), it includes weighing the Dichroa febrifuga test sample, extracting with a solvent to obtain an extract, separating the solid and liquid, and taking the liquid to obtain the test sample solution.

[0013] Optionally, step (1) also satisfies any one or more of the following A - E:

[0014] A. The mass - volume ratio of the Dichroa febrifuga test sample to the solvent is 0.1 - 0.2 g:25 mL;

[0015] B. The extraction method includes ultrasonic extraction or reflux extraction; optionally, it is ultrasonic extraction; further optionally, the power of the ultrasonic extraction is 200 - 300 W, and the frequency is 30 - 50 kHz;

[0016] C. The extraction time is 30 - 60 min; optionally, the extraction time is 45 - 60 min;

[0017] D. The solid - liquid separation is selected from centrifugation or filtration;

[0018] E. The solvent is selected from one or more of methanol or aqueous methanol solution; optionally, it is an aqueous solution of methanol with a volume percentage of 30% - 70%; further optionally, it is an aqueous solution of methanol with a volume percentage of 70%.

[0019] Optionally, the construction method further includes the steps of preparing a reference substance solution by using at least one of anisodamine, dichroine, 4 - hydroxyquinazoline, 7 - hydroxycoumarin and adding a solvent, and the step of detecting the reference substance solution by the high - performance liquid chromatography method in the above construction method to obtain a reference substance chromatogram.

[0020] Optionally, using the Dichroa febrifuga reference medicinal material as a reference, preparing a reference substance reference solution according to step (1) in the above construction method, and the step of detecting the reference substance reference solution by the high - performance liquid chromatography method in the above construction method to obtain a reference substance reference chromatogram.

[0021] Optionally, each reference substance in 1 mL of the reference substance solution contains 30 - 70 μg of each reference substance; optionally, each 1 mL of the reference substance solution contains 40 - 50 μg of anisodamine; optionally, each 1 mL of the reference substance solution contains 65 - 75 μg of febrifugine; optionally, each 1 mL of the reference substance solution contains 30 - 40 μg of 4-hydroxyquinazoline; optionally, each 1 mL of the reference substance solution contains 40 - 50 μg of 7-hydroxycoumarin.

[0022] Optionally, the solvent used in the preparation of the reference substance solution is an aqueous methanol solution with a volume percentage of 30% - 70%.

[0023] Optionally, the characteristic fingerprint of the dichroa febrifuga and its preparations has 6 common characteristic peaks. Peak 2 corresponds to the retention time of the febrifugine reference substance peak. The peak corresponding to the febrifugine reference substance peak is the S peak. The relative retention times of peaks 1, 3 - 6 with respect to the S peak are within the range of ±10% of the specified values; the specified values are: 0.83 (peak 1), 1.10 (peak 3), 1.43 (peak 4), 1.64 (peak 5), 2.77 (peak 6).

[0024] The present invention provides the application of the method for constructing the characteristic fingerprint of the dichroa febrifuga and its preparations in the quality inspection of dichroa febrifuga pharmaceutical preparations.

[0025] A method for quality inspection of dichroa febrifuga and its preparations, including the step of comparing the characteristic fingerprint of the dichroa febrifuga product to be tested with the reference characteristic fingerprint of dichroa febrifuga and its preparations; the characteristic fingerprint of the dichroa febrifuga product to be tested is constructed by using the dichroa febrifuga product to be tested according to the above construction method. The reference characteristic fingerprint of dichroa febrifuga and its preparations is selected from any one of the following (1) - (3):

[0026] (1) It has 6 common characteristic peaks. Peak 2 corresponds to the retention time of the febrifugine reference substance peak. The peak corresponding to the febrifugine reference substance peak is the S peak. The relative retention times of peaks 1, 3 - 6 with respect to the S peak are within the range of ±10% of the specified values; the specified values are: 0.83 (peak 1), 1.10 (peak 3), 1.43 (peak 4), 1.64 (peak 5), 2.77 (peak 6);

[0027] (2) The characteristic fingerprint of dichroa febrifuga and / or its preparations obtained by using a single batch or multiple batches of dichroa febrifuga and / or its preparations according to the above construction method;

[0028] (3) The characteristic fingerprint obtained by using multiple batches of dichroa febrifuga and / or its preparations according to the above construction method is made into a reference characteristic fingerprint by the average value or median method.

[0029] The technical solution of the present invention has the following advantages:

[0030] 1. The method for constructing the characteristic chromatogram of Dichroa febrifuga and its preparations provided by the present invention uses octadecylsilane-bonded silica gel as the filler, and the mobile phase includes an aqueous solution containing ammonium acetate and acetonitrile. Through a specific gradient elution program, 6 common characteristic peaks are obtained, among which the separation of common characteristic peaks including the dichroine peak is achieved, and the elution program is simple. The obtained characteristic chromatogram has a stable baseline, good peak shapes of characteristic peaks, and high resolution, providing a basis for the quality inspection and control of Dichroa febrifuga and its preparations. It can also accurately locate the peaks of anomalodichroine, dichroine, 4-hydroxyquinazoline, and 7-hydroxycoumarin, fully reflecting the integrity and characteristics of Dichroa febrifuga and its preparations.

[0031] 2. The method for constructing the characteristic chromatogram of Dichroa febrifuga and its preparations provided by the present invention examines extraction conditions such as optimizing chromatographic conditions, detection wavelength, extraction solvent, extraction method, and extraction time, and determines the optimal extraction process and chromatographic conditions, resulting in higher peak areas and better separation effects, enabling more comprehensive quality monitoring of Dichroa febrifuga and its preparations. Moreover, the preparation of the test sample in this method is simple, the chromatographic conditions are easy to achieve, the operation is simple, the results are accurate, and the method has good reproducibility.

