A method for constructing a fingerprint spectrum of a throat-clearing hard candy, a method for detecting its component content, and its application.

Abstract

This invention discloses a method for constructing a fingerprint spectrum of a throat-clearing hard candy and a method for detecting its component content, belonging to the field of component detection technology. In this invention, the throat-clearing hard candy sample solution is analyzed by high-performance liquid chromatography (HPLC) to obtain a fingerprint spectrum of the candy; and the content information of the components is obtained through the external standard method, realizing qualitative and quantitative analysis of the components of the throat-clearing hard candy. Specifically, it involves the simultaneous qualitative and quantitative analysis of the following 10 components: gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-glucose-7-O-rhamnoside, rutin, luteolin, isochlorogenic acid B, isorhamnetin-3-O-glucose, glycyrrhizic acid, and kaempferol, providing a reliable and quantifiable standard for the quality assessment of throat-clearing hard candy. The chromatogram obtained by the method of this invention has the characteristics of stable baseline, high peak resolution, strong specificity, and good peak symmetry.

Description

Technical Field

[0001] This invention belongs to the field of component detection technology, and particularly relates to a method for constructing a fingerprint spectrum of a throat-clearing hard candy and a method for detecting its component content and its application. Background Technology

[0002] Health foods formulated with traditional Chinese medicine as a base and compounded through multiple prescriptions present significant challenges in identifying key and characteristic components due to the complexity of their components. Perfect Brand Throat-Clearing Hard Candy is made from Chrysanthemum indicum, Lepidium apetalum, Lonicera japonica, Phyllanthus emblica, Platycodon grandiflorus, and Glycyrrhiza uralensis. These ingredients are formulated, extracted with water, spray-dried into powder, and then combined with peppermint and sucrose before being boiled and shaped into a hard candy product. This product has heat-clearing and throat-soothing effects and is rich in phenolic acids and flavonoids, including gallic acid, chlorogenic acid, luteolin, rutin, and glycyrrhizic acid. Due to the complexity of compound components, the identification of marker and characteristic components in products is quite difficult. Existing technologies are mostly based on the requirements of the pharmacopoeia for single raw materials, incorporating one or two marker components into the product quality standards. Moreover, different marker components require different detection methods. For example, the pharmacopoeia specifies the determination methods for gallic acid, chlorogenic acid, and glycyrrhizic acid, which are unique components in honeysuckle, licorice, amla, and platycodon, but the specified detection methods are inconsistent, causing many inconveniences for monitoring the unique component indicators of compound preparations. Furthermore, notoginseng and purslane are not listed in the pharmacopoeia. In other words, existing technologies have the following shortcomings: First, they cannot quickly and accurately identify marker components in multiple formulations; second, the monitored components are relatively singular, which is not conducive to product quality control; third, multiple indicators require multiple detection methods, resulting in long detection times and low efficiency; and fourth, there is a lack of methods for the overall simultaneous determination of the product's active ingredients and the overall quality control evaluation. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for constructing a fingerprint spectrum of a throat-clearing hard candy and a method for detecting its component content and its application. In particular, it relates to the qualitative and quantitative analysis of 10 components in the throat-clearing hard candy: gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-gluco-7-O-rhamnoside, rutin, luteolin, isochlorogenic acid B, isorhamnetin-3-O-glucoside, glycyrrhizic acid and kaempferol.

[0004] This invention uses high performance liquid chromatography (HPLC) to prepare the fingerprint spectrum of throat-clearing hard candy and analyze the content of its components, providing a reference for its quality control.

[0005] To achieve the above objectives, in a first aspect, the present invention provides a method for constructing a fingerprint spectrum of a throat-clearing hard candy, the method comprising the following steps: preparing a throat-clearing hard candy test solution, performing high-performance liquid chromatography analysis on the throat-clearing hard candy test solution, and obtaining a fingerprint spectrum of the throat-clearing hard candy;

[0006] The conditions for the high-performance liquid chromatography are as follows:

[0007] Mobile phase: Mobile phase A is methanol, and mobile phase B is a 0.1% formic acid aqueous solution.

[0008] Gradient elution conditions:

[0009]

[0010] Detector wavelength conditions:

[0011]

[0012] Chromatographic column: C18 column, 5μm × 4.6mm × 250mm.

