A method for determining the UPLC fingerprint of Xiaoer Qingre Zhike granules

Abstract

The invention discloses a method for determining the UPLC fingerprint of pediatric Qingre Zhike granules, and belongs to the technical field of drug analysis. The invention performs UPLC chromatographic analysis on pediatric Qingre Zhike granules, establishes a UPLC fingerprint containing 16 common characteristic peaks including ephedrine hydrochloride, pseudoephedrine hydrochloride, amygdalin, liquiritin, baicalin, and baicalein, and can more comprehensively evaluate the overall quality of pediatric Qingre Zhike granules, providing a new technical idea for the quality control and quality evaluation of pediatric Qingre Zhike granules.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for determining a UPLC fingerprint of children's heat-clearing and cough-relieving granules. Background Art

[0002] Pediatric Qingre Zhike Granules are made with ephedra, bitter almond (fried), gypsum, licorice, scutellaria, isatis root, and northern bean root as the main ingredients. It has the effects of clearing heat, promoting lung function, and relieving asthma. It is mostly used for fever, chills, cough with yellow sputum, shortness of breath, dry mouth, hoarseness, and sore throat caused by exogenous factors in children.

[0003] At present, the quality control method of pediatric Qingre Zhike Granules is mainly to determine the content of ephedrine hydrochloride according to the current version of the standard of "Pediatric Qingre Zhike Granules". Ephedrine hydrochloride comes from ephedra, but the raw materials of pediatric Qingre Zhike Granules are diverse and the ingredients are complex. So far, the quality control research on pediatric Qingre Zhike Granules includes the determination of baicalin content by HPLC, the determination of bitter almonds and scutellaria by thin layer chromatography, and the determination of ephedrine hydrochloride and pseudoephedrine hydrochloride by reversed-phase HPLC. The above methods cannot fully reflect the overall quality of the granules.

[0004] The fingerprint or characteristic spectrum of traditional Chinese medicine is a comprehensive and quantifiable identification method that can systematically, holistically and exclusively characterize the intrinsic characteristics of traditional Chinese medicine. It can be used to evaluate the quality of traditional Chinese medicine materials, traditional Chinese medicine preparations and semi-finished products, and is an effective means of overall quality control of traditional Chinese medicine. Ultra-high performance liquid chromatography (UPLC) uses the theory and principle of HPLC, covers new technologies such as small particle fillers, very low system volume and rapid detection methods, increases the analysis throughput and chromatographic peak capacity, and has the characteristics of simplicity, accuracy, high sensitivity, good repeatability, and a variety of detectors. It is one of the important methods for constructing fingerprints. At present, there is no report on the use of UPLC fingerprints or characteristic spectra for quality control analysis of Xiaoer Qingre Zhike Granules. Summary of the invention

[0005] The purpose of the present invention is to provide a method for determining the UPLC fingerprint of pediatric Qingre Zhike granules to solve the problems existing in the above-mentioned prior art. The present invention performs UPLC chromatographic analysis on pediatric Qingre Zhike granules to establish a UPLC fingerprint containing 16 common characteristic peaks including ephedrine hydrochloride, pseudoephedrine hydrochloride, amygdalin, liquiritin, baicalin, and baicalein, which can more comprehensively evaluate the overall quality of pediatric Qingre Zhike granules and provide a new technical idea for the quality control and quality evaluation of pediatric Qingre Zhike granules.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for determining the UPLC fingerprint of Xiaoer Qingre Zhike Granules, comprising the following steps:

[0008] (1) Prepare test sample solution and standard solution;

[0009] (2) performing UPLC chromatography analysis on the test solution and the standard solution to obtain a UPLC fingerprint of the pediatric Qingre Zhike granules consisting of common characteristic peaks of the samples;

[0010] The conditions of the UPLC chromatographic analysis are: octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B; detection wavelength is 210 nm; elution procedure is:

[0011] Time / min Mobile phase A / % Mobile phase B / % 0～4 5 95 4～15 5→15 95→85 15～30 15→30 85→70 30～40 30→70 70→30

[0012] Preferably, the preparation method of the test solution is: accurately weigh 1.5 g of the pediatric Qingre Zhike granules sample, add 25 ml of 30% methanol, and ultrasonicate for 15 min at 1100 W and 40 KHz to obtain the test solution.

