Method for establishing and detecting characteristic chromatogram of compound Haqing injection

Abstract

The invention belongs to the technical field of quality detection of traditional Chinese medicines, and particularly relates to a specific chromatogram establishment and detection method of a compound Haqing injection. The method for establishing the characteristic spectrum comprises the following steps: a, preparing a reference substance solution, namely precisely weighing an amygdalin reference substance, a calycosin-7-glucoside reference substance and a praeruptorin A reference substance, and adding methanol for dissolving to obtain the reference substance solution; b, preparation of a test solution: precisely measuring the compound Haqing injection, and preparing the test solution by adopting a solid-phase extraction instrument; and c, measuring: respectively measuring the test solution and the reference solution, injecting into a liquid chromatograph, and recording a chromatogram to obtain a characteristic spectrum formed by 10 common characteristic peaks. The method is stable, high in precision and good in reproducibility, a rapid and comprehensive detection means is provided for quality control of the compound Haqing injection, and a scientific experimental basis is provided for research of a quality evaluation system of the compound Haqing injection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine quality detection, and particularly relates to a characteristic spectrum establishment and detection method of compound Heqing injection. BACKGROUND

[0002] The main components of compound Heqing injection are toad, astragalus, ginkgo, bitter almond, purple yam, radix peucedani, schisandra chinensis, aconite, black pepper, etc. Among them, astragalus has the effects of tonifying qi and consolidating the exterior, and toad, bitter almond and purple yam have the effects of relieving cough and asthma, and radix peucedani, schisandra chinensis and aconite have the effects of warming and resolving phlegm and fluid. In particular, toad contains catecholamine compounds and indole alkyl compounds, and has the effects of slowing heart rate, strengthening heart and hormone-like anti-inflammatory. In clinical practice, it is widely used for lung deficiency cough, asthma and sputum, old chronic bronchitis, emphysema, asthmatic bronchitis, etc., and also has a preventive effect on recurrent colds.

[0003] Compound Heqing injection contains many prescription medicinal materials, and the components are complex and diverse. The existing drug standard only includes thin layer identification items, and an effective quality control method has not been established, so the quality of compound Heqing injection cannot be comprehensively reflected.

[0004] Therefore, it is necessary to establish a simple and easy, accurate, reproducible and comprehensive detection method for the components of compound Heqing injection, which is very necessary for the quality control and evaluation of compound Heqing injection. SUMMARY

[0005] The purpose of the present application is to provide a method for establishing the characteristic spectrum of compound Heqing injection, which is beneficial to comprehensive quality control.

[0006] The present application provides a method for establishing the characteristic spectrum of compound Heqing injection, which comprises the following steps:

[0007] a. Preparation of reference solution: accurately weigh bitter almond glycoside reference substance, calycosin-7-glucoside reference substance and praeparata peucedani radix reference substance, mix and dissolve in methanol to obtain the reference solution;

[0008] b. Preparation of test solution: accurately weigh compound Heqing injection, and prepare the test solution by using a solid phase extraction instrument;

[0009] c. Determination: inject the test solution and the reference solution into a liquid chromatograph, and record the chromatogram to obtain a characteristic spectrum composed of 10 common characteristic peaks.

[0010] In the method for establishing the characteristic spectrum of compound Heqing injection, in step a, 1 mL of the reference solution contains 0.03-0.5 mg of bitter almond glycoside, 5-100 μg of calycosin-7-glucoside and 10-100 μg of praeparata peucedani radix.

[0011] Further, the reference solution contains 0.3 mg of amygdalin, 25 μg of calycosin-7-glucoside and 15 μg of praeparatoside A per 1 mL of the solution.

[0012] In the method for establishing the characteristic spectrum of the compound Gehqing injection, in step b, the preparation of the test solution is as follows: the compound Gehqing injection is placed in a sample tube of a solid phase extraction instrument, and a solid phase extraction column is used to sequentially perform activation, sample loading, elution, and elution, the eluate is concentrated to dryness, dissolved, filtered, and the filtrate is collected, thereby obtaining the test solution.

[0013] Further, the solid phase extraction column is any one of Sep-Pak Vac 6cc (1g) C18 Cartridge, SelectCore C18, INN OTEG C18, SelectCore HR-C18, SelectCore PA SPE Cartridge, Bond Elut HLB, or SelectCore HLB SPE Cartridge.

[0014] Preferably, the solid phase extraction column is SelectCore HLB SPE Cartridge.

[0015] Further, in step b, the eluate is concentrated at a temperature of 60-80°C.

[0016] Preferably, the eluate is concentrated at a temperature of 75°C.

[0017] In the method for establishing the characteristic spectrum of the compound Gehqing injection, in step c, the liquid chromatograph is an ultra-high performance liquid chromatograph.