[0013] Flow rate: 0.8-1.0 mL / min

[0014] Injection volume: 10 μL

[0015] Column oven temperature: 35±1℃.

[0016] The inventors of this invention discovered in extensive fingerprint studies of throat-clearing hard candy that by using methanol as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B, and eluting under the specific detection wavelength and flow rate described above, and under the specific elution conditions, a fingerprint spectrum containing characteristic peaks of gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-gluco-7-O-rhamnoside, rutin, luteolinoside, isochlorogenic acid B, isorhamnetin-3-O-glucoside, glycyrrhizic acid, and kaempferol can be obtained. The characteristic peaks of gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-gluco-7-O-rhamnoside, rutin, luteolinoside, isorhamnetin B, isorhamnetin-3-O-glucoside, glycyrrhizic acid, and kaempferol exhibit high purity and good peak separation, and do not contain other impurity peaks.

[0017] The fingerprint spectrum fully demonstrates the formulation and chemical composition information of the throat-clearing hard candy, providing a basis for the determination, detection, and identification of its component content.

[0018] The inventors discovered that the specific selection of the mobile phase, gradient elution conditions, and flow rate during chromatographic detection significantly affects the number, purity, and resolution of characteristic peaks in the fingerprint spectrum. Specifically, using different mobile phases or different gradient elution conditions can result in the absence of relevant peaks for gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-glucose-7-O-rhamnoside, rutin, luteolinoside, isochlorogenic acid B, isorhamnetin-3-O-glucose, glycyrrhizic acid, and kaempferol. Consequently, it becomes impossible to simultaneously determine these 10 components.

[0019] As a preferred embodiment of the construction method of the present invention, the preparation method of the throat-clearing hard candy test solution is as follows: the throat-clearing hard candy is placed in a methanol aqueous solution, heated to dissolve, cooled, and diluted to a fixed volume, and then filtered to obtain the test solution.

[0020] In a preferred embodiment of the construction method of the present invention, the mass-to-volume ratio of the throat-clearing hard candy to the methanol aqueous solution is (4-6) g: 40 mL; the mass percentage of methanol in the methanol aqueous solution is 40-60%; and the heating temperature is 40±5℃.

[0021] In a second aspect, the present invention provides a method for detecting the content of components in a throat-clearing hard candy, the method comprising the following steps: preparing a throat-clearing hard candy test solution; performing high-performance liquid chromatography analysis on the throat-clearing hard candy test solution to obtain a fingerprint spectrum of the throat-clearing hard candy; and calculating the content of the component to be tested by means of the peak area of ​​the fingerprint spectrum using the external standard method.

[0022] The high-performance liquid chromatography conditions are as follows:

[0023] Mobile phase: Mobile phase A is methanol, and mobile phase B is a 0.1% formic acid aqueous solution.

[0024] Gradient elution conditions:

[0025]

[0026] Detector wavelength conditions:

[0027]

[0028] Chromatographic column: C18 column, 5μm × 4.6mm × 250mm.

[0029] Flow rate: 0.8-1.0 mL / min

[0030] Injection volume: 10 μL

[0031] Column oven temperature: 35±1℃.

[0032] As a preferred embodiment of the detection method of the present invention, the component to be tested is at least one of gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-gluco-7-O-rhamnoside, rutin, luteolin, isochlorogenic acid B, isorhamnetin-3-O-glucoside, glycyrrhizic acid and kaempferol.

[0033] As a preferred embodiment of the detection method of the present invention, the preparation method of the throat-clearing hard candy test solution is as follows: the throat-clearing hard candy is placed in a methanol aqueous solution, heated to dissolve, cooled, and diluted to a fixed volume, and then filtered to obtain the test solution.

[0034] In a preferred embodiment of the detection method of the present invention, the mass-to-volume ratio of the throat-clearing hard candy to the methanol aqueous solution is (4-6) g: 40 mL; the mass percentage of methanol in the methanol aqueous solution is 40-60%; and the heating temperature is 40±5℃.

[0035] As a preferred embodiment of the detection method of the present invention, the external standard method calculates the peak area of ​​high performance liquid chromatography and uses it as the ordinate (y), with the concentration of its components as the abscissa (x), to establish a linear regression equation for each component.

