Construction method of children seven-star tea particle fingerprint spectrum and fingerprint spectrum of children seven-star tea particle fingerprint spectrum

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis, in particular to a construction method of a fingerprint spectrum of infantile seven-star tea granules and the fingerprint spectrum of the infantile seven-star tea granules. The construction method comprises the following steps: preparation of a test solution and preparation of a reference solution; respectively injecting the reference substance solution and the test substance solution into a high performance liquid chromatograph for determination to obtain a reference substance spectrum and a test substance spectrum; liquiritin is selected as a reference peak in the atlas of the test sample; importing the fingerprint spectrum of the test sample into a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system to generate a control fingerprint spectrum, and establishing a common mode to obtain 46 common peaks; and calculating the relative retention time and the relative peak area of each common peak according to the selected reference peak. The construction method disclosed by the invention is high in sensitivity, good in accuracy, strong in repeatability and rapid in analysis, 46 common peaks are determined for 15 batches of samples, and the similarity between the samples and a control fingerprint spectrum is high.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis, and specifically relates to a method for constructing a fingerprint of Xiao'er Qixingcha Granules and the fingerprint thereof. Background Art

[0002] Xiao'er Qixingcha Granules is a traditional Chinese medicine preparation with the effects of promoting appetite and relieving stagnation, clearing heat and calming endogenous wind, and is mainly used for treating symptoms such as indigestion in children, anorexia, unsmooth defecation and urination, and restlessness at night. The main ingredients include: Coix Seed, Germinated Barley, Hawthorn Fruit, Lophatherum Gracile, Uncaria Rhynchophylla, Cryptotympana pustulata Fabricius, and Licorice Root.

[0003] Chinese Patent CN115290768B discloses a method for detecting the active ingredients of Xiao'er Qixingcha Granules. First, prepare the sample injection solution to be detected and the mixed standard solution, and then perform the detection according to the following method: Inject the sample with an injection volume of 1 - 10 μL into a Waters Acquity BEH C18 chromatographic column with a column temperature of 20 - 40 °C, then use the mobile phase composed of Phase A and Phase B to perform gradient elution at a flow rate of 0.10 - 0.45 mL / min according to the specified program, and at the same time use tandem time-of-flight mass spectrometry for detection to obtain the spectra of the standard product and the detected substance, and construct the common mode fingerprint of the detected substance by using the traditional Chinese medicine fingerprint similarity evaluation system; finally, compare the spectra of the detected substance and the standard product, and use the software-assisted fragment ion prediction function to deduce the possible structural information of the compound, and compare it with the mass spectrometry fragmentation information provided by the standard product and the existing literature reports to further determine 31 active ingredients. However, the characteristic common peaks of this patent are few, and using a high-resolution mass spectrometer as the detector does not have universality, and the precision, stability and repeatability of its method are not investigated.

[0004] Zhu Huanrong et al. disclosed a method for constructing the fingerprint of Xiao'er Qixingcha Granules in the literature (DOI: 10.13422 / j.cnki.syfjx.2014130093). Using reversed-phase high-performance liquid chromatography, Symmetry C18 chromatographic column, gradient elution with acetonitrile - 0.2% acetic acid solution, column temperature 30 °C, flow rate 1.0 mL·min -1 , detection wavelength 254 nm. However, only 20 common peaks were established, and the attributed peaks could only confirm which raw material provided them, and the specific substance attribution could not be determined, and the chromatographic peaks of Coix Seed, Germinated Barley, and Cryptotympana pustulata Fabricius could not be attributed accordingly. Summary of the Invention

[0005] In view of the above technical problems, the present invention provides a method for constructing a fingerprint of Xiao'er Qixingcha Granules and the fingerprint thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] On the one hand, the present invention provides a method for constructing a fingerprint of Xiao'er Qixingcha Granules, which is characterized by comprising the following steps:

[0008] (1) Prepare a test solution from Xiao'er Qixingcha Granules;

[0009] (2) Respectively take reference substances and prepare reference substance solutions;

[0010] (3) Detection: Inject the reference substance solution and the test solution respectively, and inject them into a high performance liquid chromatograph for determination to obtain a reference substance chromatogram and a test solution chromatogram;