[0032] 3. The quality inspection method for Dichroa febrifuga and its preparations described in the present invention can comprehensively, clearly, and effectively conduct quality inspection on Dichroa febrifuga and its preparations by comparing the characteristic chromatogram of the tested Dichroa febrifuga and its preparation products with the control characteristic chromatogram of Dichroa febrifuga and its preparations. Description of the Drawings

[0033] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 It is the chromatogram of the Dichroa febrifuga formula granules obtained in Example 1 of the present invention;

[0035] Figure 2 It is the chromatogram of the mixed reference substances obtained in Example 1 of the present invention;

[0036] Figure 3 It is the chromatogram obtained with acetonitrile as mobile phase A in the selection of the organic phase system in Experimental Example 1 of the present invention;

[0037] Figure 4 It is the chromatogram obtained with methanol as mobile phase A in the selection of the organic phase system in Experimental Example 1 of the present invention;

[0038] Figure 5It is the chromatogram obtained with 0.1% acetic acid solution as mobile phase B in the selection of the aqueous phase system in Experimental Example 1 of the present invention;

[0039] Figure 6 It is the chromatogram obtained with 0.05 mol / L potassium dihydrogen phosphate as mobile phase B in the selection of the aqueous phase system in Experimental Example 1 of the present invention;

[0040] Figure 7 It is the chromatogram obtained with 0.05 mol / L ammonium acetate as mobile phase B in the selection of the aqueous phase system in Experimental Example 1 of the present invention;

[0041] Figure 8 It is the chromatogram obtained under gradient 1 in the selection of the mobile phase gradient in Experimental Example 1 of the present invention;

[0042] Figure 9 It is the chromatogram obtained under gradient 2 in the selection of the mobile phase gradient in Experimental Example 1 of the present invention;

[0043] Figure 10 It is the chromatogram obtained under gradient 3 in the selection of the mobile phase gradient in Experimental Example 1 of the present invention;

[0044] Figure 11 It is the chromatogram obtained under gradient 4 in the selection of the mobile phase gradient in Experimental Example 1 of the present invention;

[0045] Figure 12 It is the chromatogram obtained with a flow rate of 0.25 ml / min in the selection of the flow rate in Experimental Example 1 of the present invention;

[0046] Figure 13 It is the chromatogram obtained with a flow rate of 0.30 ml / min in the selection of the flow rate in Experimental Example 1 of the present invention;

[0047] Figure 14 It is the chromatogram obtained with a flow rate of 0.35 ml / min in the selection of the flow rate in Experimental Example 1 of the present invention;

[0048] Figure 15 It is the chromatogram obtained at a column temperature of 25 °C in the selection of the column temperature in Experimental Example 1 of the present invention;

[0049] Figure 16 It is the chromatogram obtained at a column temperature of 30 °C in the selection of the column temperature in Experimental Example 1 of the present invention;

[0050] Figure 17 It is the chromatogram obtained at a column temperature of 35 °C in the selection of the column temperature in Experimental Example 1 of the present invention;

[0051] Figure 18 It is the chromatogram obtained at a wavelength of 226 nm in the selection of the wavelength in Experimental Example 1 of the present invention;

[0052] Figure 19It is the chromatogram obtained at a wavelength of 235 nm in the selection of wavelengths in Experimental Example 1 of the present invention;

[0053] Figure 20 It is the chromatogram obtained at a wavelength of 274 nm in the selection of wavelengths in Experimental Example 1 of the present invention;

[0054] Figure 21 It is the chromatogram obtained at a wavelength of 304 nm in the selection of wavelengths in Experimental Example 1 of the present invention;

[0055] Figure 22 It is the chromatogram obtained at gradient 5 in the further optimization of the mobile phase gradient in Experimental Example 1 of the present invention;

[0056] Figure 23 It is the chromatogram obtained at gradient 6 in the further optimization of the mobile phase gradient in Experimental Example 1 of the present invention;

[0057] Figure 24 It is the chromatogram obtained under ultrasonic extraction in the selection of extraction methods in Experimental Example 2 of the present invention;

[0058] Figure 25 It is the chromatogram obtained under heating reflux extraction in the selection of extraction methods in Experimental Example 2 of the present invention;

[0059] Figure 26 It is the chromatogram obtained at an extraction time of 30 min in the selection of extraction times in Experimental Example 2 of the present invention;

[0060] Figure 27 It is the chromatogram obtained at an extraction time of 45 min in the selection of extraction times in Experimental Example 2 of the present invention;

[0061] Figure 28 It is the chromatogram obtained at an extraction time of 60 min in the selection of extraction times in Experimental Example 2 of the present invention;

[0062] Figure 29 It is the chromatogram obtained using ethanol as the extraction solvent in the selection of extraction solvents in Experimental Example 2 of the present invention;

[0063] Figure 30 It is the chromatogram obtained using methanol as the extraction solvent in the selection of extraction solvents in Experimental Example 2 of the present invention;

[0064] Figure 31 It is the chromatogram obtained using 70% vol methanol as the extraction solvent in the selection of extraction solvents in Experimental Example 2 of the present invention;

[0065] Figure 32 It is the chromatogram obtained using 50% vol methanol as the extraction solvent in the selection of extraction solvents in Experimental Example 2 of the present invention;

[0066] Figure 33It is the chromatogram obtained with 30% vol methanol as the extraction solvent in the selection of the extraction solvent in Experimental Example 2 of the present invention;

[0067] Figure 34 It is the chromatogram obtained with a sampling amount of 0.1 g in the selection of the sampling amount in Experimental Example 2 of the present invention;

[0068] Figure 35 It is the chromatogram obtained with a sampling amount of 0.2 g in the selection of the sampling amount in Experimental Example 2 of the present invention;

[0069] Figure 36 It is the chromatogram obtained with a sampling amount of 0.5 g in the selection of the sampling amount in Experimental Example 2 of the present invention;

[0070] Figure 37 It is the chromatogram obtained with an injection volume of 1 μl in the selection of the injection volume in Experimental Example 2 of the present invention;

[0071] Figure 38 It is the chromatogram obtained with an injection volume of 2 μl in the selection of the injection volume in Experimental Example 2 of the present invention;

[0072] Figure 39 It is the chromatogram obtained with an injection volume of 3 μl in the selection of the injection volume in Experimental Example 2 of the present invention;

[0073] Figure 40 It is the chromatogram obtained with an injection volume of 5 μl in the selection of the injection volume in Experimental Example 2 of the present invention;

[0074] Figure 41 It is the characteristic chromatogram of multiple batches of Dichroa febrifuga obtained in Experimental Example 3 of the present invention. Among them, S1 - S18 from bottom to top are: the standard decoction BT1, BT2, BT3, BT4, BT5, BT6, BT7, BT8, BT9, BT10, BT11, BT12, BT13, BT14, BT16 of Dichroa febrifuga cut pieces, and the formulated granules KL1, KL2, KL3 of Dichroa febrifuga;

[0075] Figure 42 It is the control characteristic chromatogram of Dichroa febrifuga obtained in Experimental Example 3 of the present invention;

[0076] Figure 43 It is the locator chromatogram of reference substances obtained in Experimental Example 3 of the present invention. Among them, from bottom to top are: the formulated granules of Dichroa febrifuga; the reference substance of febrifugine; the reference substance of isofebrifugine; 4 - hydroxyquinazoline; 7 - hydroxycoumarin;

[0077] Figure 44 It is the chromatogram of the negative control solution obtained in the specificity investigation of Experimental Example 4 of the present invention. Detailed implementation mode

[0078] The following embodiments are provided to better further understand the present invention. It is not limited to the described best mode, and does not constitute a limitation on the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other prior arts falls within the protection scope of the present invention. For those embodiments where specific experimental steps or conditions are not indicated, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments whose manufacturers are not indicated, they are all conventional reagent products that can be obtained through commercial purchase.