[0036] Preferably, the external standard method includes a reference solution, which is a mixed reference solution.

[0037] By analyzing the HPLC chromatograms of the test solution and the mixed reference solution, the chemical composition information of the throat-clearing hard candy can be obtained. The chemical composition includes at least one of gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-gluco-7-O-rhamnoside, rutin, luteolin, isochlorogenic acid B, isorhamnetin-3-O-glucoside, glycyrrhizic acid, and kaempferol.

[0038] Preferably, the mixed reference solution is prepared by accurately weighing a certain amount of gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-gluco-7-O-rhamnoside, rutin, luteolin, isochlorogenic acid B, isorhamnetin-3-O-glucoside, glycyrrhizic acid and kaempferol and dissolving them in methanol. Then, accurately pipette a certain amount of the above solution into a 5 mL volumetric flask and dilute to volume with methanol to obtain the mixed reference solution.

[0039] Preferably, in the mixed reference solution, the concentrations of gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-gluco-7-O-rhamnoside, rutin, luteolin, isochlorogenic acid B, isorhamnetin-3-O-glucoside, glycyrrhizic acid, and kaempferol are 537.8 μg / mL, 16.4 μg / mL, 588.6 μg / mL, 44.7 μg / mL, 5.1 μg / mL, 7.7 μg / mL, 41.7 μg / mL, 188.5 μg / mL, 963.9 μg / mL, and 137.3 μg / mL, respectively.

[0040] In a third aspect, the present invention provides the application of the method for constructing the fingerprint spectrum of the throat-clearing hard candy or the method for detecting the component content of the throat-clearing hard candy in the quality control of the throat-clearing hard candy.

[0041] The construction or detection method of the fingerprint spectrum of throat-clearing hard candy can determine the information of its traditional Chinese medicine components and chemical components, especially the qualitative and quantitative analysis of 10 chemical components, including gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-gluco-7-O-rhamnoside, rutin, luteolin, isochlorogenic acid B, isorhamnetin-3-O-glucoside, glycyrrhizic acid and kaempferol. This method has good prospects for application in the quality control of throat-clearing hard candy.

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0043] (1) The present invention establishes a fingerprint spectrum of throat-clearing hard candy, which provides a reference for quality control.

[0044] (2) The method of the present invention can efficiently and accurately perform qualitative and quantitative analysis on gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-glucose-7-O-rhamnoside, rutin, luteolin, isochlorogenic acid B, isorhamnetin-3-O-glucose, glycyrrhizic acid and kaempferol in throat-clearing hard candy.

[0045] (3) The chromatograms obtained by the method of the present invention have stable baselines, good peak separation, symmetrical peak shapes, and strong specificity.

[0046] (4) The method of the present invention can conveniently and quickly detect and identify the quality of throat-clearing hard candy, providing a reliable and quantitative standard for its quality control. Attached Figure Description

[0047] Figure 1 This is a comparison chromatogram of the test sample and the blank sample in the specificity investigation of Example 3;

[0048] Figure 2 This is a graph showing the results of seven repeated measurements of the same sample in the precision study of Example 3;

[0049] Figure 3 The above are comparison spectra before and after spiking in the accuracy evaluation of Example 3;

[0050] Figure 4 The fingerprint chromatograms are shown for 14 batches of throat-clearing hard candy samples in Example 4;

[0051] Figure 5 This is the chromatogram of the licorice negative control sample in Example 4;

[0052] Figure 6 This is the chromatogram of the honeysuckle negative control sample in Example 4;

[0053] Figure 7 This is the chromatogram of the negative control sample of *Gnaphalium affine* in Example 4;

[0054] Figure 8 This is the chromatogram of the negative control sample of Chrysanthemum in Example 4;

[0055] Figure 9 This is the chromatogram of the Phyllanthus emblica negative control sample in Example 4;

[0056] Figure 10 This is the chromatogram of the control sample for the compound validation in Example 4;

[0057] Figure 11 This is a graph showing the effect of different flow rates in Example 5;

[0058] Figure 12 This is a graph showing the effects of different mobile phases B in Example 5;

[0059] Figure 13 The graph shows the effect of different gradient elution procedures in Example 5. Detailed Implementation

[0060] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0061] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in the field.

