Fingerprint spectrum determination method of endothelium corneum gigeriae galli and chrysanthemum mixed plant solid beverage

Abstract

The invention relates to a fingerprint establishment method and a detection method of a traditional Chinese medicine composition pink drink. The traditional Chinese medicine composition comprises the following raw materials: endothelium corneum gigeriae galli, pawpaw, hawthorn, chrysanthemum, orange peel, peach kernel and medlar. The fingerprint establishment method comprises the following steps: preparing a reference solution and a test solution; detecting the reference substance solution and the test solution by high performance liquid chromatography; a mobile phase adopted by the high performance liquid chromatography is an acetonitrile and phosphoric acid aqueous solution, and the elution mode is gradient elution.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine quality analysis, and particularly relates to a high performance liquid chromatography (HPLC) fingerprint determination method of a Scammonia sinensis and chrysanthemum mixed plant solid beverage. BACKGROUND

[0002] The Scammonia sinensis and chrysanthemum mixed plant solid beverage is named as “Taohong drink”, which is composed of seven traditional Chinese medicines of Scammonia sinensis, Papaya, Crataegus pinnatifida Bunge, chrysanthemum, orange peel, peach kernel and medlar, and has the effects of activating blood and resolving stasis, dredging qi and blood, and unblocking meridians. Scammonia sinensis, belonging to the spleen, stomach, small intestine and bladder meridians, has the pharmacological activities of invigorating the stomach, resolving stasis, and relieving edema. Papaya, belonging to the liver and spleen meridians, can be used for treating dampness and arthralgia, and relieving pain in the waist and knees. Crataegus pinnatifida Bunge, belonging to the spleen, stomach and liver meridians, is often used for treating meat accumulation, abdominal fullness, diarrhea and abdominal pain, and blood stasis. Chrysanthemum, belonging to the lung and liver meridians, has the effects of dispelling wind and clearing heat, calming the liver and improving eyesight, and clearing heat and resolving toxins, and chlorogenic acid and luteoloside are the main active components of chrysanthemum. Orange peel, also known as dried tangerine or dried orange peel, is the dried mature fruit peel of Rutaceae, and belongs to the lung and spleen meridians, and has the effects of regulating qi and invigorating the spleen, and drying dampness and resolving phlegm. Peach kernel, belonging to the heart, liver and large intestine meridians, has the effects of activating blood and resolving stasis, moistening the intestines and relieving constipation, and relieving cough and asthma. Medlar, belonging to the liver and kidney meridians, has the effects of nourishing the liver and kidney, and benefiting the eyesight, and can be used for treating deficiency and essence deficiency, lumbago and knee pain, dizziness and tinnitus, impotence and premature ejaculation, and internal heat and polydipsia.

[0003] Taohong drink (Scammonia sinensis and chrysanthemum mixed plant solid beverage) is a product newly developed by the applicant, which is made of various main raw materials, has complex components, and has few quality detection methods. There is still a lack of very rigorous quality control standards, so the production is only based on the actual situation, resulting in different production processes, which will have certain adverse effects on the standardized production of Taohong drink.

[0004] Traditional Chinese medicine compositions contain various plant medicines with complex components, and the efficacy of traditional Chinese medicine compositions is not from a single active ingredient, but the result of the joint action of the whole raw materials of traditional Chinese medicine compositions. The content and composition ratio of active ingredients in traditional Chinese medicine compositions will affect the efficacy. Based on the complexity of the components of traditional Chinese medicine compositions, any single active ingredient or index component cannot effectively evaluate the quality of traditional Chinese medicine, and a detection method suitable for traditional Chinese medicine compositions and their preparations should be used to provide rich identification information.

[0005] Traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method, which is based on the systematic study of chemical components of traditional Chinese medicine, mainly used to evaluate the authenticity, superiority and stability of traditional Chinese medicine and semi-finished product quality of traditional Chinese medicine preparation, including two aspects: (1) through the characteristic of fingerprint, the authenticity of traditional Chinese medicine can be effectively identified; (2) through the area and proportion of main characteristic peaks of fingerprint, the quality of traditional Chinese medicine can be effectively controlled. The establishment of traditional Chinese medicine fingerprint can comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine and its preparation, and then the overall description and evaluation of drug quality are carried out.