[0079] Experimental drugs and instruments

[0080] Among the test drugs, the Dichroa febrifuga formula granules and the standard decoction of Dichroa febrifuga cut pieces can be prepared by conventional methods in this field. For example, the Dichroa febrifuga formula granules in the present invention are prepared according to the following steps: Take Dichroa febrifuga medicinal materials, remove impurities according to the provisions of the Chinese Pharmacopoeia, separate them by size, soak, moisten thoroughly, cut into thin slices, dry and roast to make Dichroa febrifuga cut pieces, extract by heating under reflux twice. For the first time, add 9 times the weight of water and soak for 30 minutes, extract by heating under reflux for 30 minutes, filter. For the second time, add 7 times the weight of water and extract for 20 minutes, filter. Concentrate the filtrate to a flowable extract with a relative density of 1.02 - 1.10 g / ml (65 ± 5 °C), dry, add an appropriate amount of auxiliary materials (maltodextrin) (so that 5.88 g of granules are obtained from every 100 g of cut pieces), and granulate to obtain. Prepare 3 batches of formula granules respectively, numbered KL1, KL2, and KL3. The standard decoction (lyophilized powder) of Dichroa febrifuga cut pieces is prepared according to the following steps: Take Dichroa febrifuga medicinal materials, remove impurities according to the provisions of the Chinese Pharmacopoeia, separate them by size, soak, moisten thoroughly, cut into thin slices, dry and roast to make Dichroa febrifuga cut pieces, decoct twice. For the first time, add 9 times the weight of water and soak for 30 minutes, first bring to a boil over high heat, then decoct over low heat for 30 minutes, filter. For the second time, add 7 times the weight of water, bring to a boil over high heat, then decoct over low heat for 20 minutes, filter. Combine the filtrates, concentrate the filtrates (65 °C) to a thick extract with a relative density of about 1.08 - 1.12 g / ml, place in a freeze dryer for freeze drying, and crush into powder to obtain. Prepare 16 batches of standard decoctions respectively, numbered BT1, BT2, BT3, BT4, BT5, BT6, BT7, BT8, BT9, BT10, BT11, BT12, BT13, BT14, and BT16.

[0081] Acetonitrile is of chromatographic purity, and water is ultrapure water; other reagents are all of analytical purity.

[0082] Dichroine reference substance: Batch number: 030040 - 202207, purchased from Shanghai Hongyong Biotechnology Co., Ltd.

[0083] Isodichroine reference substance: Batch number: 250335 - 202111, purchased from Shanghai Hongyong Biotechnology Co., Ltd.

[0084] 4-Hydroxyquinazoline reference substance: batch number: 170213-202301, purchased from Shanghai Hongyong Biotechnology Co., Ltd.

[0085] 7-Hydroxycoumarin reference substance: batch number: 111739-200501, purchased from China Food and Drug Inspection Institutes.

[0086] Chromatograph: Thermo Vanquish chromatography system; Agilent 1290 chromatography system.

[0087] Chromatographic columns: ACQUITY UPLC CSH C18 (2.1×100 mm, 1.7 μm); ACQUITY UPLC BEH C18 (2.1×100 mm, 1.7 μm).

[0088] Electronic balance: METTLER TOLEDO (Swiss Mettler-Toledo) XPE56 (one part per million); XS204, XSE205, XS205 (one part per hundred thousand); Sartorius ME36S (one part per million).

[0089] Ultrasonic instrument: KQ-500DE CNC ultrasonic instrument produced by Kunshan Ultrasonic Instrument Co., Ltd.

[0090] Example 1

[0091] This embodiment provides a method for constructing a characteristic spectrum of Changshan formula granules, comprising the following steps:

[0092] Preparation of the test solution: Take the Radix Dichroae granules, grind them into powder, take 0.2g of powder, add 25ml of 70% vol methanol, ultrasonically treat (power 250W, frequency 40kHz) for 45 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0093] Preparation of reference substance solution: Take appropriate amounts of abnormal shan alkaloid reference substance, febrifugine reference substance, 4-hydroxyquinazoline reference substance, and 7-hydroxycoumarin, add 70% vol methanol to make a mixed solution containing 44 μg of abnormal shan alkaloid, 70 μg of febrifugine, 34 μg of 4-hydroxyquinazoline, and 45 μg of 7-hydroxycoumarin per 1 ml, as the reference substance solution.

[0094] Determination: Inject 2 μl each of the test solution and the reference substance solution of the Dichroa febrifuga formula granules into a Thermo Vanquish liquid chromatograph (unless otherwise specified hereinafter, this chromatograph is used and will not be specifically mentioned again). The chromatographic conditions are as follows: Use ACQUITY UPLC CSH C18 (2.1×100 mm, 1.7 μm) as the chromatographic column, use acetonitrile as mobile phase A, and use 0.05 mol / L ammonium acetate solution as mobile phase B, and perform gradient elution according to the regulations in Table 1; the flow rate is 0.25 ml per minute, the column temperature is 25 °C, and the detection wavelength is 235 nm. The number of theoretical plates calculated based on the febrifugine peak should be not less than 5000. The chromatogram of the Dichroa febrifuga formula granules obtained is shown in Figure 1 , and the chromatogram of the mixed reference substances is shown in Figure 2 . Table 2 shows the peak system suitability parameters of the reference substances, and Table 3 shows the peak system suitability parameters of the Dichroa febrifuga formula granules. It can be seen that the test solution shows 6 peaks, and the retention time of Peak 2 corresponds to that of the peak in the reference substance solution of febrifugine. Moreover, in the chromatogram of the test solution, the characteristic peaks have good peak shapes, high resolution, and short detection and analysis time.

[0095] Table 1

[0096]

[0097]

[0098] Table 2

[0099]

[0100] Table 3

[0101]

[0102] Investigation of the construction method in Experimental Example 1

[0103] 1. Selection of the organic phase system

[0104] Prepare the test solution using the same method as in Example 1.

[0105] Determination: Inject 2 μl of the above-mentioned test solution into the liquid chromatograph. The chromatographic conditions are as follows: Use ACQUITY UPLC CSH C18 (2.1×100 mm, 1.7 μm) as the chromatographic column, and perform gradient elution according to the regulations in Table 4; the flow rate is 0.25 ml per minute, the column temperature is 30 °C, and the detection wavelength is 226 nm.