[0011] (4) Establish a fingerprint: Select liquiritin as the reference peak in the test solution chromatogram obtained in step (3); Import the fingerprint of the test solution into the "Recognition Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" to generate a reference fingerprint, and establish a common pattern to obtain 46 common peaks; According to the selected reference peak, set both its retention time and peak area to 1.0, calculate the relative retention time and relative peak area of each common peak, and obtain the reference fingerprint of Xiao'er Qixingcha Granules;

[0012] The reference substances are: coixenolide, vitexin, vitexin-2-O-β-D-glucoside, isoorientin, rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine, glycyrrhizic acid, ammonium glycyrrhizinate, liquiritin, isoliquiritin, liquiritigenin, licorinchalcone B, isoliquiritigenin, echinatin, neoliquiritin, apiosylisoliquiritin, apiosylglycyin, quercetin, catechin, chlorogenic acid, apigenin, and xanthoceraside;

[0013] The elution gradient in step (3) is:

[0014] Time (min) Phase A (%) Phase B (%) 0 100 0 4 97 3 8 95 5 17 89 11 25 85 15 35 81 19 50 76 24 77 55 45 80 55 45

[0015] The Xiao'er Qixingcha Granules include coix seed, germinated rice, hawthorn, lophatherum gracile, uncaria rhynchophylla, cryptotympana pustulata, and licorice.

[0016] Further, the preparation method of the test solution is: Grind Xiao'er Qixingcha Granules, add a solvent, shake well, ultrasonicate, let stand, take the supernatant, centrifuge, and take the supernatant to obtain it.

[0017] Further, the material-liquid ratio of the Xiao'er Qixingcha Granules to the solvent is (1-4) g: 10 mL.

[0018] Further, the conditions of the ultrasonication are: power 400-600 W, frequency 40-60 kHz, time 10-90 min.

[0019] Preferably, the time is 10 min.

[0020] Further, the concentration of the reference substance solution in step (2) is 1-1.5 mg / mL.

[0021] Preferably, the concentration of the reference substance solution in step (2) is 1 mg / mL.

[0022] Furthermore, the preparation of the reference substance solution in step (2) is completed under light-proof conditions.

[0023] Furthermore, the chromatographic column in step (3) is one of Hypersil GOLD, GOWON core-shell C18 chromatographic column or Shim-pack GIST C18-AQ.

[0024] Preferably, the chromatographic column is Shim-pack GIST C18-AQ.

[0025] Furthermore, the mobile phase A in step (3) is 0.1-0.4% formic acid water.

[0026] Preferably, the mobile phase A in step (3) is 0.1% formic acid water.

[0027] Furthermore, the mobile phase B in step (3) is acetonitrile.

[0028] Furthermore, the column temperature in step (3) is 25-45 °C.

[0029] Preferably, the column temperature in step (3) is 30 °C.

[0030] Furthermore, the injection volume in step (3) is 5 μL.

[0031] Furthermore, the detection wavelength in step (3) is: 254 nm.

[0032] Furthermore, the construction method further includes tandem mass spectrometry.

[0033] In the second aspect, the present invention provides a fingerprint, and there are 46 common peaks in the fingerprint.

[0034] Furthermore, for the 46 common peaks, in ascending order of retention time, the 21st peak is chlorogenic acid, the 29th peak is isoorientin, the 33rd peak is vitexin glucoside, the 34th peak is liquiritin, the 35th peak is coix seed, the 38th peak is rhynchophylline, the 39th peak is uncarine, the 40th peak is liquiritigenin, the 42nd peak is vobtusin, the 43rd peak is hirsuteine, and the 45th peak is glycyrrhizic acid.

[0035] In the third aspect, the present invention provides an application of a fingerprint in the identification of active ingredients of Xiao'er Qixingcha Granules and / or the production of Xiao'er Qixingcha Granules.

[0036] Furthermore, the application is the quality control of the production of Xiao'er Qixingcha Granules.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention optimizes the extraction solvent, extraction time, mobile phase, mobile phase additive, chromatographic column, column temperature, injection volume, detection wavelength and gradient elution program, and the constructed fingerprint results show that the similarity between 15 batches of samples and the control fingerprint is very high, all greater than 0.940. At the same time, the method has high sensitivity, good accuracy, strong repeatability and fast analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Separation chromatograms for samples with different extraction solvents.