[0013] Preferably, the flow rate of the UPLC chromatography analysis is 0.5 ml per minute, the column temperature is 35° C., and the sample loading volume is 1 μl.

[0014] Preferably, the UPLC chromatographic analysis includes the step of identifying the common characteristic peaks of the samples in the chromatogram of the test solution using the chromatogram of the standard solution as a reference.

[0015] Preferably, the standard solution uses 30% methanol as solvent, and includes a mixed standard solution of ephedrine hydrochloride and pseudoephedrine hydrochloride, amygdalin standard solution, liquiritin standard solution, baicalin standard solution and baicalein standard solution.

[0016] Preferably, the UPLC fingerprint of the pediatric Qingre Zhike granules contains 16 common characteristic peaks of the samples, among which peak No. 1 is ephedrine hydrochloride, peak No. 2 is pseudoephedrine hydrochloride, peak No. 4 is amygdalin, peak No. 8 is liquiritigenin, peak No. 12 is baicalin, and peak No. 16 is baicalin.

[0017] The present invention also provides the application of the UPLC fingerprint of the pediatric Qingre Zhike granules determined by the above determination method in the quality control and quality evaluation of the pediatric Qingre Zhike granules.

[0018] The present invention discloses the following technical effects:

[0019] The present invention adopts ultra-high performance liquid chromatography technology, prepares the pediatric clearing and relieving cough granules into a test solution, takes the chromatograms of single-flavor medicinal material decoction solutions and standard solutions as controls, attributes and identifies the chromatographic peaks in the test solution, and constructs a UPLC fingerprint containing 16 common characteristic peaks including ephedrine hydrochloride, pseudoephedrine hydrochloride, amygdalin, liquiritigenin, baicalin and baicalein, which provides a new technical solution for comprehensively evaluating the overall quality of the pediatric clearing and relieving cough granules, and provides a new technical idea for the quality control and quality evaluation of the pediatric clearing and relieving cough granules.

[0020] The analytical method established in the present invention has the characteristics of good precision, stability and repeatability. The chromatographic conditions are optimized, the detection speed is fast, the injection volume is small, there is no need to change the detection wavelength or flow rate, all characteristic substances can be separated in one step, and the operation is simpler and more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The chromatograms under different elution conditions;

[0023] Figure 2 The chromatograms under different mobile phase components;

[0024] Figure 3 The chromatograms are for different acid types;

[0025] Figure 4 Chromatogram at a flow rate of 0.2 ml / min;

[0026] Figure 5 Chromatogram at a flow rate of 0.3 ml / min;

[0027] Figure 6 Chromatogram at a flow rate of 0.4 ml / min;

[0028] Figure 7 Chromatogram at a flow rate of 0.5 ml / min;

[0029] Figure 8 The chromatogram is at 30°C column temperature;

[0030] Fig. 9 This is the chromatogram at a column temperature of 32°C;

[0031] Fig.10 The chromatogram is at 35°C column temperature;

[0032] Fig.11 The chromatogram at a wavelength of 195 nm;

[0033] Fig.12 The chromatogram at a wavelength of 203 nm;

[0034] Fig.13 The chromatogram at a wavelength of 210 nm;

[0035] Fig.14 The chromatogram at a wavelength of 230 nm;

[0036] Fig.15 The chromatogram is at a wavelength of 250 nm;

[0037] Fig.16 The chromatogram at a wavelength of 270 nm;

[0038] Fig.17 The chromatogram at a wavelength of 300 nm;

[0039] Fig.18 The chromatogram at a wavelength of 320 nm;

[0040] Fig.19 Examining chromatograms for maximum information;

[0041] Fig. 20 The chromatograms of different extraction reagents (methanol, 80% methanol, 50% methanol, 30% methanol, and 10% methanol) of the test solution;

[0042] Fig.21 The chromatograms of different extraction reagents (water, anhydrous ethanol, 80% ethanol, 50% ethanol, 30% ethanol, and 10% ethanol) of the test solution;

[0043] Fig. 22 The chromatograms of the test solution in different extraction methods are shown;

[0044] Fig.23 The chromatograms of the test solution at different extraction times;

[0045] Fig.24 The chromatograms of the test samples with different sample weights;

[0046] Fig.25 Chromatograms of the test solution at different extraction times

[0047] Fig.26 This is the comparison chromatogram of the peak attribution of medicinal flavor - Ephedra;

