A method for constructing the fingerprint of Qingning Powder freeze-dried powder and the fingerprint

Abstract

The present invention provides a method for constructing a fingerprint of Qingning powder freeze-dried powder and the fingerprint, which includes the following steps: S1. Take the Qingning powder freeze-dried powder, add a methanol aqueous solution, and perform ultrasonic extraction to obtain a test solution; S2. Take the test solution, inject it into a high-performance liquid chromatograph, perform chromatographic analysis, and record the chromatogram; S3. Perform mass spectrometry analysis on the test solution to obtain a mass spectrometry result diagram; S4. According to the chromatogram and the mass spectrometry result diagram, construct a fingerprint. The method of the present invention comprehensively detects the types and quantities of the main chemical components contained in the traditional Chinese medicine Qingning powder. Based on this, the establishment of the fingerprint analysis and detection of Qingning powder is fast, convenient and accurate, objectively and comprehensively characterizes the quality of Qingning powder, and the method has the advantages of simplicity, good stability, high precision and good reproducibility.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine detection, and particularly relates to a method for constructing a fingerprint of Qingning Powder freeze-dried powder and the fingerprint thereof. Background Art

[0002] "Qingning Powder" is derived from Volume 1 of "Zhizhi Xiao'er Fang", and is clinically used to treat diseases such as fright and cough caused by the accumulation of heat in the heart and lungs of children. According to the specific pathogenesis of children's cough with phlegm-heat stagnating in the lungs, based on the conventional Western medicine treatment, the addition and subtraction of Qingning Powder is adopted, and satisfactory treatment effects are obtained. The prescription composition of "Qingning Powder": Mulberry Bark (fried with honey), Sweet Semen Lepidii (slightly fried), Red Poria (fried with wine), Plantain Seed (fried), each in equal parts, and Licorice Root (roasted) is halved. It has the effects of clearing heat and detoxifying, etc. Clinically, it is generally used to treat symptoms such as cough caused by the accumulation of heat in the lungs of children, and proper use can also play the role of promoting urination. However, at present, there are few studies on the component analysis of Qingning Powder, and no basis for quality control and evaluation can be provided. Summary of the Invention

[0003] In order to solve the above problems of the prior art, the present invention provides a method for constructing a fingerprint of Qingning Powder freeze-dried powder and the fingerprint thereof, which has important significance for the component identification, quality evaluation and quality standard formulation of Qingning Powder.

[0004] The present invention is realized through the following technical solutions:

[0005] A method for constructing a fingerprint of Qingning Powder freeze-dried powder, comprising the following steps:

[0006] S1. Take the Qingning Powder freeze-dried powder, add a methanol aqueous solution, and perform ultrasonic extraction to obtain a test solution;

[0007] S2. Take the test solution, inject it into a high performance liquid chromatograph, perform chromatographic analysis, and record the chromatogram;

[0008] S3. Perform mass spectrometry analysis on the test solution to obtain a mass spectrometry result diagram;

[0009] S4. According to the chromatogram and the mass spectrometry result diagram, construct a fingerprint.

[0010] Preferably, in S1, the volume concentration of the methanol aqueous solution is 80%.

[0011] Preferably, in S1, the ultrasonic time is 30 - 60 min, and more preferably 60 min.

[0012] Preferably, in S2, the chromatographic column is a Kromasil 100 - 5 - C18 chromatographic column.

[0013] Preferably, in S2, the chromatographic conditions are as follows: the mobile phase is acetonitrile - 0.1% phosphoric acid aqueous solution; ultraviolet-visible absorption detector is used for multi-wavelength switching detection, and the detection wavelengths are: 0 - 15 min, 254 nm; 15 - 43 min, 280 nm; 43 - 120 min, 254 nm; the gradient elution program is: acetonitrile volume 10% from 0 to 10 min; acetonitrile volume 10% - 20% from 10 to 20 min; acetonitrile volume 20% - 23% from 20 to 30 min; acetonitrile volume 23 - 30% from 30 to 40 min; acetonitrile volume 30% - 40% from 40 to 60 min; acetonitrile volume 40% - 45% from 60 to 80 min; acetonitrile volume 45% - 58% from 80 to 100 min; acetonitrile volume 58% - 63% from 100 to 120 min, when good resolution of each chromatographic peak in the fingerprint can be achieved.

[0014] Preferably, in S2, the flow rate of the mobile phase during chromatographic analysis is 0.6 - 1 mL / min, more preferably 0.6 mL / min.