[0106] Table 4

[0107]

[0108] Under Condition 1, acetonitrile was used as mobile phase A, and 0.05 mol / L potassium dihydrogen phosphate solution was used as mobile phase B. The obtained chromatogram is shown in Figure 3 ; Under Condition 2, methanol was used as mobile phase A, and 0.05 mol / L potassium dihydrogen phosphate solution was used as mobile phase B. The obtained chromatogram is shown in Figure 4 . From Figure 3 and 4 , it can be seen that when acetonitrile was used as the mobile phase, the overall peak shape of the chromatographic peaks was good, and there was a large optimization space for the chromatographic peaks. When methanol was used as mobile phase A, the number of peaks presented was less and the peak shape was poor. Therefore, acetonitrile was selected as the organic phase in the mobile phase for subsequent method development.

[0109] 2. Selection of Aqueous Phase System

[0110] The test solution was prepared using the same method as in Example 1.

[0111] Determination: Inject 2 μl of the above test solution into the liquid chromatograph. The chromatographic conditions are as follows: ACQUITY UPLCCSH C18 (2.1×100 mm, 1.7 μm) was used as the chromatographic column, acetonitrile was used as mobile phase A, and gradient elution was carried out according to the regulations in Table 5; the flow rate was 0.3 ml per minute, the column temperature was 30 °C, and the detection wavelength was 235 nm.

[0112] Table 5

[0113]

[0114] Under Condition 1, 0.1% acetic acid solution was used as mobile phase B. The obtained chromatogram is shown in Figure 5 ; Under Condition 2, 0.05 mol / L potassium dihydrogen phosphate was used as mobile phase B. The obtained chromatogram is shown in Figure 6 ; Under Condition 3, 0.05 mol / L ammonium acetate was used as mobile phase B. The obtained chromatogram is shown in Figure 7 . By comparison Figures 5 - 7 it was found that when acetic acid solution was used as mobile phase B, wrapped peaks and peak bifurcation phenomena would occur, the peak shape was poor, and there was more baseline interference; when potassium dihydrogen phosphate was used as mobile phase B, the peak area was small and some peaks were not obvious; when the ammonium acetate system was used in the aqueous phase system, the peak shapes of each characteristic peak were good, the baseline interference was less, and the elution time was also shorter. Finally, ammonium acetate was selected as the aqueous phase in the mobile phase for method development.

[0115] 3. Selection of Mobile Phase Gradient

[0116] The test solution was prepared using the same method as in Example 1.

[0117] Determination: Inject 2 μl of the above-mentioned test solution into the liquid chromatograph. The chromatographic conditions are as follows: Use ACQUITY UPLCCSH C18 (2.1×100 mm, 1.7 μm) as the chromatographic column, use acetonitrile as mobile phase A, use 0.05 mol / L ammonium acetate solution as mobile phase B, the flow rate is 0.3 ml per minute, the column temperature is 30 °C, and the detection wavelength is 235 nm.

[0118] In Condition 1, the elution gradient uses Gradient 1 in Table 6, and the obtained chromatogram is shown in Figure 8 ; in Condition 2, the elution gradient uses Gradient 2 in Table 6, and the obtained chromatogram is shown in Figure 9 ; in Condition 3, the elution gradient uses Gradient 3 in Table 7, and the obtained chromatogram is shown in Figure 10 ; in Condition 4, the elution gradient uses Gradient 4 in Table 7, and the obtained chromatogram is shown in Figure 11 . It can be seen from the figure that under Gradients 1 and 2, the separation effect of the main chromatographic peaks is poor. Under the condition of Gradient 3, there is a peak wrapping phenomenon for the chromatographic peak at 9 - 10 minutes, while under Gradient 4, the main chromatographic peaks have basically achieved baseline separation. Subsequently, the flow rate and column temperature will be determined under this gradient.

[0119] Table 6

[0120]

[0121] Table 7

[0122]

[0123] 4. Selection of Flow Rate

[0124] Prepare the test solution using the same method as in Example 1.

[0125] Determination: Inject 2 μl of the above-mentioned test solution into the liquid chromatograph. The chromatographic conditions are as follows: Use ACQUITY UPLCCSH C18 (2.1×100 mm, 1.7 μm) as the chromatographic column, use acetonitrile as mobile phase A, use 0.05 mol / L ammonium acetate solution as mobile phase B, perform gradient elution according to the regulations in Gradient 4 of Table 7; the column temperature is 30 °C, and the detection wavelength is 235 nm.

[0126] In Condition 1, the flow rate is 0.25 ml per minute, and the obtained chromatogram is shown in Figure 12 ; in Condition 2, the flow rate is 0.30 ml per minute, and the obtained chromatogram is shown in Figure 13 ; in Condition 3, the flow rate is 0.35 ml per minute, and the obtained chromatogram is shown in Figure 14As can be seen from the figure, at a flow rate of 0.25 ml / min, the signal peak at 21 min on the chromatogram is not a single component and is not completely separated. At flow rates of 0.30 ml / min and 0.35 ml / min, this peak is completely merged together, indicating that the separation effect is better at a flow rate of 0.25 ml / min.

[0127] 5. Selection of Column Temperature

[0128] The test solution was prepared using the same method as in Example 1.

[0129] Determination: Inject 2 μl of the above test solution into the liquid chromatograph. The chromatographic conditions are as follows: Use ACQUITY UPLC CSH C18 (2.1×100 mm, 1.7 μm) as the chromatographic column, use acetonitrile as mobile phase A, and use 0.05 mol / L ammonium acetate solution as mobile phase B, and perform gradient elution according to the regulations in Gradient 4 in Table 7; the flow rate is 0.30 ml per minute, and the detection wavelength is 235 nm.

[0130] In Condition 1, the column temperature is 25 °C, and the obtained chromatogram is shown in Figure 15 ; in Condition 2, the column temperature is 30 °C, and the obtained chromatogram is shown in Figure 16 ; in Condition 3, the column temperature is 35 °C, and the obtained chromatogram is shown in Figure 17 As can be seen from the figure, the signal peak at 20 min is completely separated at a column temperature of 25 °C, this peak is completely merged together at a column temperature of 30 °C, and it cannot be completely separated at a column temperature of 35 °C, indicating that the separation effect is better at a column temperature of 25 °C.

[0131] 6. Selection of Wavelength

[0132] The test solution was prepared using the same method as in Example 1.