[0048] Fig. 27 This is the comparison chromatogram of the peak attribution of medicinal flavor - fried bitter almonds;

[0049] Fig.28 This is the comparison chromatogram of drug flavor peak attribution-licorice;

[0050] Fig.29 This is the comparison chromatogram of the peak attribution of medicinal flavor - Scutellaria baicalensis;

[0051] Fig.30 This is the comparison chromatogram of the peak attribution of medicinal flavor - Radix scutellariae;

[0052] Fig.31 This is the comparison chromatogram of the drug flavor peak attribution-Isatis root;

[0053] Fig.32 This is the comparison chromatogram of drug flavor peak attribution-gypsum;

[0054] Fig.33 It is a comparative chromatogram for identification of chromatographic peak standards; wherein, S1 is a mixed standard solution of ephedrine hydrochloride and pseudoephedrine hydrochloride, S2 is a standard solution of amygdalin, S3 is a standard solution of liquiritin, S4 is a standard solution of baicalin, S5 is a standard solution of baicalein, and S6 is a test solution;

[0055] Fig.34 It is a stacked diagram of fingerprints of 21 batches of pediatric Qingre Zhike granules; among them, S1-S21 are 21 batches of pediatric Qingre Zhike granules samples, and R is the control fingerprint;

[0056] Fig.35 For the control fingerprint;

[0057] Fig.36 To investigate the specificity, HPLC comparative spectra were used;

[0058] Fig.37 HPLC comparative spectrum for specificity inspection; R is the reference fingerprint spectrum, S2-S7 is the precision 1-6;

[0059] Fig.38 HPLC comparison spectrum for repeatability inspection; where R is the control fingerprint spectrum, S2-S7 are repeatability 1-6;

[0060] Fig.39 is a stability test chromatogram; wherein, R is the control fingerprint, S2 is stability 0h, S3 is stability 3h, S4 is stability 6h, S5 is stability 9h, S6 is stability 12h, S7 is stability 18h, S8 is stability 24h;

[0061] Fig.40 Chromatograms for different manufacturers' reagents; R is the reference fingerprint, S2 is Aston acetonitrile / Tianjin Damao phosphoric acid, and S3 is Merck acetonitrile / Sinopharm phosphoric acid;

[0062] Fig.41The chromatograms of different chromatographic columns were investigated; among them, R is the reference fingerprint, S2 is ZORBAX EclipseXDB-C18, and S3 is Hypersil GOLD. DETAILED DESCRIPTION

[0063] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0064] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0065] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0066] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0067] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0068] Example 1 Establishment of UPLC fingerprint of Xiaoer Qingre Zhike granules

[0069] 1. Establishment of chromatographic conditions

[0070] Preparation of test solution: Take a sample of pediatric Qingre Zhike granules (Heilongjiang Jiren Pharmaceutical Co., Ltd., batch number 221204), grind it into powder, accurately weigh 1 g, put it in a stoppered conical flask, accurately add 25 ml of 50% (v / v) methanol, ultrasonically treat (1100 W, 40 kHz) for 15 minutes, take it out, cool, shake well, and filter.

[0071] 1.1 Exploration of elution conditions

[0072] Octadecylsilane bonded silica gel was used as filler (ZORBAX Eclipse XDB-C18 2.1×100mm, 1.8μm); acetonitrile was used as mobile phase A, 0.1% (v / v) phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to Table 1; the flow rate was 0.3 ml per minute; the column temperature was 30°C; and the detection wavelength was 203nm. Comparing the chromatograms, as shown in Figure 1 As shown, the results show that the chromatogram under mobile phase elution condition 1 has a better separation effect on the index components than the chromatogram under condition 2, and can fully reflect its main components, so this is used as the gradient elution chromatographic condition.

[0073] Table 1 Mobile phase elution conditions

[0074]

[0075] 1.2 Investigation of mobile phase components

[0076] Octadecylsilane bonded silica gel was used as the filler (ZORBAX Eclipse XDB-C18 2.1×100mm, 1.8μm); acetonitrile and methanol were used as mobile phase A, 0.1% (v / v) phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to condition 1 in Table 1; the flow rate was 0.3ml per minute; the column temperature was 30°C; and the detection wavelength was 203nm. Comparing the chromatograms, as shown in Figure 2 As shown, the results show that the chromatogram baseline of the methanol-0.1% (v / v) phosphoric acid system is not stable, and the chromatographic peak response value is small, so the component of the mobile phase is temporarily proposed to be acetonitrile-0.1% (v / v) phosphoric acid system.