[0015] Preferably, S4 is: importing the chromatogram obtained in S2 into the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, respectively performing data import, multi-point correction and data matching on the chromatogram of the test solution, obtaining the fingerprint and performing similarity analysis;

[0016] According to the mass spectrometry result diagram, determine the chemical components of each chromatographic peak in the fingerprint, which are: peak 1 is isoliquiritigenin / liciritigenin, peak 2 is isorhamnetin, peak 3 is liquiritin, peak 4 is sinapine thiocyanate, peak 5 is β-daucosterol, peak 6 is pachymic acid, peak 7 is dehydrotumulosic acid / 3-epidehydrotumulosic acid, peak 8 is ammonium glycyrrhizinate, peak 9 is quercetin, peak 10 is neochlorogenic acid / chlorogenic acid, peak 11 is licorinchalcone A, peak 12 is morin G, peak 13 is geniposidic acid, peak 14 is sanggenin, peak 15 is apigenin.

[0017] The fingerprint of Qingning Powder freeze-dried powder obtained by the described construction method.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention discloses a fingerprint detection method for Qingning Powder freeze-dried powder, a classic pediatric formula. According to the structural and property characteristics of the active ingredients contained in Qingning Powder, by screening conditions such as extraction method, mobile phase, column temperature, and flow rate, a fingerprint determination method was established and methodological investigations were carried out. A fingerprint of Qingning Powder was established based on multiple batches of samples, including a fingerprint of 15 characteristic peaks for the five traditional Chinese medicines of Mori Cortex, Lepidii Semen, Poria Rubra, Plantaginis Semen, and Glycyrrhizae Radix et Rhizoma in the formula. The obtained chromatograms have a relatively high similarity; the stability, repeatability, and precision are calculated using their relative retention times and relative peak areas, and the RSD values of the relative retention times and relative peak areas are both less than 5.0%. The fingerprint detection method for Qingning Powder provided by the present invention has good separation of each chromatographic peak, a stable baseline, and a good peak shape, and can comprehensively reflect the types and quantities of chemical components contained therein. Using the method of the present invention to comprehensively detect the types and quantities of the main chemical components contained in the traditional Chinese medicine Qingning Powder, the fingerprint analysis and detection of Qingning Powder are established quickly, conveniently, accurately, objectively and comprehensively characterize the quality of Qingning Powder, and the method has the advantages of simplicity, good stability, high precision, and good reproducibility. Description of the Drawings

[0020] Figure 1 It is the HPLC chromatogram of the test sample of Qingning Powder after extraction by ultrasonic for 60 min with 80% methanol in Example 1 of the present invention;

[0021] Figure 2 It is the HPLC chromatogram of the test sample of Qingning Powder obtained at a detection wavelength of 254 nm in Example 2 of the present invention;

[0022] Figure 3 It is the HPLC chromatogram of the test sample of Qingning Powder obtained at a detection wavelength of 275 nm in Example 3 of the present invention;

[0023] Figure 4 It is the HPLC chromatogram of the test sample of Qingning Powder obtained at a detection wavelength of 280 nm in Example 4 of the present invention;

[0024] Figure 5 It is the HPLC chromatogram of the test sample of Qingning Powder obtained at a flow rate of 0.6 mL / min in Example 5 of the present invention;

[0025] Figure 6 It is the HPLC chromatogram of the test sample of Qingning Powder obtained at a flow rate of 1.0 mL / min in Example 6 of the present invention;

[0026] Figure 7 It is the HPLC chromatogram of the test sample of Qingning Powder obtained in Example 7 during the optimization process of the present invention;

[0027] Figure 8 It is the HPLC chromatogram of the test sample of Qingning Powder obtained in Example 8 during the optimization process of the present invention;

[0028] Figure 9 This is the HPLC chromatogram obtained from Example 9 during the optimization process of the Qingning Powder test sample of the present invention;

[0029] Figure 10 This is the mass spectrum of isoliquiritigenin / licoricein of the present invention;

[0030] Figure 11 This is the mass spectrum of rhamnetin of the present invention;

[0031] Figure 12 This is the mass spectrum of liquiritin of the present invention;

[0032] Figure 13 This is the mass spectrum of sinapine thiocyanate of the present invention;

[0033] Figure 14 This is the mass spectrum of β - daucosterol of the present invention;

[0034] Figure 15 This is the mass spectrum of pinetumic acid of the present invention;