[0062] Throat-clearing hard candy: Perfect (Guangdong) Daily Necessities Co., Ltd., batch number: YFQY2210-01;

[0063] Gallic acid: China National Institutes for Food and Drug Control, batch number: 110831-201906, content: 91.5%;

[0064] New chlorogenic acid: Anpu, batch number: CDAA-280180, content: 97.4%;

[0065] Chlorogenic acid: China National Institutes for Food and Drug Control, batch number: 20180410, content: 98%;

[0066] Isorhamnetin-3-O-glucose-7-O-rhamnoside: Green Leaf Biotechnology, batch number: B50166, content: 97%;

[0067] Rutin: China National Institutes for Food and Drug Control, batch number: 100080-201811, content: 91.7%;

[0068] Luteolin: China National Institutes for Food and Drug Control, batch number: 111720-201609, content: 94.9%;

[0069] Isochlorogenic acid B: Anpu, batch number: CDAA-28018, content: 97.5%;

[0070] Isorhamnetin-3-O-glucoside: Yuanye Biotechnology, batch number: B21556, content: 98%;

[0071] Glycyrrhizic acid: Source Leaf Biotechnology, batch number: B20417, content: 98%;

[0072] Kaempferol: China National Institutes for Food and Drug Control, 110861-201812, content: 93.8%.

[0073] Example 1

[0074] 1. Preparation of the test solution:

[0075] Take 2 throat-clearing hard candies and place them in a 50mL Erlenmeyer flask. Weigh them and accurately add 40mL of methanol-water (mass ratio 1:1) solution. Heat in a 40℃ water bath until completely dissolved. Remove and cool. Transfer the solution to a 25mL volumetric flask and dilute to volume with methanol-water (mass ratio 1:1) solution. Shake well and filter through a 0.22μm filter membrane. Collect the filtrate to obtain the test solution.

[0076] 2. Preparation of mixed reference solution:

[0077] Accurately weigh gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-glucose-7-O-rhamnoside, rutin, luteolin, isochlorogenic acid B, isorhamnetin-3-O-glucose, glycyrrhizic acid, and kaempferol according to the sample quantities in Table 1. Then, dissolve them in methanol in brown volumetric flasks of the corresponding volumes, shake well, and dilute to the mark. Next, accurately pipette the corresponding volumes of sample solution according to the volumes in Table 1 and place them in 5 mL volumetric flasks. Dilute to 5 mL with methanol to obtain a mixed reference solution.

[0078] Table 1. Preparation of Mixed Control Solution

[0079]

[0080]

[0081] 3. Preparation of negative sample solution:

[0082] The preparation method of the corresponding negative sample hard candy obtained in the preparation process of the negative sample solution provided by this invention is exactly the same as the preparation method of the test sample hard candy, except for the difference in the raw material mass ratio; specifically, it includes the preparation of the following negative sample solution:

[0083] 3.1 Preparation of licorice negative sample solution: The ratio of raw materials of Chrysanthemum indicum: Bryophyllum indicum: Lonicera japonica: Phyllanthus emblica: Platycodon grandiflorus: Glycyrrhiza uralensis was 1:2:2:2:1:0. After water extraction and spray drying, etc., the throat-clearing hard candy was obtained. The corresponding licorice negative sample solution was prepared by following the preparation process of the test sample solution.

[0084] 3.2 Preparation of honeysuckle negative sample solution: The ratio of raw materials of Chrysanthemum indicum: Bryophyllum indicum: honeysuckle: Phyllanthus emblica: Platycodon grandiflorus: Glycyrrhiza uralensis was 1:2:0:2:1:2. After water extraction and spray drying, the honeysuckle hard candy was prepared. The honeysuckle negative sample solution was prepared according to the preparation process of the test sample solution.

[0085] 3.3 Preparation of negative sample solution of *Lepidium apetalum*: The feed ratio of *Lepidium apetalum*: *Lonicera japonica*: *Phyllanthus emblica*: *Platycodon grandiflorus*: *Glycyrrhiza uralensis* was 1:0:2:2:1:2. After water extraction and spray drying, the *Lepidium apetalum* hard candy was obtained. This hard candy was then used to prepare the corresponding negative sample solution of *Lepidium apetalum* according to the preparation process of the test sample solution.