[0133] Determination: Inject 2 μl of the above test solution into the liquid chromatograph. The chromatographic conditions are as follows: Use ACQUITY UPLC CSH C18 (2.1×100 mm, 1.7 μm) as the chromatographic column, use acetonitrile as mobile phase A, and use 0.05 mol / L ammonium acetate solution as mobile phase B, and perform gradient elution according to the regulations in Gradient 4 in Table 7; the column temperature is 30 °C, and the flow rate is 0.30 ml per minute. Perform 3D full wavelength scanning and select 4 wavelengths with more peak information: 226 nm ( Figure 18 ), 235 nm ( Figure 19 ), 274 nm ( Figure 20 ), 304 nm ( Figure 21) A comparison was made. As can be seen from the figure, the chromatographic peak signal is the largest at the absorption wavelength of 226 nm, but the baseline is uneven. At the absorption wavelength of 235 nm, the main chromatographic peak signal is more obvious and the baseline is relatively flat. At wavelengths of 274 nm and 304 nm, the absorption of the main chromatographic peaks in the first half is relatively small. Considering comprehensively, 235 nm is preferably selected as the absorption wavelength.

[0134] 7. Further optimization of the mobile phase gradient

[0135] The test solution was prepared using the same method as in Example 1.

[0136] Determination: Inject 2 μl of the above test solution into the liquid chromatograph. The chromatographic conditions are as follows: Use ACQUITY UPLC CSH C18 (2.1×100 mm, 1.7 μm) as the chromatographic column, use acetonitrile as mobile phase A, use 0.05 mol / L ammonium acetate solution as mobile phase B, the flow rate is 0.25 ml per minute, the column temperature is 25 °C, and the detection wavelength is 235 nm.

[0137] In Condition 1, the elution gradient uses Gradient 5 in Table 8, and the obtained chromatogram is shown in Figure 22 , and the chromatographic peak system suitability parameters are shown in Table 9; in Condition 2, the elution gradient uses Gradient 6 in Table 8 (the same as Table 1), and the obtained chromatogram is shown in Figure 23 , and the chromatographic peak system suitability parameters are shown in Table 10; it can be seen that under Gradient 5, the distance between Peak 6 and the adjacent impurity peak is relatively close and it is easily interfered. Under Gradient 6, the resolution of each chromatographic peak is better, the baseline is stable, and the interference of impurity peaks is small. Therefore, Gradient 6 is preferably selected as the mobile phase elution gradient.

[0138] Table 8

[0139]

[0140] Table 9

[0141]

[0142] Table 10

[0143]

[0144] 8. Selection of salt concentration

[0145] The test solution was prepared using the same method as in Example 1.

[0146] Determination: Inject 2 μl of the above-mentioned test solution into the liquid chromatograph, and the chromatographic conditions are as follows: Use ACQUITY UPLC CSH C18 (2.1×100 mm, 1.7 μm) as the chromatographic column, use acetonitrile as mobile phase A, and perform gradient elution according to the regulations in gradient 6 of Table 8; the column temperature is 25°C, the flow rate is 0.25 ml per minute, and the detection wavelength is 235 nm. In Condition 1, use 0.050 mol / L ammonium acetate solution as mobile phase B, and the chromatographic peak system suitability parameters are shown in Table 11; in Condition 2, use 0.025 mol / L ammonium acetate solution as mobile phase B, and the chromatographic peak system suitability parameters are shown in Table 12. It can be seen that when using 0.050 mol / L ammonium acetate solution, the separation effect of each chromatographic peak is slightly better than that of 0.025 mol / L ammonium acetate solution. It is preferred to use 0.050 mol / L ammonium acetate solution as the mobile phase.

[0147] Table 11

[0148]

[0149] Table 12

[0150]

[0151] Investigation on the pretreatment method of the test sample in Experimental Example 2

[0152] 1. Selection of extraction method

[0153] Preparation method of test sample 1: Take Dichroa febrifuga formula granules, grind them finely, take 0.2 g of the powder, add 25 ml of 70% vol methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain.

[0154] Preparation method of test sample 2: Take Dichroa febrifuga formula granules, grind them finely, take 0.2 g of the powder, add 25 ml of 70% vol methanol, heat in a water bath (95°C) and reflux for 30 minutes, let it cool, shake well, filter, and take the subsequent filtrate to obtain.

[0155] Determination: Inject 2 μl of each test sample solution into the liquid chromatograph, and the chromatographic conditions are as follows: Use octadecylsilane-bonded silica gel as the filler (ACQUITY UPLC CSH C18, 2.1×100 mm, 1.7 μm); use acetonitrile as mobile phase A and 0.050 mol / L ammonium acetate solution as mobile phase B, and perform gradient elution according to the regulations in Table 1; the flow rate is 0.25 ml per minute, the column temperature is 25°C, and the detection wavelength is 235 nm. The number of theoretical plates calculated based on the febrifugine peak should be not less than 5000. Figure 24 It is the chromatogram obtained under the ultrasonic extraction method. Figure 25It is the chromatogram obtained under the heating reflux method. The comparison of the chromatographic peak system suitability parameters is shown in Table 13. The two methods have little impact on the total peak area. Considering the convenience of operation of the two methods comprehensively, the ultrasonic extraction method is selected.

[0156] Table 13

[0157]

[0158] 2. Selection of extraction time

[0159] Preparation method of test sample: Take the Dichroa febrifuga formula granules, grind them finely, take 3 portions of the powder, each 0.2 g, add 25 ml of 70% vol methanol, and ultrasonically treat (power 250 W, frequency 40 kHz) for 30, 45, and 60 minutes respectively. Let it cool, shake well, filter, and take the continuous filtrate to obtain the test sample solution.

[0160] Determination: Inject 2 μl of each test sample solution into the liquid chromatograph. The chromatographic conditions are as follows: Using octadecylsilane-bonded silica gel as the filler (ACQUITY UPLC CSH C18, 2.1×100 mm, 1.7 μm); Using acetonitrile as mobile phase A and 0.050 mol / L ammonium acetate solution as mobile phase B, perform gradient elution according to the regulations in Table 1; The flow rate is 0.25 ml per minute, the column temperature is 25 °C, and the detection wavelength is 235 nm. The number of theoretical plates calculated based on the dichroine peak should be not less than 5000. Figure 26 It is the chromatogram obtained when the extraction time is 30 minutes. Figure 27 It is 45 minutes. Figure 28 It is 60 minutes. The comparison of the chromatographic peak system suitability parameters is shown in Table 14. It is found that the chromatographic peak area is larger and the system suitability parameters are relatively better when the extraction time is 45 - 60 minutes. The extraction time of 45 minutes is selected.