[0035] Figure 16 This is the mass spectrum of dehydrotumulosic acid / 3 - epi - dehydrotumulosic acid of the present invention;

[0036] Figure 17 This is the mass spectrum of ammonium glycyrrhizinate of the present invention;

[0037] Figure 18 This is the mass spectrum of quercetin of the present invention;

[0038] Figure 19 This is the mass spectrum of neochlorogenic acid / chlorogenic acid of the present invention;

[0039] Figure 20 This is the mass spectrum of licorinchalcone A of the present invention;

[0040] Figure 21 This is the mass spectrum of morusin G of the present invention;

[0041] Figure 22 This is the mass spectrum of geniposidic acid of the present invention;

[0042] Figure 23 This is the mass spectrum of sanggenin of the present invention;

[0043] Figure 24 This is the mass spectrum of apigenin of the present invention;

[0044] Figure 25 This is the fingerprint chromatogram of 15 batches of test samples based on Qingning Powder of the present invention. Detailed implementation manners

[0045] To enable those skilled in the art to better understand the solution of the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0046] The classical pediatric famous prescription Qingning Powder of the present invention is made from five medicinal materials: Cortex Mori (fried with honey), Semen Lepidii (slightly fried), Poria Rubra (fried with wine), Plantago Seed (fried), and Glycyrrhiza Radix Preparata.

[0047] The method for constructing the fingerprint spectrum of the freeze-dried powder of the classical pediatric famous prescription Qingning Powder of the present invention includes the following steps:

[0048] S1. Preparation of the test solution:

[0049] Precisely weigh the freeze-dried powder of Qingning Powder in different batches, place it in a stoppered conical flask, add 20 mL of methanol solution, extract by ultrasonic wave, take the filtrate and filter it through a 0.45 μm microporous filter membrane to obtain the test solution;

[0050] S2. Precisely pipette 15 μL of the test solution in S1 and inject it into a high-performance liquid chromatograph, and record the chromatogram from 0 to 120 min;

[0051] S3. Export the chromatogram of the test solution obtained in S2 and import it into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (version 2004A). After data import, multi-point correction and data matching for the chromatogram of the test solution respectively, the fingerprint spectrum is obtained and similarity analysis is carried out;

[0052] S4. To determine the chemical components in the fingerprint, the above-mentioned test solution was subjected to mass spectrometry analysis using a DIONEX UltiMate 3000 ultra-high performance liquid chromatography-Orbitrap mass spectrometer. The test conditions were electrospray ionization (ESI), with a spray voltage of 3500 V, a sheath gas flow rate of 40 arb, an auxiliary gas flow rate of 10 arb, an auxiliary gas temperature of 300 °C, a capillary temperature of 300 °C, a full scan mode, and a mass-to-charge ratio scanning range of m / z 100 - 1300. The total ion current chromatogram and the mass spectrometry result chromatograms of 15 chemical components were obtained. The detection data were imported into the Xcalibur software and entered the Qual Browser interface, and data analysis was performed according to the peak emergence of the chemical components. Respectively, peak 1 was isoliquiritigenin / liciritigenin at 14.90 min, peak 2 was isorhamnetin at 21.68 min, peak 3 was liquiritin at 24.71 min, peak 4 was sinapine thiocyanate at 25.98 min, peak 5 was β-daucosterol at 27.39 min, peak 6 was pinetumic acid at 34.35 min, peak 7 was dehydrotumulosic acid / 3-epidehydrotumulosic acid at 43.27 min, peak 8 was ammonium glycyrrhizinate at 45.97 min, peak 9 was quercetin at 53.32 min, peak 10 was neochlorogenic acid / chlorogenic acid at 56.32 min, peak 11 was licinchalcone A at 58.35 min, peak 12 was morin G at 61.37 min, peak 13 was geniposidic acid at 70.99 min, peak 14 was sanggenon O at 85.24 min, and peak 15 was apigenin at 102.64 min.

[0053] The preparation method of the test solution described in S1 is as follows: Weigh 1.0 g of the Qingning powder freeze-dried powder in 15 batches accurately and place it in a stoppered conical flask. Add 20 mL of 80% methanol solution, extract ultrasonically for 60 min, take the filtrate and filter it through a 0.45 μm microporous filter membrane to obtain the test solution.