[0086] 3.4 Preparation of negative sample solution of Chrysanthemum indicum: The ratio of Chrysanthemum indicum: Bryophyllum indicum: Lonicera japonica: Phyllanthus emblica: Platycodon grandiflorus: Glycyrrhiza uralensis was 0:2:2:2:1:2. After water extraction and spray drying, etc., the throat-clearing hard candy was obtained. The corresponding negative sample solution of Chrysanthemum indicum was prepared by following the preparation process of the test sample solution.

[0087] 3.5 Preparation of Phyllanthus emblica negative sample solution: The feed ratio of Chrysanthemum indicum: Achyranthes bidentata: Lonicera japonica: Phyllanthus emblica: Platycodon grandiflorus: Glycyrrhiza uralensis was 1:2:2:2:0:2. After water extraction and spray drying, etc., the Phyllanthus emblica negative sample solution was prepared. This hard candy was then used to prepare the corresponding Phyllanthus emblica negative sample solution according to the preparation process of the test sample solution.

[0088] 4. Chromatographic conditions

[0089] Mobile phase: Mobile phase A is methanol, and mobile phase B is a 0.1% formic acid aqueous solution.

[0090] Gradient elution conditions:

[0091]

[0092] Detector wavelength conditions:

[0093]

[0094] Column: Agilent AQ-C18 column, 5μm × 4.6mm × 250mm.

[0095] Flow rate: 0.8 mL / min

[0096] Injection volume: 10 μL

[0097] Column oven temperature: 35℃.

[0098] Example 2

[0099] This invention provides a method for screening characteristic components in throat-clearing hard candy, specifically including the following steps:

[0100] The test solution prepared in Example 1 was diluted 100-fold and analyzed using ultra-high performance liquid chromatography-time-of-flight mass spectrometry; wherein...

[0101] The mass spectrometry parameters were as follows: capillary voltage: 3.0 kV / -2.5 kV; ion source temperature: 120℃ (positive) / 110℃ (negative); cone voltage: 40 kV (positive) / 50 kV (negative); cone gas flow rate: 50 L / h; desolvation gas temperature: 350℃; desolvation gas flow rate: 1000 L / h; scan range: 50-1200 m / z; scan frequency: 0.5 s; low-energy collision voltage: 6 eV; high-energy collision voltage: 20-40 eV; leucine enkephalin real-time correction positive ion mode ([M+H)) was selected. + =556.2771) and negative ion mode ([MH] - =554.2615);

[0102] The chromatographic conditions were as follows: column: Waters, ACQUITYUPLC HSS T3 1.8μm, 2.1mm×100mm; mobile phase flow rate: 0.3mL / min; injection volume: 1μL; mobile phase: mobile phase A was 0.1% formic acid acetonitrile, and mobile phase B was 0.1% formic acid aqueous solution; the gradient elution program of the mobile phase is shown in Table 2.

[0103] Table 2. Ultra-high performance liquid chromatography-time-of-flight mass spectrometry flow rate elution gradient.

[0104]

[0105]

[0106] The test data was imported into the UNIFI software. In the software, the databases of Chinese medicinal herbs such as honeysuckle, ginseng, licorice, and platycodon were selected for comparative analysis. After the analysis was completed, the data was exported and deduplicated. A total of 1852 components were identified. The top 30 components with the largest number of ions and a mass error of less than 10 ppm were selected, as shown in Table 3 below.

[0107] Table 3. Results of Ultra-High Performance Liquid Chromatography Time-of-Flight Mass Spectrometry Identification Data

[0108]

[0109]

[0110]

[0111] Based on Table 3, high performance liquid chromatography was used to confirm 30 components using a single standard substance under the chromatographic conditions in Example 1. Ultimately, 10 components were confirmed. The 10 components were gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-glucose-7-O-rhamnoside, rutin, luteolin, isochlorogenic acid, isorhamnetin-3-O-glucose, glycyrrhizic acid, and kaempferol.

[0112] Example 3

[0113] The embodiments of this invention conduct methodological investigations on the test methods for gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-glucose-7-O-rhamnoside, rutin, luteolin, isochlorogenic acid, isorhamnetin-3-O-glucose, glycyrrhizic acid, and kaempferol.