[0077] 1.3 Choice of acid type

[0078] Octadecylsilane bonded silica gel was used as the filler (ZORBAX Eclipse XDB-C18 2.1×100mm, 1.8μm); acetonitrile was used as the mobile phase A, and 0.1% (v / v) phosphoric acid solution, 0.1% (v / v) formic acid solution, and 0.1% (v / v) glacial acetic acid solution were investigated respectively, and gradient elution was performed according to condition 1 in Table 1; the flow rate was 0.30ml per minute; the column temperature was 30℃; and the detection wavelength was 203nm. Comparing the chromatograms, as shown in Figure 3 As shown, the results show that the baseline of acetonitrile-0.1% (v / v) phosphoric acid solution is relatively stable. Taking into account the absorption of formic acid and glacial acetic acid at low wavelengths and their characteristic volatility, 0.1% (v / v) phosphoric acid solution is temporarily proposed as the mobile phase.

[0079] 1.4 Flow rate selection

[0080] Octadecylsilane bonded silica gel was used as filler (ZORBAX Eclipse XDB-C18 2.1×100mm, 1.8μm); acetonitrile was used as mobile phase A, 0.1% (v / v) phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to condition 1 in Table 1; the flow rates were 0.2ml / min, 0.3ml / min, 0.4ml / min, and 0.5ml / min, respectively; the column temperature was 30°C; and the detection wavelength was 203nm. Comparing the chromatograms, as shown in Figure 4-Figure 7 As shown in the figure, the results show that different flow rates have a great influence on the overall chromatographic peak shape. When the flow rate is 0.2ml~0.4ml per minute, the overall chromatographic peak shape is not good, so the flow rate is temporarily set to 0.5ml per minute.

[0081] 1.5 Selection of column temperature

[0082] Octadecylsilane bonded silica gel was used as filler (ZORBAX Eclipse XDB-C18 2.1×100mm, 1.8μm); acetonitrile was used as mobile phase A, 0.1% (v / v) phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 0.5ml; the column temperature was tested at 30℃, 32℃, and 35℃ respectively; the detection wavelength was 203nm. Comparing the chromatograms, as shown in the following table Figure 8-Figure 10 As shown in the figure, the results show that different column temperatures have little effect on the overall chromatographic peak shape. Therefore, the column temperature is temporarily set at 35℃.

[0083] 1.6 Selection of detection wavelength

[0084] Accurately pipette the sample solution into the liquid chromatograph and use a diode array detector to perform a full wavelength scan at 190-400nm. Extract the chromatograms at different detection wavelengths for comparison. Figure 11-Figure 18 As shown, the results show that when the detection wavelength is 195-210nm, the overall chromatographic peak information is more; therefore, the detection wavelength is temporarily set to 210nm.

[0085] 1.7 Information maximization study

[0086] In order to investigate whether the main components of the test sample are reflected in the spectrum and whether the principle of maximizing effective information is met, acetonitrile is used as mobile phase A, 0.1% (v / v) phosphoric acid solution is used as mobile phase B, the flow rate is 0.5 ml / min; the column temperature is 35°C; the detection wavelength is 210 nm; gradient elution is performed according to Table 2, and the acquisition time is increased to 120 minutes. Inject the test solution and record the chromatogram, such as Fig.19 As shown, the results show that by extending the elution time of high-proportion acetonitrile, there are no other chromatographic peaks in the test solution, and the information collected by this method meets the principle of information maximization.

[0087] Table 2 Mobile phase elution conditions

[0088] Time (min) Mobile phase A (%) Mobile phase B (%) 0～4 5 95 4～15 5→15 95→85 15～40 15→70 85→30 40～120 70 30