[0006] At present, traditional Chinese medicine fingerprint technology has involved many methods, including thin layer scanning (TLCS), high performance liquid chromatography (HPLC), gas chromatography (GC) and high performance capillary electrophoresis (HPCE) and other chromatographic methods, as well as ultraviolet spectroscopy (UV), infrared spectroscopy (IR), mass spectrometry (MS), nuclear magnetic resonance (NMR) and X-ray diffraction method.

[0007] Taohongyin contains many traditional Chinese medicine components, and the detectable active ingredients may reach dozens of kinds or even more. If different active ingredients in Taohongyin are identified and determined one by one, a large amount of screening work is needed. At present, there is no technical report on the establishment of fingerprint spectrum of Taohongyin to detect the quality, so it brings more technical obstacles to the establishment of Taohongyin fingerprint spectrum detection method.

[0008] Therefore, at present, a method for monitoring the quality of Taohongyin product by fingerprint spectrum method is needed, and a standardized production method based on the quality monitoring method is needed. SUMMARY

[0009] In order to improve the above technical problems, the present application provides a method for constructing or detecting the fingerprint spectrum of chicken gizzard gold and chrysanthemum mixed plant solid beverage, the medicinal material raw materials of the chicken gizzard gold and chrysanthemum mixed plant solid beverage are chicken gizzard gold, papaya, hawthorn, chrysanthemum, orange peel, peach kernel and medlar, and the method for establishing fingerprint spectrum comprises the following steps:

[0010] 1) preparing a control solution;

[0011] 2) preparing a test solution;

[0012] 3) taking the control solution and the test solution for high performance liquid detection;

[0013] 4) preparing a fingerprint spectrum.

[0014] The method is accurate, reliable and simple to operate, and provides a basis for the quality control of the solid beverage.

[0015] The fingerprint detection method of the chicken ginseng chrysanthemum mixed plant solid beverage comprises: taking a test product solution for high performance liquid detection to obtain a fingerprint, and the chromatographic conditions used are: a C18 chromatographic column, 0.1% phosphoric acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, gradient elution, and the gradient elution program is:

[0016]

[0017] In order to make the fingerprint more accurate, the control solution can be detected under the same liquid phase condition before and after the test product is detected, and the chromatographic peaks are identified by the chromatogram of the control product. The selection of the control product is based on the UPLC-Q-Exactive-Orbitrap-MS technology detection result of the chicken ginseng chrysanthemum mixed plant solid beverage carried out by the inventor in the early stage, and then the literature is consulted to screen the index components of each medicine in the chicken ginseng chrysanthemum mixed plant solid beverage, and finally determined through experimental test and other steps. Finally, the orange peel glycoside peak with good peak shape, no impurity peak interference before and after, and complete separation in the chromatogram of the test product solution of the chicken ginseng chrysanthemum mixed plant solid beverage is selected as the reference peak of the fingerprint.

[0018] The preparation method of the control solution is to dissolve the control product orange peel glycoside and the solvent that can dissolve the orange peel glycoside. In the present application, methanol and / or ethanol are preferably used, and methanol is most preferred. There is no special requirement for the concentration, and it can be prepared under the condition of meeting the upper and lower limit detection requirements of the instrument equipment. The concentration prepared in the present application is 95 μg / mL to 110 μg / mL, specifically 100.05 μg / mL.

[0019] The preparation process of the test product solution is to extract the chicken ginseng chrysanthemum mixed plant solid beverage, which is as follows: taking the chicken ginseng chrysanthemum mixed plant solid beverage, adding a solvent for extraction. The solvent can be a solvent commonly used in the field for the extraction of natural compounds, including but not limited to alcohol or aqueous alcohol solution, such as methanol, ethanol. In some embodiments of the present application, methanol, 60%-80% ethanol aqueous solution is used, specifically methanol. The extraction method can use the conventional natural compound extraction method in the field, such as ultrasonic extraction, reflux extraction. The ultrasonic extraction process is specifically used in the present application, and the test is ultrasonically extracted for 1 hour. It is found that the chromatogram baseline is more stable, the chromatographic peak is higher, and the peak shape is better by comparing several different extraction methods, which is the best way.