[0040] Figure 2 Separation chromatograms for samples with different extraction times.

[0041] Figure 3 Chromatograms were determined for samples at different detection wavelengths.

[0042] Figure 4 Chromatographic spectra of samples separated by different chromatographic columns.

[0043] Figure 5 Chromatograms of samples separated by different mobile phase systems.

[0044] Figure 6 Separation sample chromatograms for different mobile phase additives.

[0045] Figure 7 The chromatograms are of samples separated at different column temperatures.

[0046] Figure 8 Chromatograms of separated samples at different injection volumes.

[0047] Figure 9 This is the chromatogram of the sample after optimized gradient elution.

[0048] Figure 10 This is the spectrum of the test product (1: liquiritin, Rt=37.147min).

[0049] Figure 11 These are the chromatogram matching patterns for 15 batches of Xiaoer Qixing Tea Granule samples.

[0050] Figure 12 This is the reference fingerprint of Children's Seven Star Tea Granules.

[0051] The present invention Figures 1 - 10 This is the spectrum of batch number S1. DETAILED DESCRIPTION

[0052] The present invention will be described below through specific embodiments to make the technical solution of the present invention easier to understand and master. However, the present invention is not limited thereto. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0053] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein. Unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" as used herein include both singular and plural referents. Numerical ranges expressed by endpoints include all values and fractions within the corresponding ranges, as well as the expressed endpoints.

[0054] In the following embodiments, the prescription of the Pediatric Qixingcha Granules used is as follows: Coix Seed: 893 g, Germinated Barley: 893 g, Hawthorn Fruit: 446 g, Lophatherum Gracile: 670 g, Uncaria Rhynchophylla: 335 g, Cryptotympana pustulata Fabricius: 112 g, Licorice Root: 112 g.

[0055] Reference substances: Coixol (CAS#: 532-91-2), Vitexin (CAS#: 3681-93-4), Vitexin-4”-O-glucoside (CAS#: 178468-00-3), Luteolin-6-C-glucoside (CAS#: 4261-42-1), Rhynchophylline (CAS#: 76-66-4), Isorhynchophylline (CAS#: 6859-1-4), Corynoxeine (CAS#: 630-94-4), Isocorynoxeine (CAS#: 51014-29-0), Glycyrrhizic acid (CAS#: 1405-86-3), Glycyrrhizic acid ammonium salt (CAS#: 53956-04-0), Liquiritin (CAS#: 551-15-5), Isoliquiritoside (CAS#: 5041-81-6), Liquiritigenin (CAS#: 578-86-9), Licochalcone B (CAS#: 58749-23-8), Isoliquiritigenin (CAS#: 961-29-5), Echinatin (CAS#: 34221-41-5), Neo-Liquiritin (CAS#: 5088-75-5), Isoliquiritinapioside (CAS#: 120926-46-7), Liquiritin apioside (CAS#: 74639-14-8), Quercetin (CAS#: 117-39-5), Hirsutine (CAS#: 7729-23-9), Catechin (CAS#: 154-23-4), N-acetyldopamine dimers A (CAS#: 1519015-73-6), Chlorogenic acid (CAS#: 327-97-9), Apigenin (CAS#: 520-36-5), Vincosamide (CAS#: 23141-27-7), all purchased from Chengdu Must Biotechnology Co., Ltd. and with a purity of ≥ 97%,All reference substances were confirmed by ultraviolet absorption spectroscopy, high-resolution mass spectrometry, and multi-stage fragmentation data before use.

[0056] All batches of finished product samples of Xiao'er Qixingcha Granules in the experiment were provided by Guangzhou Wanglaoji Pharmaceutical Co., Ltd., as shown in Table 1.