[0089] 2. Investigation of the preparation method of test solution

[0090] 2.1 Investigation of extraction reagents

[0091] Take a sample, grind it into powder, accurately weigh 1g, put it in a stoppered conical flask (11 parallel portions), accurately add 25ml of water, 10% (v / v) methanol, 30% (v / v) methanol, 50% (v / v) methanol, 80% (v / v) methanol, methanol, anhydrous ethanol, 10% (v / v) ethanol, 30% (v / v) ethanol, 50% (v / v) ethanol, 80% (v / v) ethanol, respectively, weigh it, ultrasonically treat it (1100W, 40kHz) for 15 minutes, take it out, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and you are done. Accurately pipette each of the above test sample solutions into a liquid chromatograph, and collect the chromatogram. Figure 20-21 As shown, the results show that when water, anhydrous ethanol and 80% ethanol are used as solvents, chromatographic peaks are lost, so they are not included in the peak area comparison. There is no obvious difference in the chromatographic peaks of other solvent chromatograms. The peak area of ​​each solvent is statistically analyzed, as shown in Table 3. The results show that the RSD value of solvents with different concentrations is 1.98%, which is less than 2%, indicating that there is no obvious difference. Combined with the peak shape of each chromatographic peak and the convenience of operation, it is temporarily planned that the extraction solvent for the test solution is 30% methanol.

[0092] Table 3 Comparison of peak areas of fingerprints of different solvents

[0093]

[0094] 2.2 Investigation of extraction methods

[0095] Take a sample, grind it into powder, weigh 1g accurately, put it in a stoppered conical flask (2 parallel parts), accurately add 25ml of 30% (v / v) methanol, weigh it, examine ultrasonic treatment (1100W, 40kHz) and heating reflux, extract for 15 minutes, take it out, cool it, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and you are done. Accurately pipette the above test sample solutions into a liquid chromatograph, collect chromatograms, and count the peak areas of the two extraction methods. Fig. 22 As shown in Table 4, the results show that the chromatographic peak information and response values ​​of the two extraction methods are basically the same, and the RAD value is 0.6%, which is less than 2%. Combined with the convenience and feasibility of the operation during the preparation of the test solution, ultrasound is temporarily proposed as the extraction method for the test solution.

[0096] Table 4 Comparison of fingerprint peak areas under different extraction methods

[0097]

[0098] 2.3 Investigation of extraction time

[0099] Take a sample, grind it into powder, accurately weigh 1g, put it in a stoppered conical flask (3 parallel copies), accurately add 25ml of 30% (v / v) methanol, weigh the weight, ultrasonically treat (1100W, 40kHz) for 15 minutes, 30 minutes, and 45 minutes, take it out, cool it, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and you are done. Accurately pipette the above test sample solutions into a liquid chromatograph, collect chromatograms, and count the peak areas at different extraction times. Fig.23 As shown in Table 5, the results show that the information content and response value of the chromatographic peaks with extraction time of 15 to 45 minutes are basically the same. Its RAD value is 0.6%, which is less than 2%. Combined with the convenience of operation during the preparation of the test solution, 15 minutes is temporarily set as the extraction time of the test solution.

[0100] Table 5 Comparison of fingerprint peak areas at different extraction times

[0101]

[0102] 2.4 Sample weighing inspection

[0103] Take this product, grind it into powder, take 0.5g, 1.0g, 1.5g, 2.0g, 3.0g respectively, weigh accurately, put them in stoppered conical flasks respectively, add 25ml of 30% (v / v) methanol accurately, weigh the weight, ultrasonically treat (1100W, 40kHz) for 15 minutes, take out, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and obtain. Accurately pipette the above test sample solutions into liquid chromatograph, collect chromatograms, and count the peak areas of different sample amounts. Fig.24 As shown in Table 6, the results show that when the sample weight reaches 1.5 g, the overall response value of the chromatographic peak is good; and from Table 6, the concentration ratio is saturated, and 1.5 g is temporarily set as the sample weight of the test solution.

[0104] Table 6 Comparison of peak areas of fingerprints of different weighing samples

[0105] Sample weight (g) Total peak area Sample weight (g) Total peak area / sample weight 0.5 3839943 0.5075 7566390 1.0 7530769 1.0034 7505251 1.5 11464408 1.5051 7617041 2.0 14558620 2.0071 7253560 3.0 20591833 3.0009 6861886

[0106] 2.5 Investigation of extraction times

[0107] Take this product, grind it into powder, take about 1.5g, weigh it accurately, put it in a stoppered conical bottle, accurately add 25ml of 30% (v / v) methanol, weigh it, extract it twice, ultrasonically treat it (1100W, 40kHz) for 15 minutes each time, take it out, cool it, weigh it again, make up the lost weight with the corresponding solvent, shake it well, filter it, and you are done. Accurately pipette the above test solution into the liquid chromatograph, collect the chromatogram, such as Fig.25 As shown, the results showed that when the test sample was extracted for the second time, the overall response value was slightly lower, and one extraction was tentatively proposed as the number of extractions for the test sample solution.