[0114] 1. Specificity assessment

[0115] The mixed reference solution, test solution, and blank solution were analyzed by high-performance liquid chromatography (HPLC) under the same chromatographic conditions as in Example 1. The results are as follows: Figure 1 As shown, from Figure 1 As can be seen, the blank solvent did not cause any interfering chromatographic peaks, therefore the specificity of the method provided by this invention meets the requirements.

[0116] 2. Precision test

[0117] By repeating the preparation method of the test solution in Example 1 for the same batch of sample YFQY2210-01, 7 sample solutions were prepared. Then, the chromatographic conditions in Example 1 were used for testing. The common content of the 7 sample solutions was confirmed by referring to the retention time of the mixed reference solution. The RSD% of the content of 10 active ingredients (n=7) was calculated by peak area. The results are shown in Table 4.

[0118] Table 4 Results of Repeatability Tests

[0119]

[0120] As shown in Table 4, the peak area RSD values ​​of the 10 known active ingredients are all less than 10%, indicating that the method has good repeatability and high precision. Furthermore, during the precision test, it was also found that in addition to the 10 known active ingredients, 10 common unknown ingredients were also stably present in the product, such as... Figure 2 As shown ( Figure 2 (The symbols 1-10 represent common unknown components in the samples). Common unknown components are defined as unique components of this product.

[0121] 3. Accuracy assessment

[0122] The accuracy of this method was examined by spike recovery tests on the samples. Twenty pieces of throat-clearing hard candy were taken, and an appropriate amount of mixed reference standard was added and mixed thoroughly. Six samples were prepared repeatedly according to the preparation method of the test solution in Example 1. Then, the samples were tested according to the chromatographic conditions in Example 1, and the spike recovery rate of each reference standard was calculated. The results are shown in Table 5.

[0123] Table 5 Results of Spiked Recovery Test

[0124]

[0125] As shown in Table 5, the recoveries of each reference standard were between 90% and 110%, meeting the requirements of GB / T27417, indicating that the method has high accuracy and the detection results are accurate. The reference chromatograms before and after spiking are shown below. Figure 3 As shown.

[0126] Example 4

[0127] The present invention provides an embodiment for establishing and applying a fingerprint spectrum of throat-clearing hard candy, specifically including the following aspects;

[0128] 1. Establishment of fingerprint pattern:

[0129] Fourteen batches of samples (batch numbers QY2301-01, QY2301-02, QY2301-03, QY2302-01, QY2302-02, QY2302-03, QY2302-04, QY2302-05, QY2303-01, QY2303-02, QY2303-03, QY2303-04, QY2303-05, and QY2303-06) were analyzed. The sample solution was prepared according to the preparation method of the test solution in Example 1, and then tested according to the chromatographic conditions in Example 1. Fourteen batches of samples were collected to construct fingerprint chromatograms. Twenty common peaks were identified in the obtained fingerprint chromatograms. A reference fingerprint chromatogram was generated using the median method. Using sample 1 as the reference chromatogram, the relative retention time and relative peak area of ​​each common peak were calculated. Then, similarity rating was performed, and the similarity comparison results are shown in Table 6. The fingerprint chromatograms are as follows: Figure 4 As shown ( Figure 4 The sample numbers are numbered from bottom to top.

[0130] Table 6. Similarity Comparison Results

[0131]

[0132] The characteristic chromatogram of the test sample should show 20 characteristic peaks, and the peak corresponding to the reference peak is the S peak. The similarity between the fingerprint chromatogram of the test sample and the fingerprint chromatogram of the reference extract should be calculated according to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint chromatogram. It should not be lower than 0.95 and can be used as a standard for product quality evaluation. As can be seen from Table 6, the fingerprint chromatogram similarity of 14 batches of throat-clearing hard candy test solution is all >0.95, so fingerprint chromatogram index can be established.

[0133] Table 7 Comparison Results of 14 Batches of Samples

[0134]

[0135] As can be seen from Table 7, the batch-to-batch precision of the 14 batches of samples was good, with a maximum RSD of 10.3%. When the content of characteristic indicators in the samples was greater than 5 mg / 100g, the batch-to-batch precision could be controlled within 10%.