[0057] Table 1

[0058] Number Sample Batch Number Sample Batch S1 2408017 S9 2311016 S2 2409006 S10 2311018 S3 2409009 S11 2311020 S4 2409012 S12 2311022 S5 2409015 S13 2311024 S6 2409020 S14 2311027 S7 2409020 S15 2311030 S8 2311014

[0059] 1.1 Chromatography-Mass Spectrometry Conditions

[0060] The chromatographic column was HPLC column Shim-pack GIST C18-AQ (150 mm × 4.6 mm, 3 μm). Mobile phase A was 0.1% formic acid in water, and B was acetonitrile. Gradient elution: 0 - 4 min, 0% - 3% B; 4 - 8 min, 3% - 5% B; 8 - 17 min, 5% - 11% B; 17 - 25 min, 11% - 15% B; 25 - 35 min, 15% - 19% B; 35 - 50 min, 19% - 24% B; 50 - 77 min, 24% - 45% B; 77 - 80 min, 45% B. Flow rate: 1.0 mL / min; Column temperature: 30 °C; Injection volume: 5 μL. Detection conditions for multi-wavelength ultraviolet detector (VWD), detection wavelength: 254 nm; Sampling frequency: 5 Hz. Ionization mode: Electrospray ionization; Ion transfer tube temperature: 380 °C; Spray voltage: 5.0 kV (positive ion); -4.0 kV (negative ion); Maximum ion injection time: 500 ms for full scan in the first stage, 100 ms for multi-stage scan.

[0061] Scanning mode: Positive and negative ion full scan; Scanning range: m / z 100 - 1500; Scanning resolution: 15,000; Perform triple data-dependent scan on the top two ions with the strongest response in the full scan; Fragmentation mode: Collision-induced dissociation (CID), energy 35%.

[0062] 1.2 Preparation of Test Solution

[0063] Take an appropriate amount of Xiao'er Qixingcha Granules, grind them finely, take 2 g, weigh accurately, place it in a 50 mL centrifuge tube, accurately add 10 mL of methanol, shake well, ultrasonically treat (KQ-400DE; power / frequency, 400 W / 40 kHz) for 10 min, cool, stand still, take 2 mL of the supernatant and transfer it to a 2 mL centrifuge tube, centrifuge at high speed (14,000 rpm) for 10 min, take the supernatant, and you will get it.

[0064] 1.3 Preparation of Reference Solution

[0065] Weigh appropriate amounts of the reference substances accurately, place them in brown volumetric flasks, dissolve them with chromatographic methanol to prepare a standard stock solution (1 mg / mL), and store it in a refrigerator at 4°C for future use.

[0066] 1.4 Data processing

[0067] In the experiment, the chromatograms were processed and exported by the Chromeleon chromatographic management software (Chromeleon 7); after export, the sample chromatograms were imported into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints 2004A Edition" software (National Pharmacopoeia Commission) for fingerprint analysis to calculate the similarity of the samples.

[0068] 1.5 Selection of extraction process

[0069] 1.5.1 Selection of extraction solvent

[0070] In this experiment, the extraction solvent system of the test solution for the fingerprint of Xiao'er Qixingcha Granules was investigated, and the ultrasonic extraction efficiencies of 5 solvent systems including water, 30%, 50%, 70% and 100% methanol were compared and selected. The results are shown in Figure 1 . When water, 30% methanol, 50% methanol, and 70% methanol were used as extraction solvents, baseline drift occurred in the chromatogram within 25 - 40 minutes, and as the water content of the extraction solvent decreased, the baseline drift phenomenon weakened; when methanol was used as the extraction solvent, no baseline drift occurred in the chromatogram, and the number of chromatographic peaks had no obvious difference from that of other extraction solvents. Therefore, methanol was finally selected as the sample extraction solvent.

[0071] 1.5.2 Selection of extraction time

[0072] After selecting methanol as the extraction solvent, in order to obtain higher extraction efficiency, the ultrasonic extraction time of the test solution was investigated, including 10 min, 20 min, 30 min, 60 min, and 90 min. The results are shown in Figure 2 . As the extraction time increased, the chromatographic peak intensity did not increase significantly. Therefore, based on the principle of ensuring extraction efficiency and saving time, 10 min was preferably selected as the ultrasonic extraction time.