[0108] 3. Peak attribution and reference selection

[0109] 3.1 Attribution of drug flavor peaks

[0110] Referring to the preparation method of the test solution determined above, prepare ephedra (TianDa Chinese Medicine (China) Co., Ltd., T1012-202212-1), stir-fried bitter almond (TianDa Chinese Medicine (China) Co., Ltd., T1007-202212-4), licorice (Zhuhai Chinese Medicine Pieces Co., Ltd., 231027-020), scutellaria baicalensis (Zhuhai Chinese Medicine Pieces Co., Ltd., T1008-202301-1), northern bean root (TianDa Chinese Medicine (China) Co., Ltd., The single-flavor decoction solutions of Radix Isatidis (TianDa Chinese Medicine (China) Co., Ltd., T1001-202212-5), Gypsum (TianDa Chinese Medicine (China) Co., Ltd., T1006-202212-5), and the negative solutions corresponding to each flavor were measured according to the above-mentioned chromatographic conditions and compared with the chromatogram of the test solution of Xiaoer Qingre Zhike Granules to determine the flavor of each chromatographic peak in the sample fingerprint. The comparative chromatograms are as follows: Figure 26-Figure 32 The retention time of the chromatographic peaks in the characteristic spectrum of each medicinal piece is shown in Table 7. After comparing each single medicinal piece with the test sample, it was concluded that peaks 1 and 2 came from ephedra, peaks 3 and 4 came from fried bitter almonds, and peaks 5, 6, and 7 all came from northern bean root. Peak 8 came from liquorice. Peaks 9, 12, 13, 14, 15, and 16 all came from scutellaria baicalensis, and peaks 10 and 11 came from scutellaria baicalensis and northern bean root, which are common peaks.

[0111] Table 7 Retention time of chromatographic peaks in characteristic spectra of each medicinal decoction piece

[0112]

[0113] 3.2 Chromatographic peak identification

[0114] Using 30% (v / v) methanol as solvent, prepare 20μg / ml ephedrine hydrochloride, 20μg / ml pseudoephedrine hydrochloride mixed standard solution, 50μg / ml amygdalin standard solution, 30μg / ml liquiritin standard solution, 60μg / ml baicalin standard solution, 60μg / ml baicalein standard solution, and determine according to the above-determined chromatographic conditions. Perform chromatographic comparison between the test solution and each standard solution, and identify the chromatographic peaks. The results are as follows: Fig.33 As shown, the results show that the chromatographic peak at 2.281 minutes is the chromatographic peak of ephedrine hydrochloride, the chromatographic peak at 2.446 minutes is the chromatographic peak of pseudoephedrine hydrochloride, the chromatographic peak at 7.508 minutes is the chromatographic peak of amygdalin, the chromatographic peak at 14.842 minutes is the chromatographic peak of liquiritin, the chromatographic peak at 20.755 minutes is the chromatographic peak of baicalin, and the chromatographic peak at 25.784 minutes is the chromatographic peak of baicalein. The results of the attribution of the medicinal taste of each chromatographic peak and the identification of the standard are shown in Table 8.

[0115] Table 8 Fingerprint chromatographic peaks attribution and standard identification

[0116]

[0117]

[0118] 4. Preliminary determination of analytical methods

[0119] Determined by high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0120] (1) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler (length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm), acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to condition 1 in Table 1; the flow rate was 0.5 ml per minute; the column temperature was 35°C; and the detection wavelength was 210 nm. The theoretical plate number calculated based on the baicalin peak should be no less than 10,000.

[0121] (2) Preparation of reference solution: Take appropriate amounts of ephedrine hydrochloride, pseudoephedrine hydrochloride, amygdalin, liquiritin, baicalin, and baicalein reference substances, weigh them accurately, and add 30% (v / v) methanol to prepare a solution containing 20 μg of ephedrine hydrochloride, 20 μg of pseudoephedrine hydrochloride, 50 μg of amygdalin, 30 μg of liquiritin, 60 μg of baicalin, and 60 μg of baicalein per 1 ml, which serves as the reference solution.