[0161] Table 14

[0162]

[0163] 3. Selection of extraction solvent

[0164] Preparation method of test sample: Take the Dichroa febrifuga formula granules, grind them finely, take 5 portions of the powder, each 0.2 g, and add 25 ml of ethanol, methanol, 70% vol methanol, 50% vol methanol, and 30% vol methanol respectively. Ultrasonically treat (power 250 W, frequency 40 kHz) for 45 minutes, let it cool, shake well, filter, and take the continuous filtrate to obtain the test sample solution.

[0165] Determination: Inject 2 μl of each test solution into the liquid chromatograph under the following chromatographic conditions: Use octadecylsilane chemically bonded silica gel as the filler (ACQUITY UPLC CSH C18, 2.1×100 mm, 1.7 μm); Use acetonitrile as mobile phase A and 0.050 mol / L ammonium acetate solution as mobile phase B, and perform gradient elution according to the regulations in Table 1; The flow rate is 0.25 ml per minute, the column temperature is 25 °C, and the detection wavelength is 235 nm. The number of theoretical plates calculated based on the febrifugine peak should be not less than 5000. Figures 29 - 33 They are chromatograms obtained using ethanol, methanol, 70% vol methanol, 50% vol methanol, and 30% vol methanol as extraction solvents respectively; The comparison of chromatographic peak system suitability parameters is shown in Table 15. Among them, when ethanol is used as the extraction solvent, characteristic peaks are missing, so the system suitability parameters are not given. When methanol is used for extraction, the peak shape is poor. The peak shapes of 30% vol methanol - 70% vol methanol are better. Considering comprehensively, when 70% vol methanol is used for extraction, the peak shape is better, the system suitability parameters are relatively better, and the response of characteristic peaks is larger. 70% vol methanol is preferably selected as the extraction solvent.

[0166] Table 15

[0167]

[0168]

[0169] 4. Selection of Sampling Quantity

[0170] Preparation method of test sample: Take the Dichroa febrifuga formula granules, grind them finely, take 3 portions of the powder, each 0.1 g, 0.2 g, and 0.5 g, add 25 ml of 70% vol methanol respectively, perform ultrasonic treatment (250 W, 40 kHz) for 45 min, take out, let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0171] Determination: Inject 2 μl of each test solution into the liquid chromatograph under the following chromatographic conditions: Use octadecylsilane chemically bonded silica gel as the filler (ACQUITY UPLC CSH C18, 2.1×100 mm, 1.7 μm); Use acetonitrile as mobile phase A and 0.050 mol / L ammonium acetate solution as mobile phase B, and perform gradient elution according to the regulations in Table 1; The flow rate is 0.25 ml per minute, the column temperature is 25 °C, and the detection wavelength is 235 nm. The number of theoretical plates calculated based on the febrifugine peak should be not less than 5000. Figure 34 It is the chromatogram obtained under the sampling quantity of 0.1 g. Figure 35 It is the chromatogram obtained under the sampling quantity of 0.2 g. Figure 36It is the chromatogram obtained under a sampling amount of 0.5 g. The comparison of the chromatographic peak system suitability parameters is shown in Table 16. It can be seen that when the sampling amount is between 0.1 g and 0.2 g, the peak area increases proportionally, indicating that when the sampling amount is between 0.1 g and 0.2 g, complete extraction can be achieved. Considering the response of the chromatographic peaks comprehensively, 0.2 g is preferably selected as the sampling amount of Dichroa febrifuga formula granules.

[0172] Table 16

[0173]

[0174]

[0175] 5. Selection of injection volume

[0176] Preparation method of test sample: Take Dichroa febrifuga formula granules, grind them finely, take 0.2 g of the powder, add 25 ml of 70% vol methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 45 minutes, take out, let it cool, shake well, filter, and take the continuous filtrate to obtain the test sample solution.

[0177] Determination: Inject 1 μl, 2 μl, 3 μl, and 5 μl of the test sample solution into the liquid chromatograph respectively. The chromatographic conditions are as follows: Using octadecylsilane-bonded silica gel as the filler (ACQUITY UPLC CSH C18, 2.1×100 mm, 1.7 μm); Using acetonitrile as mobile phase A and 0.050 mol / L ammonium acetate solution as mobile phase B, perform gradient elution according to the regulations in Table 1; The flow rate is 0.25 ml per minute, the column temperature is 25 °C, and the detection wavelength is 235 nm. The number of theoretical plates calculated based on the dichroine peak should be not less than 5000. Figure 37 It is the chromatogram obtained under an injection volume of 1 μl, Figure 38 It is the chromatogram obtained under an injection volume of 2 μl, Figure 39 It is the chromatogram obtained under an injection volume of 3 μl, Figure 40 It is the chromatogram obtained under an injection volume of 5 μl. The comparison of the chromatographic peak system suitability parameters is shown in Table 17. It can be seen that when the injection volume is 5 μl, overloading occurs in some chromatographic peaks. When the injection volume is 1 - 3 μl, the peak shape is good, and the peak height and peak width are relatively moderate. Considering comprehensively, the injection volume of 2 μl is preferably selected as the injection volume of the test sample solution.

[0178] Table 17

[0179]

[0180]

[0181] Experimental Example 3 Construction method, identification of characteristic peaks and specified values of characteristic chromatogram

[0182] (1) Construction method and specified values

[0183] Take the standard decoction of Dichroa febrifuga slices and Dichroa febrifuga formula granules (batch numbers: BT1, BT2, BT3, BT4, BT5, BT6, BT7, BT8, BT9, BT10, BT11, BT12, BT13, BT14, BT16, KL1, KL2, KL3) as test samples. Prepare the test sample solutions according to the preparation method in Example 1 (when preparing the test sample of the standard decoction of Dichroa febrifuga slices, use an equal amount of the standard decoction of Dichroa febrifuga slices to replace the Dichroa febrifuga formula granules). Detect the above test sample solutions under the chromatographic conditions in Example 1. Table 18 shows the determination results of the relative retention times of each characteristic chromatogram. Figure 41 It is the characteristic chromatogram of multiple batches of Dichroa febrifuga (where S1 - S18 from bottom to top are: the standard decoction of Dichroa febrifuga slices BT1, BT2, BT3, BT4, BT5, BT6, BT7, BT8, BT9, BT10, BT11, BT12, BT13, BT14, BT16, and Dichroa febrifuga formula granules KL1, KL2, KL3). It can be seen that the relative retention times of the samples are all within the required range.