[0018] Further, the mobile phase used by the ultra-high performance liquid chromatograph is as follows:

[0019] Water is used as the mobile phase A, and methanol is used as the mobile phase B, gradient elution is adopted, and the gradient elution program is as follows:

[0020] 0-5 min, the volume fraction of the mobile phase A is kept at 95%, and the volume fraction of the mobile phase B is kept at 5%;

[0021] 5-25 min, the volume fraction of the mobile phase A decreases from 95% to 88%, and the volume fraction of the mobile phase B increases from 5% to 12%;

[0022] 25-60 min, the volume fraction of the mobile phase A decreases from 88% to 52%, and the volume fraction of the mobile phase B increases from 12% to 48%;

[0023] 60-80 min, the volume fraction of the mobile phase A decreases from 52% to 30%, and the volume fraction of the mobile phase B increases from 48% to 70%;

[0024] 80-81 min, the volume fraction of the mobile phase A decreases from 30% to 10%, and the volume fraction of the mobile phase B increases from 70% to 90%;

[0025] 81-85 min, the volume fraction of the mobile phase A is kept at 10%, and the volume fraction of the mobile phase B is kept at 90%;

[0026] 85-86 min, the volume fraction of the mobile phase A increases from 10% to 95%, and the volume fraction of the mobile phase B decreases from 90% to 5%;

[0027] 86-90 min, the volume fraction of the mobile phase A is kept at 95%, and the volume fraction of the mobile phase B is kept at 5%; the chromatographic column is filled with octadecylsilane-bonded silica gel.

[0028] Further, in step c, the detection condition of the liquid chromatography is that the flow rate is 0.2-0.5 mL / min; the column temperature is 20-40 ℃; and the detection wavelength is 200-400 nm.

[0029] Preferably, the flow rate is 0.3 mL / min; the column temperature is 35 ℃; and the detection wavelength is 0-20 min: 250 nm; 20-30 min: 220 nm; 30-62 min: 250 nm; 62-90 min: 220 nm.

[0030] The purpose of the present application is also to provide a detection method of the compound Jiaqing injection, comprising the following steps: precisely taking the compound Jiaqing injection test sample solution, using a liquid chromatograph to detect, and comparing the obtained chromatogram with the characteristic spectrum obtained by the method for establishing the characteristic spectrum of the compound Jiaqing injection.

[0031] Further, the preparation of the test sample solution is to place the compound Jiaqing injection in a solid-phase extraction instrument sample tube, and sequentially perform activation, sample loading, elution and elution procedures using a solid-phase extraction column to obtain the test sample solution.

[0032] The preparation of the test sample solution is to place the compound Jiaqing injection in a solid-phase extraction instrument sample tube, and sequentially perform activation, sample loading, elution and elution procedures using a solid-phase extraction column, collect the eluate, concentrate to dryness, dissolve, filter, and take the filtered solution to obtain the test sample solution.

[0033] Further, 10 mL of the compound Jiaqing injection is placed in a solid-phase extraction instrument sample tube.

[0034] Further, the temperature for concentrating the eluate is 60-80 ℃.

[0035] Preferably, the temperature of the eluent concentration is 75℃.

[0036] Further, the liquid chromatograph is an ultra-high performance liquid chromatograph, and the mobile phase is water as mobile phase A and methanol as mobile phase B, and a gradient elution is adopted, and the gradient elution program is as follows:

[0037] 0-5 min, the volume fraction of mobile phase A is kept at 95%, and the volume fraction of mobile phase B is 5%;

[0038] 5-25 min, the volume fraction of mobile phase A is decreased from 95% to 88%, and the volume fraction of mobile phase B is increased from 5% to 12%;

[0039] 25-60 min, the volume fraction of mobile phase A is decreased from 88% to 52%, and the volume fraction of mobile phase B is increased from 12% to 48%;

[0040] 60-80 min, the volume fraction of mobile phase A is decreased from 52% to 30%, and the volume fraction of mobile phase B is increased from 48% to 70%;

[0041] 80-81 min, the volume fraction of mobile phase A is decreased from 30% to 10%, and the volume fraction of mobile phase B is increased from 70% to 90%;

[0042] 81-85 min, the volume fraction of mobile phase A is kept at 10%, and the volume fraction of mobile phase B is 90%;

[0043] 85-86 min, the volume fraction of mobile phase A is increased from 10% to 95%, and the volume fraction of mobile phase B is decreased from 90% to 5%;

[0044] 86-90 min, the volume fraction of mobile phase A is kept at 95%, and the volume fraction of mobile phase B is 5%; and the chromatographic column is filled with octadecylsilane bonded silica gel.

[0045] Further, the detection condition of the liquid chromatography is that the flow rate is 0.2-0.5 mL / min, the column temperature is 20-40℃, and the detection wavelength is 200-400 nm.

[0046] Preferably, the flow rate is 0.3 mL / min, the column temperature is 35℃, and the detection wavelength is 0-20 min: 250 nm, 20-30 min: 220 nm, 30-62 min: 250 nm, and 62-90 min: 220 nm.

[0047] The present application has the following beneficial effects:

[0048] The application adopts the ultra-high performance liquid chromatography to establish the characteristic spectrum of compound Heqing injection, and determines the common peaks of adenosine, L-bitter apricot kernel glycoside, bitter apricot kernel glycoside, calycosin-7-glucoside, mutamycin, cytisine, bufalin, schisantherin, and praeparatum peucedani, which correspond to the medicinal materials of bitter apricot kernel, astragalus, toad, schisandra, and peucedanum, the peak separation degree is good, the characteristic peak information of the multiple medicinal materials in the compound Heqing injection can be more fully and comprehensively reflected, the method is stable, high in precision and good in reproducibility, and provides a rapid and comprehensive detection means for the quality control of the compound Heqing injection, and provides a scientific experimental basis for the research on the quality evaluation system of the compound Heqing injection.

