HPLC (High Performance Liquid Chromatography) fingerprint spectrum detection method for children lung-ventilating and cough-relieving

Abstract

The traditional Chinese medicine is prepared from twelve traditional Chinese medicinal materials including ephedra, ledebouriella cantoniensis, southwest scutellaria baicalensis, platycodon grandiflorum, semen sinapis, semen armeniacae amarae, semen lepidii, Indian kalimeris herb, astragalus membranaceus, Chinese yam, hawthorn and liquorice, has the effects of clearing away heat and relieving exterior syndrome, and is used for treating exogenous cough of children, lung-dispersing cough, lung-dispersing cough, lung-dispersing cough, lung-dispersing cough, lung-dispersing cough, lung-dispersing cough, lung-dispersing cough and lung-dispersing cough. The fingerprint spectrum established by the invention comprises 15 common characteristic peaks which respectively belong to eleven medicinal materials including semen armeniacae amarae, radix astragali seu hedysari, rhizoma dioscoreae, radix saposhnikoviae, herba ephedrae, semen lepidii, semen sinapis, radix platycodonis, southwest radix scutellariae, radix glycyrrhizae and fructus crataegi, so that the eleven medicinal materials in the traditional Chinese medicine can be identified, and meanwhile, two components in the eleven medicinal materials, namely baicalin and chlorogenic acid, are identified. The quality of the medicine can be controlled more comprehensively, the problem of fake and inferior phenomena of the medicine is solved, and the safety and effectiveness of clinical use are ensured.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine detection and relates to an HPLC fingerprint detection method for Xiaoer Xuanfei Zhike granules. Background Art

[0002] The national drug standard (WS3-422 (Z-066) -2001 (Z)) includes a traditional Chinese medicine with the effect of clearing away heat and relieving exterior symptoms, which is used for children's exogenous wind-heat cough. The name of the medicine is "Pediatric Xuanfei Zhike Granules", which is a product of Jianmin Group Yekatai Thai Pharmaceutical (Suizhou) Co., Ltd., with the approval number "National Medicine Standard Z10970029". The medicine is made of twelve traditional Chinese medicinal materials, including ephedra, saposhnikovia divaricata, southwestern scutellaria, platycodon, mustard, bitter almond, scutellaria baicalensis, amaranth, astragalus, yam, hawthorn, and licorice. It is used for children's exogenous cough, cough caused by phlegm-heat blocking the lungs, with abundant phlegm, yellow and sticky phlegm, and uncomfortable phlegm.

[0003] CN 110907550 A discloses a method for simultaneously detecting the contents of six components of children's Xuanfei Zhike granules, using a C18 chromatographic column as a stationary phase, acetonitrile and formic acid solution as mobile phases, and an ultraviolet detection wavelength of 205-230nm. The HPLC method is used to detect the contents of the six components, namely, ephedrine hydrochloride, baicalin, amygdalin, astragaloside IV, liquiritin, and cimicifuga glycoside. The method has the advantages of high detection efficiency, accuracy, sensitivity, etc., and is conducive to achieving quality control of children's Xuanfei Zhike granules. However, Chinese medicine produces its therapeutic effect through the synergistic effects of multiple components and targets. The components detected by this method can achieve the control of the six medicinal materials in the pediatric Xuanfei Zhike granules, namely ephedra, saposhnikovia divaricata, scutellaria baicalensis, bitter almond, astragalus, and licorice, but cannot achieve the control of other key medicinal materials in the prescription, including yam, scutellaria baicalensis, mustard, platycodon, and hawthorn. This provides an opportunity for the adulteration and counterfeiting of these medicinal materials, and also creates loopholes in ensuring the consistency of Chinese medicine.