[0184] Table 18

[0185]

[0186]

[0187] Six characteristic peaks should appear in the test sample chromatogram of the Dichroa febrifuga characteristic chromatogram. Among them, peak 1, peak 2, peak 3, and peak 4 should correspond to the retention times of the corresponding reference peaks of the reference substances. The peak corresponding to the reference peak of the febrifugine reference substance is the S peak. Calculate the relative retention times of each characteristic peak and the S peak, and its relative retention time should be within the range of ±10% of the specified value. The specified values are: 0.83 (peak 1), 1.10 (peak 3), 1.43 (peak 4), 1.64 (peak 5), 2.77 (peak 6). The fitting method of the control chromatogram of Dichroa febrifuga formula granules adopts Mark peak fitting, see Figure 42 ; the relative retention times of the control characteristic chromatogram are shown in Table 19, and the relative peak areas of the control characteristic chromatogram are shown in Table 20.

[0188] Table 19

[0189]

[0190] Table 20

[0191]

[0192] (2) Identification of characteristic peaks

[0193] Preparation of reference substance solution: Weigh appropriate amounts of anisodamine reference substance, febrifugine reference substance, 4-hydroxyquinazoline reference substance, and 7-hydroxycoumarin, and prepare reference substance solutions containing 44 μg of anisodamine per 1 ml, 70 μg of febrifugine per 1 ml, 34 μg of 4-hydroxyquinazoline per 1 ml, and 45 μg of 7-hydroxycoumarin per 1 ml respectively with 70% methanol.

[0194] Preparation of test solution: The test solution was prepared using the same method as in Example 1.

[0195] Determination: Inject 2 μl of each of the above-mentioned test solution and reference substance solutions into the liquid chromatograph. The chromatographic conditions are as follows: Use ACQUITY UPLC CSH C18 (2.1×100 mm, 1.7 μm) as the chromatographic column, acetonitrile as mobile phase A, and 0.05 mol / L ammonium acetate solution as mobile phase B, and perform gradient elution according to the regulations in Table 1; the flow rate is 0.25 ml per minute, the column temperature is 25 °C, and the detection wavelength is 235 nm.

[0196] The reference substance positioning chromatogram is shown in Figure 43 , in which, from bottom to top, are the febrifuge formula granules; febrifugine reference substance; anisodamine reference substance; 4-hydroxyquinazoline; 7-hydroxycoumarin. It can be seen that peaks 1, 2, 3, and 4 should correspond to the corresponding reference substance peaks of 4-hydroxyquinazoline, febrifugine, anisodamine, and 7-hydroxycoumarin. The response of the febrifugine peak is relatively high, and it is a relatively specific component in febrifuge. Therefore, febrifugine is selected as the S peak of the characteristic chromatogram of febrifuge.

[0197] Methodology verification in Experimental Example 4

[0198] 1. Precision experiment: Take the test solution of febrifuge formula granules prepared by the method in Example 1, and inject it continuously for 6 times under the chromatographic conditions in Example 1. Measure the relative retention time and relative peak area of each characteristic peak. The results show that the RSD of the relative retention time of each characteristic peak is less than 2%, and the RSD of the relative peak area is less than 5%, indicating that the precision of the instrument is good.

[0199] 2. Repeatability investigation: Take the febrifuge formula granules used in Example 1, prepare 6 test solutions in parallel according to the test solution preparation method in Example 1, inject them under the chromatographic conditions in Example 1, and measure the relative retention time and relative peak area of each characteristic peak. The results show that the RSD of the relative retention time of each characteristic peak is less than 2%, and the RSD of the relative peak area is less than 5%, indicating that the repeatability of this method is good.

[0200] 3. Intermediate precision investigation: Take the test solution of Dichroa febrifuga formula granules prepared by the method in the same Example 1. Three different personnel inject samples separately on the same instrument according to the chromatographic conditions in Example 1, and calculate the relative retention time and relative peak area of each characteristic peak. The results show that the RSD of the relative retention time of each characteristic peak is less than 2%, and the RSD of the relative peak area is less than 5%, indicating that the intermediate precision of this method among different personnel is good.

[0201] 4. Specificity investigation: Take negative granules (maltodextrin), prepare them according to the test solution preparation method in Example 1 as the negative control solution; detect according to the chromatographic conditions in Example 1, and the chromatogram of the negative control solution obtained is shown in Figure 44 , and the results show that the negative has no interference.

[0202] 5. Stability investigation: Take 6 portions of the test solution of Dichroa febrifuga formula granules prepared by the method in the same Example 1. According to the chromatographic conditions in Example 1, inject samples at 0, 2, 6, 10, 15, and 24 hours after the test solution is prepared, and measure the relative retention time and relative peak area of each characteristic peak. The results show that the RSD of the relative retention time of each characteristic peak is less than 2%, and the RSD of the relative peak area is less than 5%, indicating that the test solution is stable within 24 hours and meets the determination requirements.

[0203] 6. Robustness investigation:

[0204] (1) Different flow rates: Take 3 portions of the test solution of Dichroa febrifuga formula granules prepared by the method in the same Example 1. Detect according to the chromatographic conditions in Example 1 except for the flow rate, with different flow rates (0.25 ml / min, 0.20 ml / min, 0.30 ml / min). The results show that the RSD of the relative retention time of each characteristic peak is less than 5%, and the RSD of the relative peak area is less than 10%. The slight change in the flow rate has little influence on the separation of characteristic peaks, and this method has good robustness for different flow rates.

[0205] (2) Different column temperatures: Take 3 portions of the test solution of Dichroa febrifuga formula granules prepared by the method in the same Example 1. Detect according to the chromatographic conditions in Example 1 except for the column temperature, with different column temperatures (25 °C, 20 °C, 30 °C). The results show that the RSD of the relative retention time of each characteristic peak is less than 2%, and the RSD of the relative peak area is less than 5%. The slight change in the column temperature has little influence on the separation of characteristic peaks, and this method has good robustness for different column temperatures.

[0206] (3) Different instruments: Take the test solution of Dichroa febrifuga formula granules prepared by the method in the same Example 1. According to the chromatographic conditions in Example 1, use Agilent 1290 and Thermo Vanquish chromatographs respectively for determination, and analyze the separation effect of each characteristic peak. The results show that the RSD of the relative retention time of each characteristic peak is less than 2%, and the RSD of the relative peak area is less than 5%, indicating good reproducibility on different instruments.

[0207] (4) Different chromatographic columns: Take the test solution of Dichroa febrifuga formula granules prepared by the method in the same Example 1. According to the chromatographic conditions in Example 1, use different chromatographic columns (ACQUITY UPLC CSH C18 2.1×100mm, 1.7μm and ACQUITY UPLC BEH C18 2.1×100mm, 1.7μm) respectively for determination. The relative retention times are shown in Table 21, and the relative peak areas are shown in Table 22. Analyze the separation effect of each characteristic peak. The results show that due to the large difference in the fillers of different chromatographic columns, the separation effect of chromatographic peaks is affected. It is recommended to fix the chromatographic column as ACQUITY UPLC CSH C18 2.1×100mm, 1.7μm.