[0049] The application further provides a detection method of the compound Heqing injection, the adenosine, L-bitter apricot kernel glycoside, bitter apricot kernel glycoside, calycosin-7-glucoside, mutamycin, cytisine, bufalin, schisantherin, and praeparatum peucedani in the compound Heqing injection can be detected simultaneously by optimizing the ultra-high performance liquid chromatography detection condition and the preparation method of the test solution, the one-sidedness of determining the overall quality of the preparation by only detecting one or two chemical components is avoided, the detection method can more fully and scientifically evaluate the quality of the compound Heqing injection, so that the quality and curative effect of the product are guaranteed. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 The characteristic spectrum of the compound Heqing injection established in Example 1 (10 characteristic peaks are presented, wherein peak 1 is adenosine, peak 2 is L-bitter apricot kernel glycoside, peak 3 is bitter apricot kernel glycoside, peak 4 is calycosin-7-glucoside, peak 5 is mutamycin, peak 6 is cytisine, peak 8 is bufalin, peak 9 is schisantherin, and peak 10 is praeparatum peucedani).

[0051] Figure 2 The spectrum of the solid phase small column type investigation in Example 2

[0052] Figure 3 The spectrum of the sample loading amount investigation in Example 2.

[0053] Figure 4 The spectrum of the elution solvent amount investigation in Example 2.

[0054] Figure 5 The spectrum of the elution solvent amount investigation in Example 2.

[0055] Figure 6 The spectrum of the elution solvent amount investigation in Example 2.

[0056] Figure 7 The spectrum of the chromatographic column investigation in Example 2.

[0057] Figure 8Investigation of column temperature for Example 2.

[0058] Figure 9 Investigation of mobile phase for Example 2.

[0059] Figure 10 Characteristic peak attribution of compound Gehqing injection in Example 2.

[0060] Figure 11 Characteristic spectrum of 10 batches of samples in Example 2 (sample batch number: 230811, 230812, 230813, 230814, 230815, 230816, 250202, 250203, 250204, 231014). DETAILED DESCRIPTION

[0061] The application will be further described in detail through specific examples. The drugs and reagents involved in the specific embodiment part are all obtained by marketing.

[0062] Example 1

[0063] 1.1 Instruments and reagents

[0064] 1.1.1 Instruments

[0065] Agilent 1290 ultra-high performance liquid chromatograph, DAD detector, OpenLab 2.7 work station, Agilent Technologies (China) Co., Ltd.; chromeleon 7 work station, Thermo Fisher Scientific (China) Co., Ltd.; FS 360 full-automatic solid-phase extraction instrument, Ruike Group (Xiamen) Co., Ltd.; AutoEVA-60 full-automatic parallel concentration instrument, Ruike Group (Xiamen) Co., Ltd.

[0066] Mettler-Toledo XS105DU analytical balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.

[0067] 1L filter device, 0.2 μm microporous filter membrane, Tianjin Jinteng Experimental Equipment Co., Ltd.

[0068] 1.1.2 Reagents

[0069] Methanol, AR, batch number: 2025041501, Chengdu Kelong Chemical Reagent Co., Ltd.; water, batch number: 20241225G, Guangzhou Watsons Food and Beverage Co., Ltd.; methanol, HPLC, batch number: WXBF4280 V, Sigma Company; SelectCore HLBSPE Cartridge solid-phase small column (500 mg / 6 mL, 60 μm, batch number: HLB41025, NanoChrom Company.

[0070] Compound Heqing injection, specification: 2 mL per bottle, batch number: 230811, 230812, 230813, 230814, 230815, 230816, 250202, 250203, 250204, 231014, good doctor pharmaceutical co., LTD.

[0071] 1.1.3 Control

[0072] L-amynin (batch number: AFCB2207, purity: 99.59%) was purchased from Chengdu Epha Co., Ltd. ;

[0073] amygdalin (batch number: Y08O10K96808, purity: 98.3%) was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd. ;

[0074] calycosin (batch number: Y11J10H92789, purity: 98%) was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd. ;

[0075] bufotalin (batch number: P28M10F84299, purity: 98.2%) was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd. ;

[0076] adenosine (batch number: 110879-202204, purity: 99.4%);

[0077] calycosin glucoside (batch number: 111920-201907, purity: 96.8%);

[0078] schisantherin A (batch number: 110857-202316, purity: 99.2%);

[0079] praeruptorin A (batch number: 111711-201904, purity: 99.4%) was purchased from China Institute for Drug Control;

[0080] mutamycin (batch number: PRF9060501, purity: 98%) was purchased from Chengdu Pureway Technology Co., Ltd.

[0081] 1.2 Method and results

[0082] 1.2.1 Chromatographic conditions and system suitability

[0083] octadecylsilane bonded silica gel as the filler (column length of 100 mm, inner diameter of 2.1 mm, particle size of 1.8 μm); water as mobile phase A, methanol as mobile phase B, according to table 1 for gradient elution; flow rate of 0.3 mL / min, column temperature of 30℃; detection wavelength of multi-wavelength switching, not less than 600000 according to amygdalin.