[0136] 2. Verify the practicality of fingerprinting for product quality control:

[0137] The tests were performed according to the chromatographic conditions described in Example 1. The chromatograms after testing were then compared with those of reference sample 1 for similarity; the comparison chromatograms are shown below. Figure 5-10 As shown in Table 8, the overall similarity data is as follows;

[0138] Table 8. Overall Similarity Comparison Results

[0139]

[0140] As can be seen from Table 8, the similarity is 96.1% when the compound is not missing, and the similarity is less than 90% when a certain component of the compound is missing, indicating that the method provided by the present invention can effectively monitor the stability of product quality.

[0141] The obtained fingerprint spectrum was used to test the content of gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-gluco-7-O-rhamnoside, rutin, luteolin, isochlorogenic acid, isorhamnetin-3-O-glucoside, glycyrrhizic acid, and kaempferol in the compound verification sample. The results obtained after testing and calculation are shown in Table 9.

[0142] Table 9. Results of Component Content Test

[0143]

[0144] Further comparisons were made of the peaks, and the results are shown in Table 10.

[0145] Table 10 Comparison of similarity among characteristic peaks

[0146]

[0147]

[0148]

[0149] From the similarity matching data in Table 10, it can be seen that peak 1 is mainly affected by the amount of glycoside added, peaks 2-6 are all introduced by glycoside. Peaks 1-6 are characteristic peaks of glycoside. Therefore, when peaks 1-6 show large fluctuations in daily product quality monitoring, the influence of glycoside in the processing technology should be analyzed. Peak 9 is most affected by glycoside, followed by honeysuckle. When peak 9 shows fluctuations in daily product quality monitoring, the influence of glycoside and honeysuckle on product quality can be analyzed. Peak 11 is greatly affected by purslane. Peaks 12-13 are most affected by licorice. Peak 13, glycyrrhizin, mainly comes from honeysuckle and glycoside. Peak 19, glycyrrhizic acid, and peak 20, kaempferol are characteristic peaks of licorice. When peaks 19-20 show large fluctuations in daily product quality monitoring, the influence of licorice should be considered.

[0150] Example 5

[0151] This invention explores the influence of parameters in liquid chromatography conditions, specifically including the following aspects:

[0152] 1. Investigation of flow velocity

[0153] This section investigates the effect of flow rate on the detection of 10 components. Flow rates of 0.8 mL / min and 1.0 mL / min were selected, with all other conditions remaining consistent with the chromatographic conditions in Example 1. The test solutions were then tested, and the resulting chromatograms are shown below. Figure 11 As shown in Table 11;

[0154] Table 11 Separation of Components in Flow Rate Study

[0155]

[0156]

[0157] From Table 11 and Figure 11 As can be seen, when the flow rate for chromatographic detection is 0.8-1.0 mL / min, the 10 components in the mixed reference solution can be separated very well.

[0158] 2. Investigation of the mobile phase

[0159] This section investigates the effect of mobile phase on the detection of 10 components. Mobile phase B was selected as pure water, 0.1% formic acid aqueous solution, and 0.2% formic acid aqueous solution, respectively. All other conditions remained consistent with the chromatographic conditions in Example 1. The test solution was tested, and the resulting chromatograms are shown below. Figure 12 As shown in Table 12, the resolution is as follows;

[0160] Table 12 Separation of Components in Mobile Phase Exploration

[0161]

[0162] From Table 12 and Figure 12 As can be seen, 0.1% formic acid aqueous solution is the best mobile phase B for detection. Under pure water conditions, the baseline fluctuation is large after 12 minutes. When 0.2% formic acid aqueous solution is used as mobile phase B, under the same elution gradient conditions, the separation degree of isorhamnetin-3-O-glucoside and isochlorogenic acid among the 10 active substances decreases to less than 1. Therefore, 0.2% formic acid aqueous solution is not suitable for sample separation, and 0.1% formic acid aqueous solution is the best, with the best separation effect between peaks.