[0020] After extraction, the extract is filtered, the filtrate is evaporated to dryness, and the residue is dissolved in a reconstitution solvent, filtered again, and the filtrate is collected. The reconstitution solvent is preferably the same as the extraction solvent. The specific concentration of the reconstituted solution does not need to be strictly within a certain range; it only needs to be sufficient for the equipment to detect the components. A suitable range can be selected as: weight of chicken gizzard and chrysanthemum mixed plant solid beverage: volume of reconstitution solvent = (0.5~1.5) g: 1 mL, for example, 0.5 g, 1 g, or 1.5 g chicken gizzard and chrysanthemum mixed plant solid beverage / mL.

[0021] Under the above conditions, the fingerprint chromatogram of the chicken gizzard and chrysanthemum mixed plant solid beverage had 15 common characteristic peaks, of which peak 10 was hesperidin. When the fingerprint chromatogram was taken as reference (S) with the chromatographic peak of hesperidin at peak 10, the relative retention times of the 14 common characteristic peaks were:

[0022]

[0023] Generally, once the fingerprint spectrum identifies the characteristic peaks in a traditional Chinese medicine product, the presence of the characteristic peaks at their respective positions is sufficient to meet the requirements. However, when higher requirements are placed on the fingerprint spectrum, not only must the position of the characteristic peaks meet the requirements, but the area of ​​the characteristic peaks must also meet certain requirements to indicate that the content of each component in the composition meets certain standards.

[0024] When the fingerprint chromatogram uses the chromatographic peak of hesperidin (peak 10) as a reference (S), the relative peak areas of each common characteristic peak are:

[0025]

[0026] In preparing the fingerprint chromatogram, to achieve greater accuracy, chromatographic peaks can be identified using the chromatogram of a reference standard. Fingerprint chromatograms of the chicken gizzard and chrysanthemum mixed plant solid beverage are prepared using chromatograms from different batches of the beverage. Ideally, at least five batches should be used, preferably at least ten. The fingerprint chromatogram of the chicken gizzard and chrysanthemum mixed plant solid beverage to be tested is compared with the fingerprint chromatogram constructed according to this invention. A relative retention time within ±5% of the specified value indicates a qualified product. A similarity evaluation is performed between the fingerprint chromatogram and the test chromatogram; a similarity greater than 0.8, preferably greater than 0.90, indicates a qualified product.

[0027] The fingerprinting method of this invention uses a standard C18 column. For more optimized conditions, a 4.6 mm × 150 mm, 3-5 μm column is used. According to the high-performance liquid chromatography (HPLC) detection method of this invention, the C18 column can be an InertSustain column. ® AQ-C18 or XBridge ®C18 column, in one embodiment of the application, XBridge ® C18 column, for example C 18 column (4.6 mm x 150 mm, 5 μm). The theoretical plate number should not be less than 5000 calculated by hesperidin, and chromatograms of different batches of Jixingjiahu mixed plant solid beverage and the chromatogram of the reference substance are obtained. In the present application, InertSustain® AQ-C18 column (4.6 mm x 150 mm, 5 μm) and XBridge® C18 column (4.6 mm x 150 mm, 5 μm) are used in succession, and it is found that the chromatogram obtained by separation on the XBridge® C18 column (4.6 mm x 150 mm, 5 μm) has better peak shape of each component and the best separation degree. Thus, it can be used as the preferred chromatographic column for subsequent research.

[0028] In the wavelength range of 280-290 nm, the peaks of various components can be basically realized, which can meet the preparation of the fingerprint. Taking the reference substance of the present application as an example, the absorption effect of hesperidin is the best at 283 nm.

[0029] The present application does not have temperature-sensitive substances and operations, and thus there is no special limitation on the column temperature, and normal detection can be realized under normal laboratory conditions. Therefore, it is recommended that the high performance liquid chromatography detection environment is 20-40 ℃, for example, 30 ℃, 25 ℃, etc. in the embodiments of the present application.

[0030] The flow rate of the mobile phase can be adjusted according to the performance of the equipment and the working efficiency under the condition of good separation of each component peak. Too high flow rate leads to large equipment loss, and too low flow rate leads to long time consumption. In the embodiments of the present application, the most commonly used flow rate 1 ml.min -1 is used according to the condition of the instrument and the working efficiency, and the detection is carried out near the flow rate in the range of 0.8-1.3 ml.min -1 .