[0084] Table 1 mobile phase gradient elution conditions

[0085]

[0086] 1.2.2 Preparation of the test solution:

[0087] Take 10 mL of Compound Clam Shell Injection, batch number: 231014, and place it in the sample tube of the solid phase extraction instrument. Place the corresponding solid phase extraction column (SelectCore HLB SPE Cartridge 500mg / 6ml (60μm)) in the corresponding position, and perform the column activation, sample loading, rinsing, and elution procedures in sequence according to the program in Table 2.

[0088] Table 2 Solid Phase Extraction Elution Procedure

[0089] Command Solvent Drain Flow rate mL / min Volume (mL) Wash sample channel Methanol / / / Wash plunger Methanol / / / Activate Methanol Organic waste 2 10 Activate Water Waste water 2 10 Load / Waste water 1 10 Wash sample vial Water Waste water 1 10 Rinse Water Waste water 1 40 Wash syringe pump Methanol / 60 3 Elute Methanol Collect 1 20 End / / / /

[0090] Collect the eluent and concentrate it to dryness using a fully automated parallel concentrator under nitrogen blowing (water bath temperature 75℃). Dissolve it in methanol and bring the volume to 2 mL. Filter the solution and collect the filtrate to obtain the final product.

[0091] 1.2.3 Preparation of the reference solution

[0092] Take appropriate amounts of amygdalin reference standard, verbascoside glucoside reference standard, and angelica sinensis reference standard, accurately weigh them, and add methanol to prepare a mixed solution containing 0.3 mg amygdalin, 25 μg verbascoside glucoside, and 15 μg angelica sinensis per 1 mL, as the reference solution.

[0093] 1.2.4 Determination Method

[0094] Accurately pipette 1 μL each of the test solution and the reference solution into the ultra-high performance liquid chromatograph, measure and record the chromatograms to obtain the characteristic chromatogram of Compound Clam Shell Injection. The results are shown in the figure. Figure 1 .

[0095] Example 2: Investigation of process parameters

[0096] 2.1 Investigation of the preparation method of the test solution

[0097] 2.1.1 Investigation of Solid Column Types

[0098] The batch number of Compound Clam Shell Injection is 231014.

[0099] This invention investigated the purification and enrichment effects of seven types of solid-phase columns on the test samples, as follows:

[0100] Take 25 mL of Compound Clam Shell Injection and place it in the sample tube of the solid phase extraction instrument. Place the Sep-Pak Vac6cc(1g) C18 Cartridge solid phase column in the corresponding position and prepare the test solution according to "1.2.2".

[0101] Take Compound Fageqing Injection 25 mL, place it in the solid phase extraction instrument sample tube, place SelectCore C18 solid phase column at the corresponding position, and prepare the test solution according to the method in item 1.2.2.

[0102] Take Compound Fageqing Injection 25 mL, place it in the solid phase extraction instrument sample tube, place INN OTEC C18 solid phase column at the corresponding position, and prepare the test solution according to the method in item 1.2.2.

[0103] Take Compound Fageqing Injection 25 mL, place it in the solid phase extraction instrument sample tube, place SelectCore HR-C18 solid phase column at the corresponding position, and prepare the test solution according to the method in item 1.2.2.

[0104] Take Compound Fageqing Injection 25 mL, place it in the solid phase extraction instrument sample tube, place SelectCore PA SPE Cartridge solid phase column at the corresponding position, and prepare the test solution according to the method in item 1.2.2.

[0105] Take Compound Fageqing Injection 25 mL, place it in the solid phase extraction instrument sample tube, place Bond Elut HLB solid phase column at the corresponding position, and prepare the test solution according to the method in item 1.2.2.

[0106] Take Compound Fageqing Injection 25 mL, place it in the solid phase extraction instrument sample tube, place SelectCore HLB SPE Cartridge solid phase column at the corresponding position, and prepare the test solution according to the method in item 1.2.2.

[0107] The above detection chromatographic conditions use acetonitrile and 0.1% phosphoric acid solution as the mobile phase, 0-60 min, the volume fraction of acetonitrile rises from 10% to 80%, the volume fraction of 0.1% phosphoric acid solution drops from 90% to 20%, the flow rate is 0.3 mL / min, the column temperature is 30°C, and the detection wavelength is 254 nm.

[0108] The results show that the SelectCore HLB SPE Cartridge solid phase column retains relatively more characteristic peaks, so SelectCore HLB SPE Cartridge is selected as the solid phase column for sample purification of the characteristic spectrum. The results are shown in Table 2. Figure 2 .

[0109] 2.1.2 Sample amount investigation

[0110] The batch number of Compound Fageqing Injection is 231014.

[0111] The application investigates three sample loading amounts as follows:

[0112] Take compound Haqing injection, filter, and take the filtrate, namely the compound Haqing injection.

[0113] Take 10 mL of compound Haqing injection, place it in a solid phase extraction instrument sample tube, place the corresponding solid phase column at the corresponding position, collect the "sample loading" effluent, and obtain 10 mL of effluent.

[0114] Take 20 mL of compound Haqing injection, place it in a solid phase extraction instrument sample tube, place the corresponding solid phase column at the corresponding position, collect the "sample loading" effluent, and obtain 20 mL of effluent.

[0115] Take 30 mL of compound Haqing injection, place it in a solid phase extraction instrument sample tube, place the corresponding solid phase column at the corresponding position, collect the "sample loading" effluent, and obtain 30 mL of effluent.

[0116] The results show that a sample loading amount of 10 mL can meet the detection of the characteristic spectrum. Therefore, the sample loading amount is selected to be 10 mL. See Figure 3 .