[0073] 1.5.3 Selection of detection wavelength

[0074] Xiao'er Qixingcha Granules are composed of 7 traditional Chinese medicines including Coix Seed, Hawthorn Fruit, Germinated Rice, Lophatherum Gracile, Uncaria Rhynchophylla, Cicada Slough, and Licorice Root. There are many chemical components, with various quantities, properties, and types. Therefore, the fingerprint established in this experiment should obtain as much chemical component information as possible. In this experiment, a VWD ultraviolet detector was used to compare the chromatograms at common detection wavelengths (254 nm, 280 nm). The results are as Figure 3As shown in the figure, at different wavelengths, the sample component signal response is strong and the sensitivity is high. Therefore, the VWD UV detector was selected for the study of the fingerprint spectrum of Xiaoer Qixing Tea granules. At the detection wavelength of 280nm, the number of visible peaks of the polar components and small polar components of the spectrum is small and the signal intensity is low; the signal intensity of the medium polar components is high; and when the detection wavelength is changed to 254nm, although the intensity of some chromatographic peaks of the medium polar components is reduced to a certain extent, the number of chromatographic peaks of the polar components and small polar components is significantly increased and the signal response intensity is high. The consistency of the UV absorption intensity range of all components is strong. Therefore, the UV detection wavelength of 254nm was selected in this experiment.

[0075] 1.5.4 Selection of chromatographic columns

[0076] This experiment investigated three different types of chromatographic columns under the same chromatographic conditions, including Hypersil GOLD (150 mm × 2.1 mm, 1.9 μm), GOWON core-shell C18 column (150 mm × 4.6 mm, 2.7 μm) and HPLC column Shim-pack GIST C18-AQ (150 mm × 4.6 mm, 3 μm). The results are shown in Figure 2. Figure 4 , Shim-pack GIST C18-AQ chromatographic column has stronger selectivity for polar compounds, better separation and peak shape, stable baseline, low system pressure and can be applied to most HPLC instruments. Therefore, Shim-pack GIST C18-AQ chromatographic column was selected for subsequent fingerprint research.

[0077] 15.5 Choice of Mobile Phase

[0078] In the experiment, methanol-water and acetonitrile-water were first investigated as mobile phases. The results showed that more chromatographic peaks were obtained when acetonitrile-water was used as the mobile phase for gradient elution, with better peak shape and higher sensitivity. Therefore, the acetonitrile-water system was selected for research ( Figure 5 ). At the same time, in order to further improve the peak shape, increase the chromatographic peak separation, and enhance the signal response intensity of the detector, the experiment investigated different concentrations of formic acid as a mobile phase additive, including acetonitrile-0.1% formic acid water, acetonitrile-0.2% formic acid water, and acetonitrile-0.4% formic acid water. Figure 6 It can be seen that under different additive concentration conditions, there is no obvious difference in the chromatograms, but in the acetonitrile-0.1% formic acid water mobile phase system, the separation effect of the chromatographic peaks within 5-15 minutes is better. At the same time, considering the pH tolerance of the chromatographic column and the repeatability of the experiment, acetonitrile-0.1% formic acid water is preferably used as the mobile phase for gradient elution analysis.

[0079] 1.5.6 Column temperature selection

[0080] The temperature of the chromatographic column has a great influence on the separation effect of structurally similar compounds. In this experiment, the effects of different chromatographic column temperatures (25 °C, 30 °C, 35 °C, 40 °C, 45 °C) on sample separation were investigated respectively. The results are as Figure 7 , as the temperature of the chromatographic column increases, the sample separation time gradually shortens, the separation efficiency increases, but the resolution of two adjacent chromatographic peaks of the overall components, especially the strongly polar components, decreases. To ensure the resolution of chromatographic peaks and the service life of the chromatographic column, the chromatographic column temperature of 30 °C was selected in this experiment.

[0081] 1.5.7 Selection of injection volume

[0082] The size of the injection volume in liquid chromatography directly affects the chromatographic peak shape, resolution and signal intensity. In this experiment, the effects of different injection volumes (2 μl, 5 μl, 10 μl, 15 μl, 20 μl) on sample separation were investigated respectively. From Figure 8 it can be seen that as the injection volume increases, the chromatographic peaks become wider and the "split peak" phenomenon appears, and the resolution decreases. However, when the injection volume is 2 μl, the signal intensity of the chromatographic peak is relatively low. When the injection volume is 5 μl, the signal intensity is moderate and the resolution is good. When the injection volume increases to 10 μl, obvious split peaks appear during 5 - 10 min. Therefore, the injection volume of 5 μl was selected in this experiment.