[0031] The injection amount of high performance liquid chromatography is usually not fixedly limited, and only needs to meet the convenient operation and meet the basic detection limit of the equipment. The injection amount can be 5.0-20.0 μL, for example, 5 μL, 10 μL, etc.

[0032] The separation effect can be ensured within the gradient elution condition range given in the foregoing of the present application, and the most preferred gradient condition is:

[0033]

[0034] In the present application, "optionally", "optionally" or "optionally present" means that the event or situation described later can but does not necessarily occur, and the description includes both cases where the event or situation occurs and does not occur.

[0035] In the present application, "comprising" and "including" are both open expressions, i.e. including the content indicated by the present application, but not excluding other aspects. It should be understood that "comprising" and "including" can cover the closed meaning, i.e. "consisting of".

[0036] Advantages of the present application:

[0037] Traditional Chinese medicine fingerprint is mainly used to evaluate the stability and reproducibility of traditional Chinese medicinal materials and their preparations. The present application quickly and intuitively identifies the effective material basis of the chicken gizzard gold chrysanthemum mixed plant solid beverage by using the chemical fingerprint quality control method, and a total of 15 common peaks are determined, which lays a foundation for the establishment of the comprehensive quality standard of the chicken gizzard gold chrysanthemum mixed plant solid beverage.

[0038] By analyzing 10 batches of chicken gizzard gold chrysanthemum mixed plant solid beverage, a HPLC fingerprint determination method of chicken gizzard gold chrysanthemum mixed plant solid beverage is established, and the quality is comprehensively evaluated. Through precision, repeatability and stability experiments, it is verified that the method of the present application has high stability and reliability, and can be used as the determination method of the fingerprint of the chicken gizzard gold chrysanthemum mixed plant solid beverage. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 A is the chromatogram of the hesperidin reference substance; Figure 1 B is the fingerprint of the chicken gizzard gold chrysanthemum mixed plant solid beverage; Figure 1 C is the superimposed chromatogram of the HPLC fingerprints of 10 batches of chicken gizzard gold chrysanthemum mixed plant solid beverage.

[0040] Figure 2 A is the chromatogram of the upper limit of gradient elution; Figure 2 B is the chromatogram of the lower limit of gradient elution.

[0041] Figure 3 A and B are the fingerprints of the chicken gizzard gold chrysanthemum mixed plant solid beverage under elution conditions one and two, respectively.

[0042] Figure 4 A and B are the fingerprints of the chicken gizzard gold chrysanthemum mixed plant solid beverage under different chromatographic column conditions, wherein A is the fingerprint of the chicken gizzard gold chrysanthemum mixed plant solid beverage under the condition of XBridge C18 chromatographic column; B is the fingerprint of the chicken gizzard gold chrysanthemum mixed plant solid beverage under the condition of InertSustain AQ-C18 chromatographic column.

[0043] Figure 5 Fingerprint of Jinejin and Juhua mixed plant solid beverage obtained by different sample extraction methods, wherein A, B and C are Jinejin and Juhua mixed plant solid beverage fingerprint obtained by extraction methods A, B and C, respectively. DETAILED DESCRIPTION

[0044] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the present application. Any technology implemented based on the above description of the present application is covered within the scope of protection intended by the present application.

[0045] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0046] Experimental instruments and reagents:

[0047] 1. Instruments

[0048] High performance liquid chromatograph (model: LC-20A, Shimadzu Corporation, Japan), magnetic stirring water bath (model: LC-WB-8+, Shanghai Libangxi Instrument Technology Co., Ltd.); XS105 electronic balance (Sartorius Science Instrument (Beijing) Co., Ltd.); Milli-Q ultrapure water purification system (Beijing Wuzhou Dongfang Science and Technology Co., Ltd.).

[0049] 2. Reagents and reagents

[0050] Hesperidin (purity ≥ 98%, batch number 110721-202220, China Institute for Food and Drug Control); 10 batches of Jinejin and Juhua mixed plant solid beverage (S1-S10), batch numbers are 202111191, 202111192, 202111201, 202111211, 202111291, 202112281, 202202101, 202203241, 202203251, 202204121, respectively, from Shanxi Yabaohexi Pharmaceutical Technology Co., Ltd.; chromatographically pure acetonitrile; chromatographically pure methanol; ultrapure water.