[0117] 2.1.3 Investigation of elution solvent types

[0118] The application investigates four elution solvents as follows

[0119] Prepare the test sample solution according to "1.2.2", namely the water sample.

[0120] Prepare the test sample solution according to "1.2.2", wherein the "elution solvent is 4% ammonia water solution", namely the 4% ammonia water sample.

[0121] Prepare the test sample solution according to "1.2.2", wherein the "elution solvent is 1% NaHCO3 solution", namely the 1% NaHCO3 sample.

[0122] Prepare the test sample solution according to "1.2.2", wherein the "elution solvent is 5% acetic acid solution", namely the 5% acetic acid sample.

[0123] The results show that water and 4% ammonia water solution have equivalent purification effects on compound Haqing injection. Considering safety and economy, water is selected as the elution solvent for the characteristic spectrum. See Figure 4 .

[0124] 2.1.4 Investigation of elution solvent amounts

[0125] The application investigates four amounts of elution solvent (water) as follows:

[0126] Prepare the test sample solution according to "1.2.2", wherein the "elution volume (mL)" is 10 mL, namely the elution-10 mL.

[0127] Prepare the test sample solution according to the item "1.2.2", wherein the "elution volume (mL)" is 20 mL, to obtain elution-20 mL.

[0128] Prepare the test sample solution according to the item "1.2.2", wherein the "elution volume (mL)" is 30 mL, to obtain elution-30 mL.

[0129] Prepare the test sample solution according to the item "1.2.2", wherein the "elution volume (mL)" is 40 mL, to obtain elution-40 mL.

[0130] The results show that the elution effect is the best when 40 mL of water is used for elution. Therefore, the amount of elution solvent (water) is selected to be 40 mL. See Figure 5 .

[0131] 2.1.5 Elution solvent amount investigation

[0132] The present application investigates three amounts of elution solvent (methanol), as follows

[0133] Prepare the test sample solution according to the item "1.2.2", wherein the "elution volume (mL)" is 10 mL, to obtain elution-10 mL.

[0134] Prepare the test sample solution according to the item "1.2.2", wherein the "elution volume (mL)" is 20 mL, to obtain elution-20 mL.

[0135] Prepare the test sample solution according to the item "1.2.2", wherein the "elution volume (mL)" is 30 mL, to obtain elution-30 mL.

[0136] The results show that all characteristic peaks can be eluted by using 10 mL of methanol for elution. In order to achieve more sufficient elution effect, the amount of elution solvent (methanol) is selected to be 20 mL. See Figure 6 .

[0137] The mobile phase of 2.1.2-2.1.5 is 0.1% phosphoric acid as mobile phase A and methanol as mobile phase B, and the gradient program is the same as that in the item "1.2.1".

[0138] 2.2 Selection of chromatographic conditions

[0139] 2.2.1 Selection of chromatographic column

[0140] The present application investigates three kinds of chromatographic columns, namely ACQUITY HSS T3 C18 2.1mmx100mm, 1.8μm; ACQUITY HSS C18 2.1mmx100mm, 1.8μm; ACQUITY BEH C18 2.1mmx100mm, 1.7μm. The results show that the above three kinds of chromatographic columns can achieve the separation effect of chromatographic peaks. Therefore, the chromatographic column takes octadecylsilane bonded silica gel as the filler (2.1x100mm, 1.8μm). See Figure 7 .

[0141] 2.2.2 Column temperature investigation

[0142] The present application investigates three different column temperatures, namely 30℃, 35℃, 40℃. The results show that the column temperature has little effect on the characteristic spectrum, and the characteristic peak separation is slightly better at 30℃. Therefore, the column temperature is selected to be 30℃. See Figure 8 .

[0143] 2.2.3 Investigation of mobile phase

[0144] The present application investigates three kinds of mobile phases, namely methanol-0.1% phosphoric acid, methanol-0.1% formic acid, methanol-water. The gradient program of the three kinds of mobile phases is according to the results of "1.2.1". The results show that the separation effect of methanol-0.1% phosphoric acid and methanol-water is equivalent. The preparation of methanol-water is simple and convenient, therefore, methanol-water is selected as the mobile phase of the characteristic spectrum. See Figure 9 .

[0145] The mobile phase of 2.2.1~2.2.2 is 0.1% phosphoric acid as mobile phase A, and methanol as mobile phase B, and the gradient program is according to "1.2.1".

[0146] 2.3 Identification of characteristic peaks

[0147] Take compound Gehqing injection (batch number: 231014), and prepare the test sample solution according to the method in "1.2.2". According to the detection method in "1.2.1", precisely take 1μL of the test sample solution, adenosine control sample solution, L-bitter apricot kernel glycoside control sample solution, bitter apricot kernel glycoside control sample solution, calycosin-7-glucoside control sample solution, mutamycin glycoside control sample solution, calycosin control sample solution, bufalin control sample solution, schisantherin control sample solution, praeroside control sample solution and 9 mixed standard control sample solution, and inject them into the liquid chromatograph, and measure and record the chromatogram. Compare the chromatograms of the above-mentioned control samples and the test sample, and identify the chemical components in the sample according to the retention time. The results show that peak 1 is adenosine, peak 2 is L-bitter apricot kernel glycoside, peak 3 is bitter apricot kernel glycoside, peak 4 is calycosin-7-glucoside, peak 5 is mutamycin glycoside, peak 6 is calycosin, peak 8 is bufalin, peak 9 is schisantherin, and peak 10 is praeroside. SeeFigure 10 .