[0208] Table 21

[0209]

[0210] Table 22

[0211]

[0212] Obviously, the above examples are only for clear illustration and not limitations on the implementation methods. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation methods here. And the obvious changes or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. A method for constructing a characteristic spectrum of Radix Dichroae and its preparations, characterized in that: The steps include: (1) Preparation of test solution; (2) The sample solution is tested by high performance liquid chromatography, using octadecylsilane bonded silica gel as the filler, the mobile phase comprises an aqueous solution containing ammonium acetate and acetonitrile, the gradient elution program comprises: 0 → 1-16 min → 35-40 min, and the volume percentage of acetonitrile in the mobile phase is: 5%-6% → 5%-11% → 28%-30%.

2. The construction method according to claim 1, characterized in that: Step (2) also satisfies at least one of the following 1) to 4): 1) The detection wavelength is 226-304 nm, the flow rate is 0.20-0.35 mL / min, and the column temperature is 20-35° C.; optionally, the detection wavelength is 235 nm, the flow rate is 0.25-0.30 mL / min, and the column temperature is 25-30° C.; 2) A chromatographic column with a size of 2.1 mm × 100 mm and 1.7 μm is used in the detection process; optionally, an ACQUITY UPLC CSHC18 or ACQUITY UPLC BEH C18 chromatographic column is used; 3) The injection volume is 1 to 5 μL; optionally, 1 to 3 μL; 4) The concentration of ammonium acetate in the aqueous solution containing ammonium acetate is 0.025-0.050 mol / L; optionally, it is 0.050 mol / L.

3. The construction method according to claim 1 or 2, characterized in that: The gradient elution program includes: 0→2min→11min→16min→40min, and the volume percentage of acetonitrile in the mobile phase is: 5%→5%→10%→11%→28%; or includes: 0→1min→13min→35min, and the volume percentage of acetonitrile in the mobile phase is: 6%→6%→10%→30%.

4. The construction method according to any one of claims 1 to 3, characterized in that: The step (1) includes weighing a Radix Dichroae test sample, adding a solvent for extraction to obtain an extract, separating the solid from the liquid, and obtaining a liquid, which is the test sample solution.

5. The construction method according to claim 4, characterized in that: The step (1) also satisfies any one or more of the following A to E: A. The mass volume ratio of Changshan test sample to solvent is 0.1-0.2 g:25 mL; B. The extraction method includes ultrasonic extraction or reflux extraction; optionally, ultrasonic extraction; further optionally, the power of the ultrasonic extraction is 200-300W and the frequency is 30-50kHz; C. The extraction time is 30 to 60 minutes; optionally, the extraction time is 45 to 60 minutes; D. The solid-liquid separation is selected from centrifugation or filtration; E. The solvent is selected from one or more of methanol or methanol aqueous solution; optionally, it is an aqueous solution of 30% to 70% by volume of methanol; further optionally, it is an aqueous solution of 70% by volume of methanol.

6. The construction method according to any one of claims 1 to 5, characterized in that: The construction method further comprises the step of preparing a reference solution using at least one solubilizing agent selected from the group consisting of anomalous shanoprine, febrifugine, 4-hydroxyquinazoline, and 7-hydroxycoumarin, and the step of detecting the reference solution by high performance liquid chromatography to obtain a reference spectrum according to the construction method of any one of claims 1 to 5; And / or, using Changshan reference medicinal material as a reference material to prepare a reference material solution according to step (1) in the construction method described in any one of claims 1 to 5, and detecting the reference material solution by high performance liquid chromatography in the construction method described in any one of claims 1 to 5 to obtain a reference material spectrum.

7. The construction method according to claim 6, characterized in that: Each 1 mL of the reference solution contains 30 to 70 μg of each reference substance; optionally, each 1 mL of the reference solution contains 40 to 50 μg of anomalous shan alkaloids; optionally, each 1 mL of the reference solution contains 65 to 75 μg of febrifugine; optionally, each 1 mL of the reference solution contains 30 to 40 μg of 4-hydroxyquinazoline; optionally, each 1 mL of the reference solution contains 40 to 50 μg of 7-hydroxycoumarin; And / or, the solvent used in the preparation of the reference solution is a methanol aqueous solution with a volume percentage of 30% to 70%.

8. The construction method according to any one of claims 1 to 7, characterized in that: The characteristic spectrum of the febrifuga and its preparation has 6 common characteristic peaks, peak 2 corresponds to the retention time of the febrifugax reference substance peak, the peak corresponding to the febrifugax reference substance peak is the S peak, and the relative retention times of peaks 1, 3 to 6 and the S peak are within the range of ±10% of the specified value; the specified value is: 0.83 (peak 1), 1.10 (peak 3), 1.43 (peak 4), 1.64 (peak 5), and 2.77 (peak 6).

9. Application of the method for constructing the characteristic spectrum of Radix Dichroae and its preparations according to any one of claims 1 to 8 in the quality inspection of Radix Dichroae pharmaceutical preparations.

10. A quality detection method for Radix Dichroae and its preparation, characterized in that: The method comprises the steps of comparing the characteristic spectrum of the Radix Dichroae product to be tested with the control characteristic spectrum of Radix Dichroae and its preparations; the characteristic spectrum of the Radix Dichroae product to be tested is constructed using the Radix Dichroae product to be tested according to the construction method described in any one of claims 1 to 8, and the control characteristic spectrum of Radix Dichroae and its preparations is selected from any one of the following (1) to (3): (1) It has 6 common characteristic peaks, peak 2 corresponds to the retention time of the febrifugine reference peak, the peak corresponding to the febrifugine reference peak is the S peak, and the relative retention times of peaks 1, 3 to 6 and the S peak are within the range of ±10% of the specified values; the specified values ​​are: 0.83 (peak 1), 1.10 (peak 3), 1.43 (peak 4), 1.64 (peak 5), and 2.77 (peak 6); (2) Characteristic spectrum of Radix Dichroae and / or its preparation obtained by the construction method according to any one of claims 1 to 8 using a single batch or multiple batches of Radix Dichroae and / or its preparation; (3) Using multiple batches of Radix Dichroae and / or its preparations to obtain characteristic spectra according to the construction method described in any one of claims 1 to 8, a control characteristic spectra is prepared by the average value or median method.

Images