[0004] In view of the multi-component and multi-target characteristics of traditional Chinese medicine and its compound preparations, the fingerprint of traditional Chinese medicine can fully reflect the chemical composition information contained in it, especially in the current situation where the effective ingredients of traditional Chinese medicine compound preparations have not been fully elucidated, it can realize the comprehensive evaluation of the intrinsic quality of traditional Chinese medicine and the effective control of its overall substances. It is an important method for quality control of traditional Chinese medicine and Chinese patent medicine recognized at home and abroad. If the HPLC fingerprint of Xiaoer Xuanfei Zhike Granule can be established, then combining the qualitative analysis of the fingerprint and the quantitative analysis of the test results will be more conducive to the comprehensive evaluation of the quality of traditional Chinese medicine, prevent counterfeiting and ensure the consistency of clinical medication. Summary of the invention

[0005] In view of the shortcomings of the current quality detection methods and evaluation systems for pediatric Xuanfei Zhike granules, the purpose of the present invention is to establish an HPLC fingerprint detection and analysis method for pediatric Xuanfei Zhike granules, aiming to monitor and evaluate the quality of the drug more comprehensively as a whole, and ensure the safety and effectiveness of the drug in clinical use.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A HPLC fingerprint detection method for children's Xuanfei Zhike granules, wherein the granules are made of twelve Chinese medicinal materials, namely, ephedra, saposhnikovia divaricata, scutellaria baicalensis, platycodon grandiflorum, mustard seeds, bitter almonds, scutellaria baicalensis, amaranth, astragalus, yam, hawthorn, and liquorice, and have the effects of clearing away heat and relieving exterior symptoms, and are used for children's exogenous cough. The detection method comprises the following steps:

[0008] (1) ultrasonically extracting the Xiaoer Xuanfei Zhike granules with methanol, filtering with a microporous filter membrane, and taking the filtrate to obtain a test solution;

[0009] (2) Weigh baicalin and chlorogenic acid reference substances separately and prepare a single reference substance solution with methanol;

[0010] (3) Accurately pipetting the test solution or the single reference solution, and detecting it with a high performance liquid chromatograph, wherein the high performance liquid chromatograph comprises an ultraviolet detector, the detection wavelength of the ultraviolet detector is set to 210-320 nm, the stationary phase of the high performance liquid chromatograph is a C18 chromatographic column, the mobile phase A is acetonitrile, the mobile phase B is methanol, the mobile phase C is an acid solution, the gradient elution, the flow rate is 0.5-2.0 ml / min, the column temperature is 25-35° C., and the theoretical plate number is not less than 3500 calculated based on baicalin;

[0011] (4) Record the chromatograms of the test solution and the single reference solution, and use the Chinese Pharmacopoeia Committee's Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System to import data, perform multi-point correction and data matching on the chromatogram of the test solution to obtain the standard fingerprint of the Chinese medicine, compare the fingerprint of the sample to be tested with the chromatogram of each single reference solution and evaluate the similarity with the standard fingerprint.

[0012] Wherein, the methanol described in steps (1) and (2) is 70% methanol, and the methanol described in step (3) is pure methanol.

[0013] The ultrasonic extraction has a power of 300 W, a frequency of 40 kHz and a time of 30 min.

[0014] Wherein, the pore size of the microporous filter membrane is 0.8 μm and 0.45 μm.

[0015] Wherein, the detection wavelength of the ultraviolet detector is set to 260nm.

[0016] Wherein, the mobile phase C is 0.2% trifluoroacetic acid solution.

[0017] The gradient elution time is 80 min, which is divided into 10 sections. The volume ratio of the mobile phase in each section is as follows:

[0018]

[0019] Wherein, the column temperature is 25°C.

[0020] Among them, each 1 ml of single reference solution contains 50 μg of chlorogenic acid and 60 μg of baicalin.

[0021] Among them, there are 15 common peaks in the standard fingerprint, among which peaks 1 and 2 belong to bitter almonds, peak 3 belongs to astragalus and yam, peaks 4 and 10 belong to saposhnikovia divaricata, peak 5 belongs to ephedra, peak 6 belongs to scutellaria baicalensis and mustard seeds, peak 7 belongs to scutellaria baicalensis, peak 8 belongs to platycodon, peaks 9, 12 and 13 belong to scutellaria baicalensis, peak 11 belongs to liquorice, peak 14 belongs to hawthorn, and peak 15 belongs to astragalus and hawthorn. After comparison with the chromatogram of a single reference solution, it was confirmed that peak 3 was chlorogenic acid and peak 9 was baicalin.