[0051] Example 1: Establishment of Standard Fingerprint of Jinejin and Juhua Mixed Plant Solid Beverage

[0052] The fingerprint was prepared under the following conditions, and the related results are shown in Figure 1 .

[0053] 1. Chromatographic conditions

[0054] The HPLC fingerprint analysis conditions used XBridge® C18 column (4.6 mm x 150 mm, 5 μm); mobile phase A phase (0.1% phosphoric acid water), B phase (acetonitrile); gradient elution; flow rate is 1.0 ml·min -1 ; column temperature 30℃; injection volume 5 μL, detection wavelength is 283 nm.

[0055] Table 1 Gradient elution method

[0056]

[0057] 2. Preparation of reference solution

[0058] Take the hesperidin reference substance, accurately weigh, add methanol to prepare a reference solution with a concentration of 100.05 μg / ml.

[0059] 3. Preparation of test solution

[0060] Take 5.0 g of the chicken gall mixed chrysanthemum plant solid beverage granules, place it in a conical flask with a stopper, add an appropriate amount of methanol, ultrasonic extraction for 1 h, shake well, filter, and evaporate the filtrate to dryness. Dissolve with 5 ml of methanol, centrifuge, take the supernatant, and perform nitrogen blowing until the volume is one-half of the original volume.

[0061] 4. HPLC fingerprint analysis of chicken gall mixed chrysanthemum plant solid beverage

[0062] 4.1 Precision test

[0063] Take the chicken gall mixed chrysanthemum plant solid beverage test sample of the same batch (batch number: 202203241), continuously inject 6 times, record the retention time and peak area of each common chromatographic peak, take the retention time and peak area of peak No. 10 hesperidin as a reference, and convert the relative retention time and relative peak area of each common peak, in order to investigate the consistency of the relative retention time and relative peak area ratio of the chromatographic peak. The results show that the relative retention time RSD of each common peak is less than 1%, and the relative peak area RSD is less than 2.5%, which indicates that the determination method is stable, the system tightness is good, and the method is stable and reliable.

[0064] 4.2 Stability test

[0065] Take the same batch (batch number: 202203241) of Jineijin chrysanthemum mixed plant solid beverage test sample, sample analysis at different time points (0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h), record the retention time and peak area of each common chromatographic peak, take the retention time and peak area of peak No. 10, hesperidin, as the reference, and convert the relative retention time and relative peak area of each common peak, to investigate the consistency of the relative retention time and relative peak area ratio of the chromatographic peak. The results show that the relative retention time RSD of each common peak is less than 1%, and the relative peak area RSD is less than 2.5%. It is shown that the Jineijin chrysanthemum mixed plant solid beverage sample of the application has good stability under the experimental conditions, and can ensure the reliability of the analysis results within 24 h.

[0066] 4.3 Reproducibility test

[0067] Take the same batch (batch number: 202203241) of Jineijin chrysanthemum mixed plant solid beverage test sample, sample analysis at different time points (0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h), record the retention time and peak area of each common chromatographic peak, take the retention time and peak area of peak No. 10, hesperidin, as the reference, and convert the relative retention time and relative peak area of each common peak, to investigate the consistency of the relative retention time and relative peak area ratio of the chromatographic peak. The results show that the relative retention time RSD of each common peak is less than 1%, and the relative peak area RSD is less than 2.5%. It is shown that the Jineijin chrysanthemum mixed plant solid beverage sample of the application has good stability under the experimental conditions, and can ensure the reliability of the analysis results within 24 h.

[0068] 4.4 Establishment of Jineijin chrysanthemum mixed plant solid beverage HPLC fingerprint and similarity evaluation

[0069] Take 10 batches of Jineijin chrysanthemum mixed plant solid beverage (batch number: 202111191, 202111192, 202111201, 202111211, 202111291, 202112281, 202202101, 202203241, 202203251, 202204121), prepare test sample solution according to the method in item 3, and sample analysis, record the chromatogram respectively. Select the representative spectrum (Jineijin chrysanthemum mixed plant solid beverage with batch number 202203241) as the control fingerprint, use the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" recommended by the China Pharmaceutical Association to analyze, take the similarity of the chromatogram as the evaluation index of the fingerprint, and use the multi-point correction full spectrum matching to generate the common mode of the fingerprint. The similarity of each batch sample to the control fingerprint is (0.998, 0.997, 0.996, 0.997, 0.996, 0.996, 0.996, 0.997, 0.999, 0.997).