[0054] The liquid chromatography conditions in S2 are as follows: Chromatographic column: Kromasil 100-5-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile (A) and 0.1% phosphoric acid aqueous solution (B), gradient elution, multi-wavelength switching detection by ultraviolet-visible absorption detector, detection wavelengths: 0 - 15 min, 254 nm; 15 - 43 min, 280 nm; 43 - 120 min, 254 nm; column temperature 30 °C; flow rate 0.6 mL / min; injection volume: 15 μL; elution program: 0 - 10 min, 10% A; 10 - 20 min, 10% - 20% A; 20 - 30 min, 20% - 23% A; 30 - 40 min, 23 - 30% A; 40 - 60 min, 30 - 40% A; 60 - 80 min, 40 - 45% A; 80 - 100 min, 45 - 58% A; 100 - 120 min, 58 - 63% A.

[0055] 2. Optimization of fingerprint detection:

[0056] S1. Optimization in the preparation of sample solution

[0057] In the present invention, different extraction methods (ultrasonic time 30 min, ultrasonic time 45 min, ultrasonic time 60 min) and different extraction solvents (ultrapure water, 80% methanol aqueous solution (volume concentration), methanol) were experimentally compared. The results showed that the chromatogram components obtained by ultrasonic extraction for 60 min were relatively comprehensive and had good resolution. Therefore, the method of ultrasonic extraction for 60 min was adopted; in the investigation of extraction solvents, it was found that the chromatogram of the 80% methanol aqueous solution extract had the most information and the highest component content; so 80% methanol aqueous solution was selected for extraction.

[0058] S2. Optimization of chromatographic conditions

[0059] In the present invention, the ultraviolet-visible absorption detector was used to investigate the detection wavelengths, and chromatograms at 254 nm, 275 nm, and 280 nm were extracted. It was found that when the detection wavelength conditions were: 0 - 15 min, 254 nm; 15 - 43 min, 280 nm; 43 - 120 min, 254 nm, the chromatogram contained the most comprehensive information and had a stable baseline. Therefore, this method was selected as the detection wavelength condition.

[0060] In the present invention, the flow rates (0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 1.0 mL / min) were screened, and it was found that when the flow rate was 0.6 mL / min, the separation effect of each substance was better. Therefore, the flow rate of 0.6 mL / min was maintained.

[0061] The present invention compared the elution effects of multiple different elution systems, namely methanol-water, acetonitrile-water, methanol-acetonitrile-water, methanol-0.1% phosphoric acid aqueous solution, acetonitrile-0.1% phosphoric acid aqueous solution, acetonitrile-0.05% phosphoric acid aqueous solution, acetonitrile-0.2% phosphoric acid aqueous solution, acetonitrile-0.1% formic acid, acetonitrile-0.1% glacial acetic acid, at different gradients. The results showed that when acetonitrile and 0.1% phosphoric acid aqueous solution were used as the mobile phase, the separation effect of each component in Qingning Powder was better. Therefore, acetonitrile and 0.1% phosphoric acid aqueous solution were finally selected as the mobile phase.

[0062] After determining the optimal mobile phase composition, the present invention screened the optimal gradient elution program through a large number of experiments. It was found that when the acetonitrile volume was 10% from 0 to 10 min; 10% - 20% from 10 to 20 min; 20% - 23% from 20 to 30 min; 23 - 30% from 30 to 40 min; 30% - 40% from 40 to 60 min; 40% - 45% from 60 to 80 min; 45% - 58% from 80 to 100 min; 58% - 63% from 100 to 120 min, good resolution of each chromatographic peak in the fingerprint could be achieved.

[0063] The following will describe the implementation scheme of the present invention in detail with reference to the examples. For those not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0064] The instruments and reagents used in the examples are as follows:

[0065] 1. Instruments and test drugs are shown in Table 1

[0066] Table 1 Instruments and test drugs used in the present invention

[0067]

[0068] 2. Drugs and reagents are shown in Table 2

[0069] Table 2 Drugs and reagents used in the present invention

[0070]

[0071] The following takes Qingning Powder as an example to illustrate the present invention in detail through specific examples.