[0022] Using the Chinese medicine chromatographic fingerprint similarity evaluation system (2004A version), the chromatogram of the sample to be tested was compared with the fitted standard liquid fingerprint to obtain the similarity. The samples with a similarity greater than 0.900 were judged to be qualified.

[0023] The beneficial effects of the present invention are:

[0024] The present invention successfully establishes a fingerprint spectrum detection and analysis method for children's Xuanfei Zhike granules. The established fingerprint spectrum includes 15 common characteristic peaks, which are attributed to eleven medicinal materials, namely, bitter almond, astragalus, yam, bamboo leaf siler, ephedra, scutellaria, mustard, platycodon, southwestern scutellaria, liquorice, and hawthorn. Therefore, the eleven medicinal materials in the product can be identified, and two components, namely, baicalin and chlorogenic acid, are also identified. Therefore, the present invention can more comprehensively control the quality of drugs, solve the problem of counterfeit and inferior drugs, and ensure the safety and effectiveness of clinical use.

[0025] The fingerprint spectrum established by the present invention has a good overall effect (large number of peaks, large main peak area, high separation, etc.), can be detected by one extraction and sampling, and the method is simple to operate, short in time, and has good reproducibility of results. It can strengthen the overall quality monitoring and evaluation of the pediatric Xuanfei Zhike granules to ensure clinical safety and effectiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the liquid chromatogram prepared under the optimal conditions.

[0027] Figure 2 is a comparative chromatogram of the extraction solvents.

[0028] Figure 3 It is a comparative chromatogram at different wavelengths.

[0029] Figure 4 This is the liquid chromatogram obtained under the mobile phase gradient elution condition (1).

[0030] Figure 5 This is the liquid chromatogram obtained under the mobile phase gradient elution condition (2).

[0031] Figure 6 This is the liquid chromatogram obtained under the mobile phase gradient elution condition (3).

[0032] Figure 7 The chromatograms are compared at different column temperatures.

[0033] Figure 8 It is an overlay of fingerprint spectra of 12 batches of samples.

[0034] Fig. 9 It is the standard fingerprint of Xiaoer Xuanfei Zhike Granules.

[0035] Fig.10 It is an identification diagram comparing the sample fingerprint and the chromatograms of each reference substance. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with the embodiments.

[0037] Instruments and reagents: Shimadzu LC-2040C CN liquid chromatograph, including diode array detector, Labsolutions workstation; PGJ-10 / 20-AS ultrapure water instrument (Wuhan Pinguan Instrument Co., Ltd.); Mettler ME203E, Mettler ME104E, Mettler TOLEDO XPE105 electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.). Agilent TC-C18 (2) chromatographic column (5 μm, 250 × 4.6 mm), the theoretical plate number calculated based on baicalin should be no less than 3500.

[0038] Chlorogenic acid reference substance (purity 96.03%, batch number 110753-20219) and baicalin reference substance (purity 97.2%, batch number 110715-202223) were purchased from China Food and Drug Inspection Institutes; acetonitrile and methanol were of chromatographic grade, n-butanol, ethyl acetate, ethanol, trifluoroacetic acid, etc. were of analytical grade, and water was purified water.

[0039] The Xiaoer Xuanfei Zhike Granules are from Jianmin Group Yekai Thai Pharmaceutical (Suizhou) Co., Ltd., with batch numbers of 220702, 220801, 220903, 221101, 221202, 230201, 230301, 230302, 230402, 230502, 230601, 230701, represented by S1-S12, and the prescription is as follows:

[0040] Ephedra 105g, Saposhnikovia divaricata 210g, Scutellaria baicalensis 210g, Platycodon grandiflorum 105g, Mustard seed 105g, Bitter almond 105g, Lemongrass seed 105g, Amaranthus 210g, Astragalus 210g, Dioscorea batatas 105g, Hawthorn 210g, Licorice 105g.