[0148] 2.4 Specification of characteristic peak

[0149] Take 10 batches of compound Gaiqing injection (batch number: 230811, 230812, 230813, 230814, 230815, 230816, 250202, 250203, 250204, 231014), prepare the test sample solution according to the method under item “1.2.2”. According to the detection method under item “1.2.1”, the retention time of each characteristic peak is determined, and the RSD value of the relative retention time is calculated. The results are shown in Table 3, Table 4, Figure 11 The relative retention time RSD is between 0.00% and 0.11%, and the stability and batch consistency of the quality of 10 batches of compound Gaiqing injection are good.

[0150] Table 3 Retention time (min) of 10 batches of samples

[0151] Lot 1 2 3 4 5 6 7 8 9 10 231014 10.904 27.217 28.031 45.613 53.470 58.921 63.768 66.622 71.222 78.639 230811 10.774 26.976 27.773 45.514 53.360 58.828 63.673 66.600 70.950 78.453 230812 10.810 27.009 27.815 45.534 53.368 58.837 63.673 66.606 70.918 78.433 230813 10.819 26.985 27.786 45.537 53.370 58.846 63.677 66.619 70.893 78.424 230814 10.865 27.022 27.823 45.553 53.375 58.856 63.384 66.636 70.869 78.410 230815 10.847 27.007 27.805 45.549 53.366 58.847 63.672 66.624 70.853 78.398 230816 10.854 27.004 27.802 45.549 53.371 58.859 63.683 66.639 70.843 78.390 250202 10.816 26.934 27.735 45.524 53.352 58.843 63.670 66.634 70.819 78.379 250203 10.838 26.953 27.746 45.517 53.333 58.843 63.652 66.620 70.804 78.362 250204 10.835 26.914 27.707 45.500 53.320 58.807 63.636 66.605 70.788 78.347

[0152] Table 4 Relative retention time of 10 batches of samples

[0153] Lot 1 2 3 4 5 6 7 8 9 10 231014 0.239 0.597 0.615 1.000 1.172 1.292 1.398 1.461 1.561 1.724 230811 0.237 0.593 0.610 1.000 1.172 1.293 1.399 1.463 1.559 1.724 230812 0.237 0.593 0.611 1.000 1.172 1.292 1.398 1.463 1.557 1.723 230813 0.238 0.593 0.610 1.000 1.172 1.292 1.398 1.463 1.557 1.722 230814 0.239 0.593 0.611 1.000 1.172 1.292 1.391 1.463 1.556 1.721 230815 0.238 0.593 0.610 1.000 1.172 1.292 1.398 1.463 1.556 1.721 230816 0.238 0.593 0.610 1.000 1.172 1.292 1.398 1.463 1.555 1.721 250202 0.238 0.592 0.609 1.000 1.172 1.293 1.399 1.464 1.556 1.722 250203 0.238 0.592 0.610 1.000 1.172 1.293 1.398 1.464 1.556 1.722 250204 0.238 0.592 0.609 1.000 1.172 1.292 1.399 1.464 1.556 1.722 Mean 0.24 0.59 0.61 1.00 1.17 1.29 1.40 1.46 1.56 1.72 RSD % 0.25 0.11 0.10 0.00 0.02 0.02 0.16 0.03 0.07 0.05

[0154] The test sample characteristic spectrum should present 10 characteristic peaks, among which peak 3, peak 4, and peak 10 should correspond to the retention time of the corresponding reference substance amygdalin, calycosin-7-glucoside, and praepariflorun A, and peak 4 corresponding to the calycosin-7-glucoside reference substance is an S peak. The relative retention time of the remaining characteristic peaks to the S peak should be within ±10% of the specified value. The specified value is peak 1 (0.24), peak 2 (0.59), peak 4 (S) (1.00), and peak 5 (1.17), peak 6 (1.29), peak 7 (1.40), peak 8 (1.46), peak 9 (1.56).

[0155] Methodology verification of Example 3

[0156] 3.1 Injection precision

[0157] According to the detection method under item “1.2.1”, take 1 μL of the three mixed reference substance solutions of amygdalin, calycosin-7-glucoside, and praepariflorun A under item “1.2.3” and continuously inject 5 times, record the peak area, and calculate the RSD value of the peak area. The results are shown in Table 5. The peak area RSD is between 0.38% and 0.93%, indicating good injection precision.

[0158] Table 5 Precision-peak area

[0159] Number Amygdalin Calycosin-7-glucoside Methyl-8-glucoside 1 565.7 184.9 179.8 2 561.9 184.3 177.4 3 567.2 186.3 180.3 4 562.8 187.1 178.0 5 563.8 188.6 179.0 Mean 564.3 186.2 178.9 RSD (%) 0.38 0.93 0.68

[0160] 3.2 Reproducibility test

[0161] Take compound Geqing injection (batch number: 231014), prepare 6 test sample solutions according to the method under item "1.2.2". Detect according to the detection method under item "1.2.1", record the retention time of each characteristic peak, calculate the RSD value of relative retention time, the results are shown in Table 6, Table 7. The relative retention time RSD is between 0.00% and 0.12%, indicating that the method has good reproducibility.