[0163] 3. Investigation of gradient elution

[0164] This section investigates the effect of gradient elution programs on the detection of 10 components. Gradient elution programs 1 and 2 were selected for elution, with all other conditions remaining consistent with the chromatographic conditions in Example 1. Gradient elution program 1 was the same as that in Example 1, and the gradient elution levels 2 are shown in Table 13. The test solution was tested, and the resulting chromatograms are shown below. Figure 13 As shown;

[0165] Table 13 Gradient Elution Table

[0166] Time / min 0 8 8.1 18 18.1 29 30 35 Mobile phase A / % 5 10 25 30 40 50 5 5 Mobile phase B / % 95 90 75 70 60 50 95 95

[0167] from Figure 13 As can be seen, when using gradient elution level 2, the chromatographic peaks obtained in the 10-15 minute range are relatively concentrated, making it impossible to separate the target peak.

[0168] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of a throat-clearing hard candy, characterized in that, The construction method includes the following steps: preparing a throat-clearing hard candy test solution, performing high-performance liquid chromatography analysis on the throat-clearing hard candy test solution, and obtaining the fingerprint spectrum of the throat-clearing hard candy; The conditions for the high-performance liquid chromatography are as follows: Mobile phase: Mobile phase A is methanol, and mobile phase B is a 0.1% formic acid aqueous solution. Gradient elution conditions: Detector wavelength conditions: Chromatographic column: C18 column, 5μm × 4.6mm × 250mm. Flow rate: 0.8-1.0 mL / min Injection volume: 10 μL Column oven temperature: 35±1℃; The throat-soothing hard candy is a Perfect brand throat-soothing hard candy made from ingredients such as Chrysanthemum indicum, Lepidium apetalum, Lonicera japonica, Phyllanthus emblica, Platycodon grandiflorus, and Glycyrrhiza uralensis. The preparation method of the throat-clearing hard candy test solution is as follows: place the throat-clearing hard candy in a methanol aqueous solution, heat to dissolve, cool, make up to volume, and then filter to obtain the test solution.

2. The construction method according to claim 1, characterized in that, The mass-to-volume ratio of the throat-clearing hard candy to the methanol-water solution is (4-6) g: 40 mL; the mass percentage of methanol in the methanol-water solution is 40-60%; and the heating temperature is 40±5℃.

3. A method for detecting the component content of a throat-soothing hard candy, characterized in that, The detection method includes the following steps: preparing a throat-clearing hard candy test solution; performing high-performance liquid chromatography analysis on the throat-clearing hard candy test solution to obtain a fingerprint spectrum of the throat-clearing hard candy; and calculating the content of the analyte by means of the peak area of ​​the fingerprint spectrum using the external standard method. The high-performance liquid chromatography conditions are as follows: Mobile phase: Mobile phase A is methanol, and mobile phase B is a 0.1% formic acid aqueous solution. Gradient elution conditions: Detector wavelength conditions: Chromatographic column: C18 column, 5μm × 4.6mm × 250mm. Flow rate: 0.8-1.0 mL / min Injection volume: 10 μL Column oven temperature: 35±1℃; The throat-soothing hard candy is a Perfect brand throat-soothing hard candy made from ingredients such as Chrysanthemum indicum, Lepidium apetalum, Lonicera japonica, Phyllanthus emblica, Platycodon grandiflorus, and Glycyrrhiza uralensis. The components to be tested are gallic acid, neochlorogenic acid, chlorogenic acid, isorhamnetin-3-O-gluco-7-O-rhamnoside, rutin, luteolin, isochlorogenic acid B, isorhamnetin-3-O-glucoside, glycyrrhizic acid, and kaempferol. The preparation method of the throat-clearing hard candy test solution is as follows: place the throat-clearing hard candy in a methanol aqueous solution, heat to dissolve, cool, make up to volume, and then filter to obtain the test solution.

4. The detection method according to claim 3, characterized in that, The mass-to-volume ratio of the throat-clearing hard candy to the methanol-water solution is (4-6) g: 40 mL; the mass percentage of methanol in the methanol-water solution is 40-60%; and the heating temperature is 40±5℃.

5. The application of the method for constructing the fingerprint spectrum of the throat-clearing hard candy as described in any one of claims 1-2 or the method for detecting the component content of the throat-clearing hard candy as described in any one of claims 3-4 in the quality control of the throat-clearing hard candy.

Images