[0072] Example 1

[0073] A method for constructing the fingerprint of freeze-dried powder of Qingning Powder, a classic pediatric formula, includes the following steps:

[0074] S1. Preparation of the test solution of Qingning Powder:

[0075] Accurately weigh 1.0 g of lyophilized powder of Qingningsan Powder from batch 15 and place it in a stoppered conical flask, add 20 mL of 80% methanol solution, perform ultrasonic extraction for 60 min, and filter the filtrate through a 0.45 μm microporous filter membrane to obtain the test solution;

[0076] S2. Chromatographic conditions

[0077] Chromatographic column: Kromasil 100-5-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% phosphoric acid aqueous solution, gradient elution, UV-visible absorption detector multi-wavelength switching detection, detection wavelength: 0-15 min, 254 nm; 15-43 min, 280 nm; 43-120 min, 254 nm; column temperature 30 ° C; flow rate 0.6 mL / min; injection volume: 15 μL; elution program is as shown in Table 3.

[0078] Table 3 Liquid chromatography elution program

[0079]

[0080] S3, the chromatogram of the Qingning powder test solution obtained in S2 was exported and imported into the Chinese medicine chromatographic fingerprint similarity evaluation system (2004A version), and the chromatogram of the test solution was subjected to data import, multi-point correction and data matching, and 15 batches of fingerprints were obtained ( Figure 25 , Figure 1 for Figure 5 one of the fingerprints), and similarity analysis was performed (Table 4);

[0081] Table 4 Similarity between each batch of samples and the common pattern

[0082]

[0083] 4. To determine the chemical components in the fingerprint, the above test solution was subjected to mass spectrometry analysis to obtain the total ion flow diagram and the mass spectrum results of 15 chemical components ( Figures 10 - 24) Import the test data into Xcalibur software, enter the Qual Browser interface, and perform data analysis according to the peak emergence of chemical components. The 1st peak is isoliquiritigenin / liciritigenin at 14.90 min, the 2nd peak is isorhamnetin at 21.68 min, the 3rd peak is liquiritin at 24.71 min, the 4th peak is sinapine thiocyanate at 25.98 min, the 5th peak is β-daucosterol at 27.39 min, the 6th peak is pinipeolide at 34.35 min, the 7th peak is dehydrotumulosic acid / 3-epi-dehydrotumulosic acid at 43.27 min, the 8th peak is ammonium glycyrrhizinate at 45.97 min, the 9th peak is quercetin at 53.32 min, the 10th peak is neochlorogenic acid / chlorogenic acid at 56.32 min, the 11th peak is licorinchalcone A at 58.35 min, the 12th peak is morin G at 61.37 min, the 13th peak is geniposidic acid at 70.99 min, the 14th peak is sanggenon C at 85.24 min, and the 15th peak is apigenin at 102.64 min.

[0084] S5. Methodological investigation

[0085] (1) Stability experiment

[0086] Take the test solution obtained in step S1, and inject samples for analysis at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h respectively according to the above chromatographic conditions. Using isoliquiritigenin, liciritigenin, isorhamnetin, liquiritin, sinapine thiocyanate, β-daucosterol, pinipeolide, dehydrotumulosic acid, 3-epi-dehydrotumulosic acid, ammonium glycyrrhizinate, quercetin, neochlorogenic acid, chlorogenic acid, licorinchalcone, morin, geniposidic acid, sanggenon C, and apigenin as reference peaks, by analyzing the peak areas and retention times of the common peaks in the HPLC fingerprint of the sample and calculating the RSD values, record the chromatogram, and the results are shown in Table 5. As can be seen from Table 5, there are no obvious changes in the relative retention times and the ratios of relative peak areas of the main chromatographic peaks, and the RSDs are 0.34% - 3.69% and 0.67% - 3.71% respectively, indicating that the components of the test solution are stable within 24 h.

[0087] (2) Precision experiment

[0088] Take the test solution obtained in step S1, and under the above chromatographic conditions, continuously measure it 6 times. Using isoliquiritigenin, liquiritigenin, isorhamnetin, liquiritin, sinapine thiocyanate, β-daucosterol, pinetumic acid, dehydrotumulosic acid, 3-epidehydrotumulosic acid, ammonium glycyrrhizinate, quercetin, neochlorogenic acid, chlorogenic acid, licorinchalcone, sanggenon C, geniposidic acid, sanggenol O, apigenin as the reference peaks, by analyzing the peak areas and retention times of the common peaks in the HPLC fingerprint of the sample and calculating the RSD values, record the chromatogram, and the results are shown in Table 5. It can be seen that there are no obvious changes in the relative retention times and the ratios of relative peak areas of the main chromatographic peaks, and the RSDs are 0.22% - 3.8% and 0.36% - 2.95% respectively, with RSD < 5.0%, indicating that the precision of the instrument is good.