[0122] (3) Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 1.5 g, weigh it accurately, place it in a stoppered conical flask, accurately add 25 ml of 30% methanol, stopper it tightly, weigh it, ultrasonically treat it (power 1100 W, 40 kHz) for 15 minutes, let it cool, weigh it again, make up the lost weight with 30% (v / v) methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0123] (4) Determination: Accurately pipette 1 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and measure.

[0124] 5. Establishment of reference fingerprint

[0125] Take 21 batches of Xiaoer Qingre Zhike Granules (Heilongjiang Jiren Pharmaceutical Co., Ltd.) that have been on the market, prepare the test solution according to the method under "4. Preliminary determination of analytical methods", and record the chromatograms in turn. The fingerprints of 21 batches of Xiaoer Qingre Zhike Granules were evaluated for similarity using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" of the Pharmacopoeia Committee. Mark peak matching was performed with the common peaks to form a common pattern diagram ( Fig.34 ) and establish a reference fingerprint ( Fig.34 R and Fig.35 ), 16 common peaks were detected in the fingerprints of 21 batches of samples. The similarity was calculated, as shown in Table 9, the similarity of 21 batches of Xiaoer Qingre Zhike Granules was 0.994-1.000.

[0126] Table 9 Similarity results of 21 batches of children's Qingrezhike granules

[0127]

[0128]

[0129] Example 2 Methodology Verification

[0130] 1. Specificity test

[0131] In order to investigate whether the blank solvent and excipients interfere with the fingerprint of the children's Qingrezhike granules, under the chromatographic conditions determined in Example 1, 30% (v / v) methanol solution, excipients, reference solution, and test solution were accurately drawn into the liquid chromatograph and the chromatogram was recorded. Fig.36 As shown, the results show that the blank solvent and excipients have no interfering peaks at the chromatographic peak position and have good specificity.

[0132] 2. Precision test

[0133] Take the test solution, inject it 6 times continuously, record the chromatogram, and compare it with the reference fingerprint ( Fig.35 ) for comparison, such as Fig.37As shown in Table 10, the similarity values ​​of the common peaks of the Children's Qingre Zhike Granules were all 1.000. The "National Drug Standard Work Manual" (4th edition) requires that the precision similarity of the fingerprint spectrum should not be less than 0.95. The results show that the instrument has good precision.

[0134] Table 10 Similarity results of precision investigation

[0135] Sample name Precision 1 Precision 2 Precision 3 Precision 4 Precision 5 Precision 6 RSD(%) Similarity 1.000 1.000 1.000 1.000 1.000 1.000 0.0

[0136] 3. Repeatability test

[0137] Take about 1.5g of this product, weigh accurately, operate according to the preparation method and chromatographic conditions of the test solution in Example 1, prepare 6 samples in parallel, record the chromatogram, and compare it with the reference fingerprint ( Fig.35 ) for comparison, such as Fig.38 As shown in Table 11, the common peak similarity values ​​were all 1.000. The National Drug Standard Work Manual (4th edition) requires that the fingerprint repeatability similarity should not be less than 0.95. The results show that the repeatability is good.

[0138] Table 11 Similarity results of repeatability test

[0139] Sample name Repeatability 1 Repeatability 2 Repeatability 3 Repeatability 4 Repeatability 5 Repeatability 6 Similarity 1.000 1.000 1.000 1.000 1.000 1.000

[0140] 4. Durability test

[0141] 4.1 Stability

[0142] Take the repetitive 1 sample and place it for 0, 3, 6, 9, 12, 18, and 24 hours after preparation, operate according to the chromatographic conditions of Example 1, record the chromatogram, and compare it with the control fingerprint ( Fig.35 ) for comparison, such as Fig.39 As shown in Table 12, the common peak similarity values ​​were all 1.000. The National Drug Standard Work Manual (4th Edition) requires that the fingerprint stability similarity should not be less than 0.95. The results show that the test solution has good stability within 24 hours.