[0041] Example 1 Screening and Optimization of Chromatographic Conditions

[0042] 1. Investigation of sample extraction solvent

[0043] Five different extraction solvents, including 70% (volume) methanol, pure methanol, ethanol, ethyl acetate, and water-saturated n-butanol, were investigated.

[0044] Take an appropriate amount of this product (batch number: 230402) under the item of filling amount, accurately weigh 5 portions, each about 5 g, and place them in stoppered conical flasks respectively. Accurately add 70% methanol, pure methanol, ethanol, ethyl acetate, and 15 ml of water-saturated n-butanol to each, weigh the weight, and treat with ultrasound (power 300 W, frequency 40 kHz) for 30 minutes. Cool, weigh the weight again, make up the lost weight with the corresponding extraction solvent, shake well, filter with 0.8μm and 0.45μm microporous filter membranes in turn, and take the filtrate to obtain.

[0045] The chromatographic conditions were as follows:

[0046] Chromatographic column: Agilent TC-C18(2) (250 mm × 4.6 mm, 5 μm);

[0047] Mobile phase: acetonitrile, methanol, 0.2% (volume) trifluoroacetic acid aqueous solution;

[0048] Gradient elution program:

[0049]

[0050]

[0051] The flow rate is 1 ml / min;

[0052] The column temperature was 25°C;

[0053] The injection volume was 20 μl;

[0054] Detection wavelength: 260nm;

[0055] The number of theoretical plates is not less than 3500. The chromatographic recording time is 80 minutes.

[0056] By comparison, it can be seen (see Figure 2 ), under the same detection conditions, there are almost no peaks in the chromatogram of the test solution prepared by extraction with ethyl acetate, the chromatogram of the test solution prepared by extraction with ethanol lacks characteristic peaks 1, 10, and 14, the chromatogram of the test solution prepared by extraction with water-saturated n-butanol lacks characteristic peaks 3 and 7, the overall effect of the chromatograms prepared by the two extraction solvents of methanol and 70% methanol solution is basically the same, the number of peaks and the separation of the main chromatographic peaks are good, among which the peak height of the main chromatographic peak in the chromatogram prepared by extraction with 70% methanol solution is stronger than that of methanol, so 70% methanol is preferably used as the extraction solvent.

[0057] 2. Investigation of detection wavelength

[0058] The 230402 batches of pediatric Xuanfei Zhike granules were tested, and the chromatographic effects at different wavelengths such as 210, 230, 245, 260, 275, 290, 305, and 320 nm were investigated. 70% methanol was used as the extraction solvent, and other chromatographic conditions were the same as in Example 1.

[0059] From the experimental results (see Figure 3 ), in general, the smaller the wavelength, the more chromatographic peaks there are, and the stronger the peak signal is. For example, at wavelengths of 275, 290, 305, and 320 nm, peaks 2, 14, and 15 are missing or have very weak peak signals, but some peaks, such as chromatographic peaks 1 and 14, have weak or even no peaks below 260 nm. Comprehensive analysis shows that the overall effects of peak number, responsiveness, baseline noise, etc. at 260 nm are better, so the wavelength of 260 nm is selected as the optimal detection wavelength for fingerprints.

[0060] 3. Optimization of mobile phase elution program

[0061] The gradient elution program was optimized with acetonitrile (A): 0.2% phosphoric acid aqueous solution (B) as the mobile phase system, and the following three gradient elution conditions were investigated:

[0062] Gradient elution condition 1:

[0063]

[0064] Gradient elution condition 2:

[0065]

[0066] Gradient elution condition 3:

[0067]

[0068] Gradient elution condition 4:

[0069]