[0162] Table 6 Reproducibility test - retention time (min)

[0163] Number 1 2 3 4(S) 5 6 7 8 9 10 1 10.904 27.217 28.031 45.613 53.470 58.921 63.768 66.622 71.222 78.639 2 10.877 27.200 28.016 45.616 53.467 58.923 63.768 66.630 71.195 78.632 3 10.854 27.155 27.969 45.598 53.442 58.900 63.747 66.625 71.132 78.598 4 10.832 27.116 27.922 45.571 53.417 58.881 63.720 66.617 71.081 78.566 5 10.904 27.149 27.955 45.578 53.419 58.887 63.720 66.624 71.049 78.535 6 10.878 27.112 27.916 45.571 53.409 58.880 63.712 66.623 71.022 78.514

[0164] Table 7 Reproducibility test - relative retention time

[0165] Number 1 2 3 4(S) 5 6 7 8 9 10 1 0.239 0.597 0.615 1.000 1.172 1.292 1.398 1.461 1.561 1.724 2 0.238 0.596 0.614 1.000 1.172 1.292 1.398 1.461 1.561 1.724 3 0.238 0.596 0.613 1.000 1.172 1.292 1.398 1.461 1.560 1.724 4 0.238 0.595 0.613 1.000 1.172 1.292 1.398 1.462 1.560 1.724 5 0.239 0.596 0.613 1.000 1.172 1.292 1.398 1.462 1.559 1.723 6 0.239 0.595 0.613 1.000 1.172 1.292 1.398 1.462 1.558 1.723 Mean 0.24 0.60 0.61 1.00 1.17 1.29 1.40 1.46 1.56 1.72 RSD (%) 0.25 0.12 0.13 0.00 0.01 0.01 0.01 0.04 0.07 0.03

[0166] 3.3 Stability test

[0167] Take compound Geqing injection (batch number: 231014), prepare test sample solution according to the method under item "1.2.2", detect according to the detection method under item "1.2.1", measure at 0h, 3h, 6h, 9h, 12h, 15h, 18h, 24h, 36h, 48h, record the retention time, peak area, and calculate the RSD value of relative retention time and peak area, the results are shown in Table 8, Table 9, Table 10. The relative retention time RSD is between 0.00% and 0.28%, the peak area RSD is between 0.83% and 2.11%, indicating that the test sample solution is stable within 48h.

[0168] Table 8 Stability test - retention time (min)

[0169] Time / h 1 2 3 4(S) 5 6 7 8 9 10 0 10.904 27.217 28.031 45.613 53.470 58.921 63.768 66.622 71.222 78.639 3 10.872 27.170 27.980 45.589 53.442 58.895 63.745 66.617 71.158 78.615 6 10.857 27.134 27.943 45.575 53.425 58.885 63.726 66.617 71.105 78.585 9 10.882 27.146 27.953 45.591 53.437 58.904 63.739 66.636 71.067 78.546 12 10.899 27.160 27.969 45.593 53.429 58.904 63.732 66.635 71.039 78.526 15 10.892 27.116 27.920 45.581 53.420 58.900 63.725 66.640 71.012 78.509 18 10.894 27.121 27.923 45.600 53.426 58.899 63.721 66.635 71.016 78.509 24 10.864 27.082 27.884 45.581 53.406 58.872 63.693 66.605 71.003 78.483 30 10.825 27.040 27.842 45.533 53.568 58.830 63.674 66.593 70.975 78.459 36 10.872 27.051 27.842 45.557 53.379 58.859 63.684 66.631 70.883 78.416 48 10.772 26.886 27.671 45.493 53.304 58.782 63.614 66.586 70.779 78.335

[0170] Table 9 Stability test - relative retention time

[0171] Time / h 1 2 3 4(S) 5 6 7 8 9 10 0 0.239 0.597 0.615 1.000 1.172 1.292 1.398 1.461 1.561 1.724 3 0.238 0.596 0.614 1.000 1.172 1.292 1.398 1.461 1.561 1.724 6 0.238 0.595 0.613 1.000 1.172 1.292 1.398 1.462 1.560 1.724 9 0.239 0.595 0.613 1.000 1.172 1.292 1.398 1.462 1.559 1.723 12 0.239 0.596 0.613 1.000 1.172 1.292 1.398 1.462 1.558 1.722 15 0.239 0.595 0.613 1.000 1.172 1.292 1.398 1.462 1.558 1.722 18 0.239 0.595 0.612 1.000 1.172 1.292 1.397 1.461 1.557 1.722 24 0.238 0.594 0.612 1.000 1.172 1.292 1.397 1.461 1.558 1.722 30 0.238 0.594 0.611 1.000 1.176 1.292 1.398 1.463 1.559 1.723 36 0.239 0.594 0.611 1.000 1.172 1.292 1.398 1.463 1.556 1.721 48 0.237 0.591 0.608 1.000 1.172 1.292 1.398 1.464 1.556 1.722 Mean 0.24 0.59 0.61 1.00 1.17 1.29 1.40 1.46 1.56 1.72 RSD % 0.29 0.26 0.28 0.00 0.12 0.01 0.03 0.06 0.12 0.06

[0172] Table 10 Stability test - peak area

[0173]

Claims

1. A method for establishing the characteristic chromatogram of compound Jiaqing injection, characterized in that, It comprises the following steps: a. Preparation of reference solution: accurately weigh amygdalin control, calycosin-7-glucoside control, and praeparatio praefuji control, and add methanol to dissolve, and then obtain; b. Preparation of test solution: accurately measure compound Fulingqing injection, and prepare test solution by using solid phase extraction instrument; c. Determination: inject test solution and reference solution into liquid chromatograph, and record chromatogram, and then obtain characteristic spectrum composed of 10 common characteristic peaks.