[0089] (3) Repeatability experiment

[0090] Take 6 portions of the test solution of Qingning Powder, and under the above chromatographic conditions, inject samples for analysis respectively. Using isoliquiritigenin, liquiritigenin, isorhamnetin, liquiritin, sinapine thiocyanate, β-daucosterol, pinetumic acid, dehydrotumulosic acid, 3-epidehydrotumulosic acid, ammonium glycyrrhizinate, quercetin, neochlorogenic acid, chlorogenic acid, licorinchalcone, sanggenon C, geniposidic acid, sanggenol O, apigenin as the reference peaks, by analyzing the peak areas and retention times of the common peaks in the HPLC fingerprint of the sample and calculating the RSD values, record the chromatogram, and the results are shown in Table 5. There are no obvious changes in the relative retention times and the ratios of relative peak areas of the main chromatographic peaks, and the RSDs are 0.39% - 3.90% and 0.40% - 4.07% respectively, with RSD < 5.0%, indicating that the repeatability of this experimental method is good.

[0091] Table 5 Results of the methodological investigation of the fingerprint of Qingning Powder

[0092]

[0093] Example 2

[0094] S1. Preparation of the test solution of Qingning Powder:

[0095] Precisely weigh 1.0 g of the freeze-dried powder of Qingning Powder and place it in a stoppered conical flask, add 20 mL of 80% methanol solution, ultrasonically extract for 60 min, take the filtrate and filter it through a 0.45 μm microporous filter membrane to obtain the test solution;

[0096] S2. Chromatographic conditions

[0097] Chromatographic column: Kromasil 100-5-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% phosphoric acid aqueous solution, gradient elution, ultraviolet-visible absorption detector for multi-wavelength switching detection, detection wavelength: 254 nm; column temperature 30 °C; flow rate 0.6 mL / min; injection volume: 15 μL; elution program is as shown in Table 6 below.

[0098] Table 6 Liquid Chromatography Elution Program

[0099]

[0100] The final experimental results are shown in Figure 2 , the detection at 36 min - 70 min of this method is better, so the detection with a detection wavelength of 254 nm was advanced during the subsequent optimization process.

[0101] Example 3

[0102] S1. Preparation of the test solution of Qingning Powder:

[0103] Precisely weigh 1.0 g of freeze-dried Qingning Powder and place it in a stoppered conical flask. Add 20 mL of 80% methanol solution, ultrasonically extract for 60 min, take the filtrate and filter it through a 0.45 μm microporous filter membrane to obtain the test solution;

[0104] S2. Chromatographic conditions

[0105] Chromatographic column: Kromasil 100-5-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% phosphoric acid aqueous solution, gradient elution, ultraviolet-visible absorption detector for multi-wavelength switching detection, detection wavelength: 275 nm; column temperature 30 °C; flow rate 0.8 mL / min; injection volume: 15 μL; elution program is as shown in Table 7 below.

[0106] Table 7 Liquid Chromatography Elution Program

[0107]

[0108] The final experimental results are shown in Figure 3 , the overall peak emergence situation is not as good as that detected at 254 nm. The results show that some substances were detected at 15 - 35 min, and the rest were not good.

[0109] Example 4

[0110] S1. Preparation of the test solution of Qingning Powder:

[0111] Precisely weigh 1.0 g of freeze-dried Qingning Powder and place it in a stoppered conical flask. Add 20 mL of 80% methanol solution, ultrasonically extract for 60 min, take the filtrate and filter it through a 0.45 μm microporous filter membrane to obtain the test solution;

[0112] S2, Chromatographic Conditions

[0113] Chromatographic column: Kromasil 100-5-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% phosphoric acid aqueous solution, gradient elution, ultraviolet-visible absorption detector with multi-wavelength switching detection, detection wavelength: 0 - 45 min, 280 nm; 45 - 85 min, 254 nm; column temperature 30°C; flow rate 0.8 mL / min; injection volume: 15 μL; elution program is as shown in Table 8 below.

[0114] Table 8 Liquid Chromatography Elution Program

[0115]

[0116] The experimental result chromatogram of this method is shown in Figure 4 , there are more peaks in the first 35 min, but the peak density is large. During optimization, continue to focus on separating the peaks in this time period. Considering the influence of the organic phase concentration of the mobile phase on the peak distance, continue to optimize the concentration gradient.