[0070] Results: Figure 4 , 5 As shown in Figures 6 and 1, under gradient elution condition (1), the peak shape is poor and some peaks are not separated; under gradient elution condition (2), the chromatographic peaks are concentrated in time and the separation of some main peaks is poor; under gradient elution condition (3), the overall effect of the chromatogram is good and the distribution of the main chromatographic peaks is appropriate, but there are many interfering peaks at chromatographic peaks 10 and 11, especially peak 11 may contain impurities; therefore, on the basis of gradient elution condition (3), a third phase is introduced into the mobile phase, namely gradient elution condition (4). Under this condition, the separation effect of chromatographic peaks 10 and 11 is significantly improved, and the peak shape and peak separation of the main chromatographic peaks are the best, so gradient elution condition (4) is preferred.

[0071] 4. Column temperature inspection

[0072] The 230402 batches of Xiaoer Xuanfei Zhike Granules were tested and the overall effect of the chromatograms of the samples measured at column temperatures of 25°C, 30°C, and 35°C were compared. Figure 7 ). The results show that when the column temperature is 30℃, the peaks emerge faster, the peak shape of peak 1 is poor, and the separation of peaks 5 and 15 is poor; when the column temperature is 35℃, the peaks emerge faster, the impact on chromatographic peaks 1-12 is greater, and the separation is poor. Overall, the column temperature of 25℃ is better, so the preferred column temperature is 25℃.

[0073] Example 2 Methodological Investigation

[0074] 1. Repeatability test

[0075] Take the same batch of Xiaoer Xuanfei Zhike Granules (batch number: 230402) for the test solution, prepare 6 test solutions according to the optimized conditions and measure them to examine the repeatability. Select the chromatographic peak No. 8 in the middle as the reference peak, and calculate the RSD value of the relative retention time and relative peak area of ​​each common peak. The results show that the relative standard deviation of the relative retention time of each chromatographic peak is less than 2.0%, and the relative standard deviation of the relative peak area of ​​each chromatographic peak is less than 3.0%, and the repeatability is good. See Tables 1 and 2.

[0076] Table 1 Relative retention time of each peak in repeatability test

[0077] Peak 1 2 3 4 5 6 average value RSD 1 0.217 0.215 0.224 0.216 0.218 0.220 0.218 1.45％ 2 0.315 0.298 0.313 0.308 0.311 0.312 0.310 1.97％ 3 0.412 0.407 0.411 0.417 0.420 0.413 0.413 1.11％ 4 0.607 0.611 0.608 0.614 0.613 0.605 0.610 0.58％ 5 0.671 0.667 0.675 0.672 0.678 0.669 0.672 0.60％ 6 0.515 0.507 0.519 0.512 0.520 0.508 0.514 1.07％ 7 0.804 0.801 0.813 0.815 0.807 0.809 0.808 0.66％ 8(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00％ 9 1.024 1.012 1.035 1.027 1.017 1.046 1.027 1.20％ 10 1.236 1.234 1.187 1.198 1.186 1.221 1.209 1.89％ 11 1.320 1.314 1.317 1.321 1.325 1.288 1.314 1.02％ 12 1.418 1.413 1.417 1.420 1.412 1.417 1.416 0.22％ 13 1.572 1.567 1.573 1.601 1.584 1.585 1.580 0.78％ 14 1.681 1.673 1.680 1.679 1.685 1.691 1.682 0.36％ 15 1.764 1.746 1.778 1.753 1.781 1.764 1.764 0.77％

[0078] Table 2 Relative peak area of ​​each peak in repeatability test

[0079]

[0080]

[0081] 2. Precision test

[0082] Take the test solution of the same batch of Xiaoer Xuanfei Zhike Granules (batch number: 230402) and repeat the injection for 6 times to examine the precision. Take the chromatographic peak No. 8 as the reference peak, and calculate the RSD of the relative retention time and relative peak area of ​​each common peak. The results show that the relative standard deviation of the relative retention time of each common peak is less than 1.20%, and the relative standard deviation of the relative peak area is less than 1.50%, with good precision. See Tables 3 and 4.