2. The method of claim 1, wherein: In step a, the reference solution contains 0.03-0.5 mg amygdalin, 5-100 μg calycosin-7-glucoside, and 10-100 μg praeparatio praefuji per 1 mL.

3. The method of claim 1, wherein: In step b, the preparation of test solution is to place compound Fulingqing injection in solid phase extraction instrument sample tube, and then activate, sample, elute, and elute by using solid phase extraction column, and then collect eluent, concentrate to dryness, dissolve, filter, and then obtain.

4. The method of claim 1, wherein: In step c, the liquid chromatograph is ultra-high performance liquid chromatograph.

5. The method of claim 1, wherein: In step c, the mobile phase used by the liquid chromatograph is: water as mobile phase A and methanol as mobile phase B, gradient elution, and the program of gradient elution is: 0-5 min, keep the volume fraction of mobile phase A at 95% and the volume fraction of mobile phase B at 5%; 5-25 min, the volume fraction of mobile phase A decreases from 95% to 88% and the volume fraction of mobile phase B increases from 5% to 12%; 25-60 min, the volume fraction of mobile phase A decreases from 88% to 52% and the volume fraction of mobile phase B increases from 12% to 48%; 60-80 min, the volume fraction of mobile phase A decreases from 52% to 30% and the volume fraction of mobile phase B increases from 48% to 70%; 80-81 min, the volume fraction of mobile phase A decreases from 30% to 10% and the volume fraction of mobile phase B increases from 70% to 90%; 81-85 min, keep the volume fraction of mobile phase A at 10% and the volume fraction of mobile phase B at 90%; 85-86 min, the volume fraction of mobile phase A increases from 10% to 95% and the volume fraction of mobile phase B decreases from 90% to 5%; 86-90 min, keep the volume fraction of mobile phase A at 95% and the volume fraction of mobile phase B at 5%; and the chromatographic column uses octadecylsilane bonded silica gel as filler.

6. The method of claim 1, wherein: In step c, the detection conditions of liquid chromatography are as follows: flow rate is 0.2-0.5 mL / min; column temperature is 20-40 ℃; and detection wavelength is 200-400 nm; preferably, flow rate is 0.3 mL / min; column temperature is 35 ℃; and detection wavelength is 0-20 min: 250 nm; 20-30 min: 220 nm; 30-62 min: 250 nm; 62-90 min: 220 nm. It comprises the following steps: accurately take compound Fulingqing injection test solution, determine by using liquid chromatograph, and compare the obtained chromatogram with the characteristic spectrum obtained by the method for establishing characteristic spectrum of compound Fulingqing injection, and then determine.

7. A detection method of compound Jieqing injection, characterized in that: ​ 8. The detection method of claim 7, wherein: The preparation of the test solution is to place the compound Heqing injection in the solid phase extraction instrument sample tube, and use the solid phase extraction column to perform the activation, sample loading, elution and elution procedures in sequence.

9. The detection method of claim 8, wherein: The liquid chromatograph is an ultra-high performance liquid chromatograph, the mobile phase is water as the mobile phase A and methanol as the mobile phase B, gradient elution is adopted, and the gradient elution procedure is as follows: 0-5 min, the volume fraction of the mobile phase A is kept at 95%, and the volume fraction of the mobile phase B is kept at 5%; 5-25 min, the volume fraction of the mobile phase A decreases from 95% to 88%, and the volume fraction of the mobile phase B increases from 5% to 12%; 25-60 min, the volume fraction of the mobile phase A decreases from 88% to 52%, and the volume fraction of the mobile phase B increases from 12% to 48%; 60-80 min, the volume fraction of the mobile phase A decreases from 52% to 30%, and the volume fraction of the mobile phase B increases from 48% to 70%; 80-81 min, the volume fraction of the mobile phase A decreases from 30% to 10%, and the volume fraction of the mobile phase B increases from 70% to 90%; 81-85 min, the volume fraction of the mobile phase A is kept at 10%, and the volume fraction of the mobile phase B is kept at 90%; 85-86 min, the volume fraction of the mobile phase A increases from 10% to 95%, and the volume fraction of the mobile phase B decreases from 90% to 5%; 86-90 min, the volume fraction of the mobile phase A is kept at 95%, and the volume fraction of the mobile phase B is kept at 5%; and the chromatographic column is filled with octadecylsilane bonded silica gel.

10. The method of claim 7, wherein: The detection conditions of the liquid chromatography are as follows: the flow rate is 0.2-0.5 mL / min; the column temperature is 20-40 ℃; and the detection wavelength is 200-400 nm; preferably, the flow rate is 0.3 mL / min; the column temperature is 35 ℃; and the detection wavelength is 0-20 min: 250 nm; 20-30 min: 220 nm; 30-62 min: 250 nm; 62-90 min: 220 nm. ​

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