[0070] 4.5 Identification of common peaks in fingerprint spectra, calculation of relative peak area and relative retention time

[0071] Using a similarity evaluation system, 15 peaks were detected in 10 batches of fingerprint chromatograms. Compared with the mixed reference standard, peak number 10 was identified as hesperidin, with a retention time of 68.625 min. Peak number 10 had a good peak shape, with no interference from impurity peaks before or after it, indicating complete separation; therefore, it was selected as the reference peak for calculating the relative peak area and relative retention time of other common peaks. The results are shown in Tables 2 and 3 below.

[0072] Table 2. Relative peak areas of 15 common peaks in 10 batches of samples (S1~S10)

[0073]

[0074] Table 3. Relative retention times of 15 common peaks in 10 batches of samples (S1~S10)

[0075]

[0076] 4.6 Range of gradient elution conditions

[0077] By adjusting the gradient elution conditions, it can be found that under the currently determined elution conditions, the ratio of mobile phases A and B can be appropriately adjusted, and the upper and lower limits of the elution conditions can be determined through experiments.

[0078] Upper limit of elution conditions:

[0079]

[0080] Lower limit elution conditions:

[0081]

[0082] See results Figure 2 , Figure 2 In this context, A represents the upper limit elution condition of the mobile phase. Figure 2 B represents the lower limit elution condition of the mobile phase. Both conditions must be met to achieve good separation and complete elution.

[0083] Example 2: Comparison of fingerprint spectral preparation methods for chicken gizzard and chrysanthemum mixed plant solid beverage

[0084] 1. Determination of chromatographic conditions and exploration of fingerprinting methods

[0085] The fingerprint chromatograms of Taohong drink were prepared under different high-performance liquid chromatography (HPLC) conditions.

[0086] (1) Elution conditions

[0087] The inventors compared several different elution conditions and proved that the mobile phase elution conditions currently selected in this invention are optimal. Due to the large number of samples, only a brief example is given below: Granules of this product were taken and prepared according to the method for preparing the test solution in Example 1. The following two elution conditions were compared, and the results are shown in [Figure 1]. Figure 3 As can be seen, under the elution conditions below, the peaks of each component are well-defined, indicating good separation. Therefore, elution condition one was selected as the elution condition for the fingerprint chromatogram of the chicken gizzard and chrysanthemum mixed plant solid beverage sample.

[0088] Washing condition one:

[0089]

[0090] Washing condition two:

[0091]

[0092] (2) Column type

[0093] Take the granules of this product and analyze them according to the preparation method and chromatographic conditions of the test solution in Example 1, using InertSustain® AQ-C18 (4.6 mm × 150 mm, 5 μm) and XBridge® C18 (4.6 mm × 150 mm, 5 μm) columns, respectively. The results are shown in the figure. Figure 4 It can be seen that both can meet the requirements for fingerprint chromatogram testing and can be used as chromatographic columns for fingerprint chromatogram preparation. The chromatograms obtained using the XBridge® C18 column (4.6 mm × 150 mm, 5 μm) showed good peak shapes and resolution for each component. Therefore, the XBridge® C18 column (4.6 mm × 150 mm, 5 μm) was selected as the chromatographic column for the fingerprint chromatogram of the chicken gizzard and chrysanthemum mixed plant solid beverage sample.

[0094] 2. Determination of Sample Extraction Method

[0095] Peach Blossom Drink has a complex composition, and different extraction solvents can affect the extraction efficiency of its active ingredients. Finding the most suitable extraction method is crucial to maximizing the extraction of more components and ensuring that they are reflected as peaks in the HPLC chromatogram, which is one of the technical challenges.