[0083] Table 3 Relative retention time of each peak in precision test

[0084] Peak 1 2 3 4 5 6 average value RSD 1 0.217 0.215 0.214 0.217 0.218 0.220 0.217 0.99％ 2 0.315 0.308 0.313 0.311 0.316 0.312 0.313 0.92％ 3 0.382 0.392 0.384 0.387 0.388 0.394 0.388 1.18％ 4 0.607 0.611 0.608 0.614 0.613 0.605 0.610 0.58％ 5 0.671 0.667 0.675 0.672 0.678 0.669 0.672 0.60％ 6 0.680 0.683 0.674 0.681 0.675 0.684 0.680 0.61％ 7 0.804 0.801 0.813 0.815 0.807 0.809 0.808 0.66％ 8(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00％ 9 1.024 1.012 1.025 1.037 1.016 1.021 1.023 0.85％ 10 1.236 1.234 1.227 1.198 1.226 1.221 1.224 1.12％ 11 1.320 1.315 1.317 1.321 1.325 1.309 1.318 0.42％ 12 1.418 1.414 1.417 1.421 1.415 1.417 1.417 0.17％ 13 1.572 1.567 1.573 1.594 1.586 1.581 1.579 0.64％ 14 1.681 1.673 1.682 1.679 1.683 1.692 1.682 0.37％ 15 1.764 1.745 1.773 1.763 1.781 1.761 1.765 0.69％

[0085] Table 4 Relative peak area of ​​each peak in precision test

[0086]

[0087]

[0088] 3. Stability test

[0089] Take the test solution of the same batch of Xiaoer Xuanfei Zhike Granules (batch number: 230402), place it at room temperature, and inject it into the liquid chromatograph at 0h, 4h, 8h, 12h, and 24h respectively. Take the chromatographic peak No. 8 as the reference peak to examine the RSD of the relative retention time and relative peak area of ​​each common peak. The results show that the relative standard deviation of the relative retention time of each chromatographic peak is less than 1.0%, and the relative standard deviation of the relative peak area of ​​each chromatographic peak is less than 2.0%, and the stability is good. See Tables 5 and 6.

[0090] Table 5 Relative retention time of each peak in stability test

[0091] Peak 1 2 3 4 5 6 average value RSD 1 0.222 0.218 0.221 0.217 0.218 0.220 0.220 0.90％ 2 0.316 0.314 0.321 0.315 0.317 0.312 0.316 0.97％ 3 0.395 0.392 0.386 0.394 0.388 0.394 0.391 0.94％ 4 0.611 0.608 0.616 0.614 0.613 0.610 0.612 0.47％ 5 0.669 0.667 0.675 0.672 0.673 0.669 0.671 0.45％ 6 0.675 0.683 0.674 0.681 0.677 0.685 0.679 0.66％ 7 0.785 0.801 0.792 0.803 0.784 0.792 0.794 0.99％ 8(S) 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.00％ 9 1.020 1.015 1.025 1.033 1.014 1.021 1.021 0.69％ 10 1.211 1.217 1.222 1.198 1.225 1.221 1.216 0.82％ 11 1.305 1.315 1.317 1.321 1.325 1.309 1.315 0.56％ 12 1.414 1.421 1.417 1.425 1.418 1.417 1.419 0.27％ 13 1.578 1.592 1.573 1.587 1.586 1.581 1.583 0.43％ 14 1.693 1.673 1.685 1.679 1.683 1.692 1.684 0.45％ 15 1.775 1.745 1.763 1.763 1.781 1.761 1.765 0.71％

[0092] Table 6 Relative peak areas of each peak in stability test

[0093]

[0094]

[0095] Example 3 Establishment and Analysis of Fingerprint

[0096] 1. Establishment of standard fingerprint and determination of common peaks