[0117] Example 5

[0118] S1, Preparation of the Test Solution of Qingning Powder:

[0119] Precisely weigh 1.0 g of Qingning Powder freeze-dried powder and place it in a stoppered conical flask. Add 20 mL of 80% methanol solution, ultrasonically extract for 60 min, take the filtrate and filter it through a 0.45 μm microporous filter membrane to obtain the test solution;

[0120] S2, Chromatographic Conditions

[0121] Chromatographic column: Kromasil 100-5-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% phosphoric acid aqueous solution, gradient elution, ultraviolet-visible absorption detector with multi-wavelength switching detection, detection wavelength: 0 - 60 min, 280 nm; 60 - 120 min, 254 nm; column temperature 30°C; flow rate 0.6 mL / min; injection volume: 15 μL; elution program is as shown in Table 9 below.

[0122] Table 9 Liquid Chromatography Elution Program

[0123]

[0124] The experimental result chromatogram of this method is shown in Figure 5 , the peaks appear well after 45 min, but the baseline drifts after 60 min. During subsequent optimization, considering the influence of the organic phase concentration of the mobile phase on the baseline, continue to optimize the concentration gradient.

[0125] Example 6

[0126] S1. Preparation of the test solution of Qingning Powder:

[0127] Precisely weigh 1.0 g of the freeze-dried powder of Qingning Powder and place it in a stoppered conical flask. Add 20 mL of 80% methanol solution, ultrasonically extract for 60 min, take the filtrate and filter it through a 0.45 μm microporous filter membrane to obtain the test solution.

[0128] S2. Chromatographic conditions

[0129] Chromatographic column: Kromasil 100 - 5 - C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% phosphoric acid aqueous solution, gradient elution, ultraviolet-visible absorption detector with multi-wavelength switching detection, detection wavelength: 0 - 40 min, 280 nm; 40 - 120 min, 254 nm; column temperature 30°C; flow rate 1.0 mL / min; injection volume: 15 μL; the elution program is as shown in Table 10 below.

[0130] Table 10 Liquid chromatography elution program

[0131]

[0132] The experimental result chromatogram of this method is shown in Figure 6 , with more and denser peaks eluting before 45 min, but almost no peaks eluting after that.

[0133] Example 7

[0134] S1. Preparation of the test solution of Qingning Powder:

[0135] Precisely weigh 1.0 g of the freeze-dried powder of Qingning Powder and place it in a stoppered conical flask. Add 20 mL of 80% methanol solution, ultrasonically extract for 60 min, take the filtrate and filter it through a 0.45 μm microporous filter membrane to obtain the test solution.

[0136] S2. Chromatographic conditions

[0137] Chromatographic column: Kromasil 100 - 5 - C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% phosphoric acid aqueous solution, gradient elution, ultraviolet-visible absorption detector with multi-wavelength switching detection, detection wavelength: 0 - 45 min, 280 nm; 45 - 85 min, 254 nm; column temperature 30°C; flow rate 0.8 mL / min; injection volume: 15 μL; the elution program is as shown in Table 11 below.

[0138] Table 11 Liquid chromatography elution program

[0139]

[0140] The experimental result chromatogram of this method is shown in Figure 7, the peak emergence is average. The number is relatively large but dense before 30 min, and the baseline moves up after 40 min.

[0141] Example 8

[0142] S1. Preparation of the test solution of Qingning Powder:

[0143] Precisely weigh 1.0 g of the freeze-dried powder of Qingning Powder and place it in a stoppered conical flask. Add 20 mL of 80% methanol solution, ultrasonically extract for 60 min, take the filtrate and filter it through a 0.45 μm microporous filter membrane to obtain the test solution;

[0144] S2. Chromatographic conditions

[0145] Chromatographic column: Kromasil 100-5-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% phosphoric acid aqueous solution, gradient elution, ultraviolet-visible absorption detector for multi-wavelength switching detection, detection wavelength: 0 - 45 min, 280 nm; 45 - 85 min, 254 nm; column temperature 30 °C; flow rate 0.6 mL / min; injection volume: 15 μL; the elution program is as shown in Table 12 below.

[0146] Table 12 Liquid chromatography elution program

[0147]

[0148] The experimental result spectrum of this method is shown in Figure 8 , the overall peak emergence is not good. There is an incomplete peak emergence at the last 89 min, and the number of peaks in the whole spectrum is small.