[0096] Samples were extracted using several different methods, A, B, and C, with the following procedures:

[0097] A: Take about 5.0 g of the product granules, accurately weigh them, place them in a stoppered conical flask, add an appropriate amount of methanol, extract by sonication for 1 hour, cool, filter, evaporate the filtrate to dryness, add 5 ml of methanol to dissolve the residue, filter, and take the filtrate to obtain the product.

[0098] B: about 5.0 g of the granules were precisely weighed, placed in a conical flask with a stopper, and refluxed with an appropriate amount of methanol in an 80-degree-Celsius water bath for 1 h. After cooling, the solution was filtered, and the filtrate was evaporated to dryness. The residue was dissolved in 5 ml of methanol, filtered, and the filtrate was collected to obtain the sample solution.

[0099] C: about 5.0 g of the granules were precisely weighed, placed in a conical flask with a stopper, and refluxed with an appropriate amount of 70% ethanol in an 80-degree-Celsius water bath for 1 h. After cooling, the solution was filtered, and the filtrate was evaporated to dryness. The residue was dissolved in 5 ml of 70% ethanol, filtered, and the filtrate was collected to obtain the sample solution.

[0100] The sample solutions prepared by the above three methods were detected according to the final determined chromatographic conditions, and the results are shown in Table 1. Figure 5 As shown in Table 1, all the three methods meet the requirements of the preparation of the fingerprint, and can be used as the extraction method for the preparation of the fingerprint or the determination of the content. However, after comparison, it is found that the method of ultrasonic extraction with methanol is the best, the chromatogram obtained by this method has the most stable baseline, the best separation effect of the chromatographic peaks, and the best peak shape.

[0101] The above describes the embodiments of the present application. However, the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A fingerprint chromatographic detection method for a traditional Chinese medicine composition, a chicken gizzard lining and chrysanthemum mixed plant solid beverage, wherein the raw materials of the traditional Chinese medicine composition are chicken gizzard lining, papaya, hawthorn, chrysanthemum, tangerine peel, peach kernel, and wolfberry. The sample solution is subjected to high-performance liquid chromatography (HPLC) to obtain the fingerprint chromatogram. The chromatographic conditions used are: a C18 column, with 0.1% phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, and gradient elution is performed. The gradient elution program is as follows: 。 2. The method as described in claim 1, characterized in that, It also includes a high-performance liquid chromatography (HPLC) step for detecting a reference solution, wherein the reference solution is hesperidin.

3. The method as described in claim 2, characterized in that, The preparation method of the reference solution is as follows: take the reference standard hesperidin, add methanol or ethanol to dissolve it, preferably the concentration of hesperidin in the reference solution is 95 μg / mL to 110 μg / mL.

4. The method as described in claim 1, characterized in that, The preparation method of the test solution is as follows: take chicken gizzard and chrysanthemum mixed plant solid beverage, add extraction solvent to extract, filter the extract, evaporate the filtrate to dryness, add extraction solvent to redissolve, filter, and take the filtrate as the test solution; Preferably, the extraction solvent is methanol or a 60%-80% aqueous solution of ethanol; Preferably, the extraction process is ultrasonic extraction.

5. The method as described in claim 1, characterized in that, The fingerprint spectrum has 15 characteristic peaks, of which peak 10 is hesperidin. Using the chromatographic peak of hesperidin (peak 10) as a reference (S), the relative retention times of the 14 characteristic peaks are: 。 6. The method as described in claim 5, characterized in that, When the fingerprint spectrum uses the chromatographic peak of hesperidin (peak 10) as a reference (S), the relative peak areas of each characteristic peak are: 。 7. The method according to any one of claims 1-6, characterized in that, The detection wavelength of the high-performance liquid chromatography is 280-290 nm, preferably 283 nm.

8. The method according to any one of claims 1-6, characterized in that, The gradient elution procedure is as follows: 。 9. The use of the method according to any one of claims 1-8 in the quality control of chicken gizzard and chrysanthemum mixed plant solid beverage: the fingerprint spectrum of the chicken gizzard and chrysanthemum mixed plant solid beverage to be tested is compared with the fingerprint spectrum of the standard chicken gizzard and chrysanthemum mixed plant solid beverage, and the relative retention time should be within ±5% of the specified value for the product to be qualified. The similarity evaluation with the standard fingerprint spectrum is performed, and the similarity is greater than 0.8 for the product to be qualified.

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