[0097] According to the determined preparation method of the test solution of the Xiaoer Xuanfei Zhike Granules and the liquid chromatography detection conditions, 12 batches of Xiaoer Xuanfei Zhike Granules samples were taken for detection and analysis. The fingerprint superposition diagram of the 12 batches of samples is shown in Figure 8 The main chromatographic peaks obtained by the determination all appeared within 80 minutes. The obtained fingerprints were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2004A Edition)" software for processing. The median method was used, the time window width was set to 0.2, and the multi-point correction method was used to perform full peak matching on the chromatogram. The standard reference fingerprint was fitted and generated, see Fig. 9 , and 15 common peaks were determined. Taking chromatographic peak No. 8 as the reference peak, the relative retention times of other common peaks in 15 batches of samples were calculated (see Table 7). The average relative retention times (peak numbers) were 0.217, 0.313, 0.388, 0.610, 0.804, 1.018, 1.226, 1.317, 1.415, 1.575, 1.679, and 1.750, respectively, and their relative deviations were all within 1.0%.

[0098] Table 7-1 Relative retention time of common peaks in 12 batches of samples of Xiaoer Xuanfei Zhike Granules

[0099]

[0100]

[0101] Table 7-2 Relative peak areas of the total peaks in 12 batches of Xiaoer Xuanfei Zhike granules samples

[0102]

[0103]

[0104]

[0105] 2. Identification of common peaks

[0106] Preparation of reference solution: Accurately weigh baicalin and chlorogenic acid reference substances respectively, and prepare a single reference solution containing 50 μg chlorogenic acid and 60 μg baicalin per 1 ml with methanol.

[0107] The chromatograms of baicalin and chlorogenic acid reference substances were compared with the sample fingerprints, and the chromatographic peaks at the same retention time as the reference substances were identified. Fig.10 The results showed that among the common peaks, peak No. 9 was identified as baicalin and peak No. 3 was identified as chlorogenic acid.

[0108] 3. Correlation between the fingerprints of medicinal materials and finished products

[0109] Preparation of medicinal material test solution: Extract each medicinal material in the prescription according to the granule preparation process of this product to obtain an extract, and assign the chromatographic peaks according to the standard fingerprint conditions. The results are shown in Table 8.

[0110] Table 8 Correlation between Xiaoer Xuanfei Zhike Granules and Medicinal Materials

[0111]

[0112]

[0113] The 15 common peaks were attributed to medicinal materials, and a total of eleven medicinal materials in the prescription were identified, among which peaks 1 and 2 were attributed to bitter almonds, peak 3 chlorogenic acid was attributed to astragalus and yam, peaks 4 and 10 were attributed to saposhnikovia divaricata, peak 5 was attributed to ephedra, peak 6 was attributed to scutellaria baicalensis and mustard seeds, peak 7 was attributed to scutellaria baicalensis, peak 8 was attributed to platycodon, baicalin 9, peaks 12 and 13 were attributed to scutellaria baicalensis, peak 11 was attributed to liquorice, peak 14 was attributed to hawthorn, and peak 15 was attributed to astragalus and hawthorn.

[0114] 4. Similarity evaluation and sample qualification determination

[0115] The chromatograms of the 12 batches of samples were compared with the standard reference fingerprints, and similarity evaluation was performed according to the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2004A Edition). See Table 9. The similarities of the 12 batches of samples were 0.975, 0.984, 0.983, 0.979, 0.967, 0.979, 0.972, 0.969, 0.952, 0.928, 0.919, and 0.922, respectively. The similarities of the 12 batches were all above 0.910, indicating that the quality stability and uniformity of the samples were good. After comprehensive consideration, the samples with a similarity greater than 0.900 were selected as qualified products.