[0149] Example 9

[0150] S1. Preparation of the test solution of Qingning Powder:

[0151] Precisely weigh 1.0 g of the freeze-dried powder of Qingning Powder and place it in a stoppered conical flask. Add 20 mL of 80% methanol solution, ultrasonically extract for 60 min, take the filtrate and filter it through a 0.45 μm microporous filter membrane to obtain the test solution;

[0152] S2. Chromatographic conditions

[0153] Chromatographic column: Kromasil 100-5-C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: acetonitrile and 0.1% phosphoric acid aqueous solution, gradient elution, ultraviolet-visible absorption detector for multi-wavelength switching detection, detection wavelength: 0 - 60 min, 280 nm; 60 - 120 min, 254 nm; column temperature 30 °C; flow rate 0.6 mL / min; injection volume: 15 μL; the elution program is as shown in Table 13 below.

[0154] Table 13 Liquid chromatography elution program

[0155]

[0156] The experimental result spectrum of this method is shown in Figure 9 , and the results are not good. The peaks mainly gather before 25 min and are relatively dense. There are fewer peaks after 30 min and the content is not high, which cannot provide highly valuable results for this research. The concentration gradient can be further optimized.

[0157] The present invention optimizes the mobile phase gradient step by step, and also optimizes different aspects such as flow rate, extraction solvent, wavelength, etc. The finally optimized scheme is Example 1.

Claims

1. A method for constructing a fingerprint of Qingning Powder freeze-dried powder, characterized in that, It includes the following steps: S1. Take Qingning powder for injection, add methanol aqueous solution, and perform ultrasonic extraction to obtain a test solution; S2. Take the test solution, inject it into a high-performance liquid chromatograph, perform chromatographic analysis, and record the chromatogram. Among them, the chromatographic column used in the high-performance liquid chromatograph is a Kromasil 100-5-C18 chromatographic column, and the chromatographic conditions are as follows: the mobile phase is acetonitrile - 0.1% phosphoric acid aqueous solution; ultraviolet-visible absorption detector is used for multi-wavelength switching detection, and the detection wavelengths are: 0 - 15 min, 254 nm; 15 - 43 min, 280 nm; 43 - 120 min, 254 nm; the gradient elution program is: 0 - 10 min, the volume of acetonitrile is 10%; 10 - 20 min, the volume of acetonitrile is 10% - 20%; 20 - 30 min, the volume of acetonitrile is 20% - 23%; 30 - 40 min, the volume of acetonitrile is 23 - 30%; 40 - 60 min, the volume of acetonitrile is 30% - 40%; 60 - 80 min, the volume of acetonitrile is 40% - 45%; 80 - 100 min, the volume of acetonitrile is 45% - 58%; 100 - 120 min, when the volume of acetonitrile is 58% - 63%, good resolution of each chromatographic peak in the fingerprint can be achieved; S3. Perform mass spectrometry analysis on the test solution to obtain a mass spectrometry result diagram; S4. According to the chromatogram and the mass spectrometry result diagram, construct a fingerprint. Among them, according to the mass spectrometry result diagram, determine the chemical components of each chromatographic peak in the fingerprint, which are: peak 1 is isoliquiritigenin / licoricidin, peak 2 is isorhamnetin, peak 3 is liquiritin, peak 4 is sinapine thiocyanate, peak 5 is β-daucosterol, peak 6 is pachymic acid, peak 7 is dehydrotumulosic acid / 3-epidehydrotumulosic acid, peak 8 is ammonium glycyrrhizinate, peak 9 is quercetin, peak 10 is neochlorogenic acid / chlorogenic acid, peak 11 is licorinchalcone A, peak 12 is morin G, peak 13 is geniposidic acid, peak 14 is sanggenon, and peak 15 is apigenin.

2. The method for constructing the fingerprint of Qingning powder freeze-dried powder according to claim 1, characterized in that In S1, the volume concentration of the methanol aqueous solution is 80%.

3. The method for constructing the fingerprint spectrum of Qingning powder freeze-dried powder according to claim 1, characterized in that, In S1, the ultrasonic time is 30 - 60 min.

4. The method for constructing the fingerprint spectrum of Qingning powder freeze-dried powder according to claim 1, wherein In S2, the flow rate of the mobile phase during chromatographic analysis is 0.6 - 1 mL / min.

5. The method for constructing the fingerprint spectrum of Qingning powder freeze-dried powder according to claim 1, characterized in that, S4 is: Import the chromatogram obtained in S2 into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints, and perform data import, multi-point correction, and data matching on the chromatogram of the test solution respectively to obtain a fingerprint and perform similarity analysis.

Images