[0083] 1.5.8 Optimization of gradient program

[0084] The Xiao'er Qixingcha Granules are composed of 7 kinds of traditional Chinese medicines, with a very large number and variety of chemical components. At the same time, the fingerprint requires separating and characterizing as many chemical components in the sample as possible. Therefore, based on the HPLC-UV technology and considering the system pressure resistance problem, a high-performance liquid chromatographic column with a particle size of 3 μm was used for chromatographic analysis of the sample. At the same time, due to the different properties of the chemical components contained, the gradient elution program was optimized to separate the largest number of chromatographic peaks in the shortest time, and finally the analysis of the sample was completed within 80 min (as Figure 9 ).

[0085] 1.6 Establishment of standard fingerprint

[0086] Take 15 batches of Xiao'er Qixingcha Granules samples, prepare the test solution, and perform HPLC-UV analysis, and record the chromatogram for 80 min.

[0087] In the chromatogram, select the chromatographic peaks with larger peak areas, moderate and stable elution times as the reference peaks. The results show that the chromatographic peak with a retention time of 37.147 min meets this condition. After comparison with the reference substance and mass spectrometry confirmation, it is the liquiritin chromatographic peak. Therefore, liquiritin was selected as the reference peak ( Figure 10 ).

[0088] The HPLC fingerprint spectra of 15 batches of samples were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A Edition", and the median method was used to automatically match the spectral peaks ( Figure 11 ), generating a reference fingerprint spectrum and establishing a common pattern ( Figure 12 ). Chromatographic peaks with good stability, strong absorption, and obvious characteristics were selected as common peaks, and 46 common peaks were determined based on the measurement results of 15 batches of samples. According to the selected reference peak of liquiritin (peak No. 34), both its retention time and peak area were set to 1.0, and the relative retention times and relative peak areas of each common peak were calculated and listed in Table 2 and Table 3 respectively.

[0089] Table 2

[0090]

[0091]

[0092]

[0093]

[0094] Table 3

[0095]

[0096]

[0097]

[0098]

[0099] 1.7 Similarity evaluation of samples

[0100] 1.7.1 Similarity evaluation of samples

[0101] The HPLC fingerprint spectra of 15 batches of samples were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A Edition" to generate a reference fingerprint spectrum of Pediatric Qixingcha Granules. Under the analysis and inspection mode, the similarities of the fingerprint spectra of 15 batches of samples were calculated. The results showed that the similarities between the 15 batches of samples and the reference fingerprint spectrum were high, all greater than 0.940 (Table 4).

[0102] Table 4

[0103]

[0104]

[0105]

[0106] 1.7.4 Identification of main fingerprint spectrum characteristic peaks

[0107] Under the same chromatographic conditions, the chromatograms of each reference substance and sample were determined. After comparison with the reference substance chromatogram and confirmation by mass spectrometry, it was shown that the 21st peak in the fingerprint chromatogram characteristic peaks was chlorogenic acid, the 29th peak was isoorientin, the 33rd peak was vitexin glucoside, the 34th peak was liquiritin, the 35th peak was coix seed, the 38th peak was rhynchophylline, the 39th peak was uncarine, the 40th peak was liquiritigenin, the 42nd peak was vincoside lactam, the 43rd peak was hirsuteine, and the 45th peak was glycyrrhizic acid.

[0108] 1.8 Methodology investigation

[0109] 1.8.1 Precision

[0110] The test solution was prepared according to the sample preparation method, and was injected continuously for 5 times for HPLC analysis. The results showed that the RSD values of the relative peak areas and relative retention times of each common peak were less than 2.98% and 0.13% respectively, indicating that the instrument precision was good and met the requirements of the fingerprint chromatogram.

[0111] 1.8.2 Stability

[0112] The test solution was prepared according to the sample preparation method, and was subjected to HPLC analysis at 0, 3, 6, 9, 12, and 24 h respectively. The results showed that the RSD values of the relative peak areas and relative retention times of each common peak were less than 4.66% and 0.23% respectively, indicating that the sample solution had good stability within 24 h.

[0113] 1.8.3 Repeatability

[0114] Five samples of the same batch were taken, the test solution was prepared according to the sample preparation method, and was subjected to HPLC analysis. The results showed that the RSD values of the relative peak areas and relative retention times of each common peak were less than 4.72% and 0.17% respectively, indicating that the method had good repeatability.