[0143] Table 12 Similarity results of stability study

[0144] Sample name Stability 0h Stability 3h Stability 6h Stability 9h Stability 12h Stability 18h Stability 24h Similarity 1.000 1.000 1.000 1.000 1.000 1.000 1.000

[0145] 4.2 Reagents from different manufacturers

[0146] Take about 1.5g of sample, weigh accurately, operate according to the preparation method and chromatographic conditions of the test solution in Example 1, investigate Aston acetonitrile / Tianjin Damao phosphoric acid, Merck acetonitrile / Sinopharm phosphoric acid, record the chromatogram, and compare it with the control fingerprint ( Fig.35 ) for comparison, such as Fig.40As shown in Table 13, the common peak similarity values ​​were all 1.000. The National Drug Standard Work Manual (4th edition) requires that the similarity of fingerprint spectra of reagents from different manufacturers should not be less than 0.90. The results show that the durability of reagents from different manufacturers is good.

[0147] Table 13 Similarity results of reagents from different manufacturers

[0148] Sample name Aston Acetonitrile / Tianjin Damao Phosphoric Acid Merck acetonitrile / Sinopharm phosphoric acid Similarity 1.000 1.000

[0149] 4.3 Different chromatographic columns

[0150] Take about 1.5g of sample, weigh accurately, operate according to the preparation method and chromatographic conditions of the test solution in Example 1, examine different brands of chromatographic columns, record the chromatogram, and compare it with the reference fingerprint ( Fig.35 ) for comparison, such as Fig.41 As shown in Table 14, the common peak similarity values ​​were measured to be 1.000 and 0.998, respectively. The National Drug Standard Work Manual (4th edition) requires that the similarity of fingerprint spectra from different chromatographic columns should not be less than 0.90. The results show that chromatographic columns of different brands have good durability.

[0151] Table 14 Similarity results of different chromatographic columns

[0152] Sample name ZORBAX Eclipse XDB-C18 HypersilGOLD Similarity 1.000 0.998

[0153] 5. Conclusion

[0154] After the above-mentioned methodological verification, the fingerprints obtained by the determination were basically consistent, the characteristic peaks in the chromatogram were sharp, and 16 main chromatographic characteristic peaks were present in the fingerprint of the test sample, which corresponded to the characteristic peaks in the chromatographic peaks of the control fingerprint. This shows that this method has good durability and can be applied to the quality control of Xiaoer Qingre Zhike Granules.

[0155] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for determining the UPLC fingerprint of Xiaoer Qingre Zhike Granules, characterized in that: The steps include: (1) Prepare test sample solution and standard solution; (2) performing UPLC chromatographic analysis on the test solution and the standard solution to obtain a UPLC fingerprint of the pediatric Qingre Zhike granules consisting of common characteristic peaks of the samples; The preparation method of the test solution is: weighing a sample of the Xiaoer Qingre Zhike granules, adding a methanol aqueous solution, and subjecting the mixture to ultrasonic treatment to obtain the test solution; The conditions of the UPLC chromatographic analysis are: octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B; detection wavelength is 210 nm; elution procedure is: 。 2. The measuring method according to claim 1, characterized in that The preparation method of the test solution is as follows: accurately weigh 1.5 g of the pediatric Qingre Zhike granule sample, add 25 ml of 30% methanol, and ultrasonicate for 15 minutes at 1100 W and 40 KHz to obtain the test solution.

3. The measuring method according to claim 1, characterized in that The flow rate of the UPLC chromatography analysis was 0.5 ml per minute, the column temperature was 35° C., and the sample loading volume was 1 μl.

4. The measuring method according to claim 1, characterized in that The UPLC chromatographic analysis includes the step of identifying the common characteristic peaks of the samples in the chromatogram of the test solution by using the chromatogram of the standard solution as a reference.

5. The measuring method according to claim 1, characterized in that The standard solution uses 30% methanol as a solvent, and includes a mixed standard solution of ephedrine hydrochloride and pseudoephedrine hydrochloride, amygdalin standard solution, liquiritin standard solution, baicalin standard solution and baicalein standard solution.

6. The measuring method according to claim 1, characterized in that The UPLC fingerprint of the pediatric Qingre Zhike granules contains 16 common characteristic peaks of the samples, among which peak No. 1 is ephedrine hydrochloride, peak No. 2 is pseudoephedrine hydrochloride, peak No. 4 is amygdalin, peak No. 8 is liquiritigenin, peak No. 12 is baicalin, and peak No. 16 is baicalein.

7. Application of the UPLC fingerprint of the Pediatric Qingre Zhike Granules determined by the determination method according to any one of claims 1 to 6 in the quality control and quality evaluation of the Pediatric Qingre Zhike Granules.

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