[0116] Table 9 Similarity evaluation of 12 batches of Xiaoer Xuanfei Zhike granules samples

[0117] batch number Similarity batch number Similarity batch number Similarity 220702 0.975 221202 0.967 230402 0.952 220801 0.984 230201 0.979 230502 0.928 220903 0.983 230301 0.972 230601 0.919 221101 0.979 230302 0.969 230701 0.922

Claims

1. A HPLC fingerprint detection method for children's Xuanfei Zhike granules, which are made of twelve Chinese medicinal materials, including ephedra, saposhnikovia divaricata, scutellaria baicalensis, platycodon grandiflorum, mustard seeds, bitter almonds, scutellaria baicalensis, amaranth, astragalus, yam, hawthorn, and liquorice, and have the effect of clearing away heat and relieving exterior symptoms, and are used for children's exogenous cough, characterized in that: The detection method comprises the following steps: (1) ultrasonically extracting the Xiaoer Xuanfei Zhike granules with methanol, filtering with a microporous filter membrane, and taking the filtrate to obtain a test solution; (2) Weigh baicalin and chlorogenic acid reference substances separately and prepare a single reference substance solution with methanol; (3) Accurately pipette the test solution or the single reference solution and detect it with a high performance liquid chromatograph, wherein the high performance liquid chromatograph comprises an ultraviolet detector, the detection wavelength of the ultraviolet detector is set to 210-320 nm, and the stationary phase of the high performance liquid chromatograph is C 18 Chromatographic column, mobile phase A is acetonitrile, mobile phase B is methanol, mobile phase C is acid solution, gradient elution, flow rate is 0.5-2.0 ml / min, column temperature is 25-35°C, theoretical plate number is not less than 3500 calculated based on baicalin; (4) Recording the chromatograms of the test solution and the single reference solution, using the Chinese Pharmacopoeia Committee Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System to import data, perform multi-point correction and data matching on the chromatogram of the test solution to obtain the standard fingerprint of the Chinese medicine, compare the fingerprint of the sample to be tested with the chromatogram of each single reference solution and evaluate the similarity with the standard fingerprint, The gradient elution time is 80 min, which is divided into 10 sections. The mobile phase volume ratios in each section are as follows:

2. The HPLC fingerprint detection method of Xiaoer Xuanfei Zhike Granules as claimed in claim 1, characterized in that: The methanol described in steps (1) and (2) is 70% methanol, and the methanol described in step (3) is pure methanol.

3. The HPLC fingerprint detection method of Xiaoer Xuanfei Zhike Granules as claimed in claim 1 is characterized in that: The power of the ultrasonic extraction is 300 W, the frequency is 40 kHz, and the time is 30 min.

4. The HPLC fingerprint detection method of the Xiaoer Xuanfei Zhike Granules as claimed in claim 1 is characterized in that: The pore sizes of the microporous filter membrane are 0.8 μm and 0.45 μm.

5. The HPLC fingerprint detection method of Xiaoer Xuanfei Zhike Granules as claimed in claim 1 is characterized in that: The detection wavelength of the ultraviolet detector is set to 260 nm.

6. The HPLC fingerprint detection method of Xiaoer Xuanfei Zhike Granules as claimed in claim 1, characterized in that: The mobile phase C is 0.2% trifluoroacetic acid solution.

7. The HPLC fingerprint detection method of Xiaoer Xuanfei Zhike Granules as claimed in claim 1 is characterized by: The column temperature was 25°C.

8. The HPLC fingerprint detection method of Xiaoer Xuanfei Zhike Granules as claimed in claim 1, characterized in that: Each 1 ml of single reference solution contains 50 μg of chlorogenic acid and 60 μg of baicalin.

9. The HPLC fingerprint detection method of Xiaoer Xuanfei Zhike Granules as claimed in claim 1, characterized in that: There are 15 common peaks in the standard fingerprint, among which peaks 1 and 2 belong to bitter almonds, peak 3 belongs to astragalus and yam, peaks 4 and 10 belong to saposhnikovia divaricata, peak 5 belongs to ephedra, peak 6 belongs to scutellaria baicalensis and mustard seeds, peak 7 belongs to scutellaria baicalensis, peak 8 belongs to platycodon, peaks 9, 12 and 13 belong to scutellaria baicalensis, peak 11 belongs to liquorice, peak 14 belongs to hawthorn, and peak 15 belongs to astragalus and hawthorn. After comparison with the chromatogram of a single reference solution, it was confirmed that peak 3 was chlorogenic acid and peak 9 was baicalin.

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