[0115] The present invention uses high performance liquid chromatography, and optimizes the extraction solvent, extraction time, detection wavelength, mobile phase, mobile phase additive, chromatographic column, column temperature, injection volume, and gradient elution program to establish the chromatographic fingerprint of the finished product of Xiao'er Qixingcha granules. Using liquiritin as the reference peak, 46 common peaks were determined according to the measurement results of 15 batches of samples, and the attribution of the common peaks was carried out to generate the reference fingerprint of Xiao'er Qixingcha granules. The results showed that the similarity between the 15 batches of samples and the reference fingerprint was very high, all greater than 0.940.

[0116] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a fingerprint of Xiao'er Qixingcha Granules, characterized in that It includes the following steps: (1) Prepare the test solution from the Xiao'er Qixingcha Granules. (2) Respectively take the reference substances and prepare the reference substance solutions. (3) Detection: Inject the reference substance solution and the test solution respectively, and inject them into a high performance liquid chromatograph for determination to obtain the reference substance chromatogram and the test solution chromatogram. (4) Establish the fingerprint: Select liquiritin as the reference peak in the test solution chromatogram in step (3); Import the fingerprint of the test substance into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" to generate the reference fingerprint, establish the common pattern to obtain 46 common peaks; According to the selected reference peak, calculate the relative retention time and relative peak area of each common peak to obtain the reference fingerprint of the Xiao'er Qixingcha Granules. The reference substances are: coixenolide, vitexin, vitexin-2-O-β-D-glucoside, isoorientin, rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine, glycyrrhizic acid, ammonium glycyrrhizinate, liquiritin, isoliquiritin, liquiritigenin, licorinchalcone B, isoliquiritigenin, echinatin, neoliquiritin, apiosylisoliquiritin, apiosylglycycoumarin, quercetin, catechin, chlorogenic acid, apigenin, and xylopicin. The elution gradient in step (3) is: 0 - 4 min, 0% → 3% mobile phase B; 4 - 8 min, 3% → 5% mobile phase B; 8 - 17 min, 5% → 11% mobile phase B; 17 - 25 min, 11% → 15% mobile phase B; 25 - 35 min, 15% → 19% mobile phase B; 35 - 50 min, 19% → 24% mobile phase B; 50 - 77 min, 24% → 45% mobile phase B; 77 - 80 min, 45% mobile phase B. The Xiao'er Qixingcha Granules include coix seed, germinated rice, hawthorn, lophatherum herb, uncaria stem with hooks, cicada slough, and licorice root.

2. The construction method according to claim 1, characterized in that The ratio of the Xiao'er Qixingcha Granules to the solvent in the test solution in step (1) is (1 - 4):10 mL.

3. The construction method according to claim 2, wherein The solvent is one of water, methanol, or methanol aqueous solution, preferably methanol.

4. The construction method according to claim 1, characterized in that The chromatographic column in step (3) is one of Hypersil GOLD chromatographic column, GOWON core-shell C18 chromatographic column, or Shim-pack GIST C18-AQ chromatographic column, preferably Shim-pack GIST C18-AQ chromatographic column.

5. The construction method according to claim 1, characterized in that The mobile phase A in step (3) is 0.1 - 0.4% formic acid water, preferably 0.1% formic acid water; the mobile phase B in step (3) is acetonitrile.

6. The construction method according to claim 1, characterized in that, The column temperature in step (3) is 25 - 45 °C.

7. The construction method according to claim 1, characterized in that The injection volume in step (3) is 5 μL.

8. The construction method according to claim 1, wherein The detection wavelength in step (3): 254 nm.

9. A fingerprint spectrum constructed by the construction method according to any one of claims 1-8, characterized in that, There are 46 common peaks in the fingerprint; According to the retention time from small to large, the 21st peak is chlorogenic acid, the 29th peak is isoorientin, the 33rd peak is vitexin-2-O-β-D-glucoside, the 34th peak is liquiritin, the 35th peak is coixenolide, the 38th peak is corynoxeine, the 39th peak is rhynchophylline, the 40th peak is liquiritigenin, the 42nd peak is xylopicin, the 43rd peak is hirsutine, and the 45th peak is glycyrrhizic acid.

10. Use of the fingerprint obtained by the construction method according to any one of claims 1-8 or the fingerprint according to claim 9 in the identification of active ingredients of Pediatric Qixingcha Granules and / or in the production of Pediatric Qixingcha Granules.

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