A method for simultaneously detecting contents of hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-jakol in Erchen decoction

Abstract

The present application relates to the technical field of drug component detection, and in particular to a method for simultaneously detecting the contents of hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol in Erchen Decoction. The present application realizes the content determination of five kinds of core active components in the medicinal materials of Chenpi (hesperidin), Banxia (uridine), Gancao (glycyrrhizic acid and glycyrrhizin) and Shengjiang (6-gingerol) in Erchen Decoction by optimizing the mobile phase system and gradient elution program through ultra-high liquid chromatography, and the obtained chromatogram has good separation degree, stable baseline and symmetrical peak shape, and can accurately determine the content of each component. By establishing the detection limit and standard curve of the five kinds of standard products, the one-sidedness of quality control caused by single component detection is avoided, and the quality status of Erchen Decoction can be comprehensively reflected, thereby providing a scientific and reliable technical means for the quality control and evaluation of Erchen Decoction.

Description

Technical Field

[0001] The present invention relates to the technical field of drug component detection, and particularly relates to a method for simultaneously detecting the contents of hesperidin, uridine, glycyrrhizic acid, liquiritin and 6-gingerol in Erchen Decoction. Background Art

[0002] Erchen Decoction is a classic traditional Chinese medicine (TCM) prescription for resolving phlegm, which is composed of six Chinese herbs, namely tangerine peel, pinellia tuber, poria cocos, licorice root, fresh ginger and smoked plum. It has the core effects of drying dampness and resolving phlegm, regulating qi and harmonizing the middle, and is clinically commonly used to treat cough with copious phlegm, chest and epigastric fullness and stuffiness, nausea and vomiting, etc. caused by retention of phlegm-dampness.

[0003] The quality control of TCM compound prescriptions is the key to ensuring the stability of clinical efficacy and the safety of drug use; Erchen Decoction has a complex composition, and its medicinal effects stem from the synergistic effects of multiple active ingredients. Therefore, a multi-index comprehensive quality control system needs to be established to comprehensively characterize its quality level. At present, the quality control research on Erchen Decoction is mostly limited to the qualitative identification or quantitative analysis of single components. For example, only hesperidin in tangerine peel or liquiritin in licorice root is detected. Such methods are difficult to comprehensively reflect the species distribution and content differences of active ingredients in the preparation, easily lead to one-sidedness in quality evaluation, and cannot effectively monitor the overall quality stability of Erchen Decoction. Summary of the Invention

[0004] In order to overcome the above problems, the present invention provides a method for simultaneously detecting the contents of hesperidin, uridine, glycyrrhizic acid, liquiritin and 6-gingerol in Erchen Decoction.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solutions: In the first aspect of the present invention, there is provided a method for simultaneously detecting the contents of hesperidin, uridine, glycyrrhizic acid, liquiritin and 6-gingerol in Erchen Decoction, comprising the following steps: Using ultra-high performance liquid chromatography to detect a standard mixture solution of hesperidin, uridine, glycyrrhizic acid, liquiritin and 6-gingerol. Taking the peak area of each standard as the ordinate and the concentration as the abscissa, establish a standard curve; Centrifuge Erchen Decoction to collect the supernatant, dry it, then dissolve it again with an aqueous methanol solution, filter to collect the filtrate, and obtain a test solution; Using ultra-high performance liquid chromatography to detect the peak area of each component in the test solution, and based on each standard curve, obtain the contents of hesperidin, uridine, glycyrrhizic acid, liquiritin and 6-gingerol in Erchen Decoction.

[0006] In one or more embodiments, the solvent in the standard mixture solution of hesperidin, uridine, glycyrrhizic acid, liquiritin and 6-gingerol is an aqueous methanol solution; Preferably, the volume fraction of methanol in the methanol-water solution is 45-55%, more preferably 50%. Using methanol-water solution as a solvent can fully dissolve the standard to prepare a standard mixed solution, ensuring the accuracy of detection.

[0007] In one or more embodiments, the preparation method of Er Chen Tang includes the following steps: According to the formula, mix dried tangerine peel powder, pinellia powder, poria powder, licorice powder, ginger powder and dried plum powder, add water, soak, and then decoct twice and combine the two filtrates to obtain Er Chen Tang.

[0008] Preferably, the total mass ratio of tangerine peel powder, pinellia powder, poria powder, licorice powder, ginger powder, and dried plum powder to water is 1:(7~9), more preferably 1:8.

[0009] Preferably, the soaking time is 20-40 minutes, and more preferably 30 minutes.

[0010] Preferably, the time for each of the two decoction processes is 0.5 to 1 hour.

[0011] The preparation method of Er Chen Tang conforms to the general preparation principles of traditional Chinese medicine compound decoction. Clarifying the preparation process of Er Chen Tang ensures the consistency and stability of the sample source, providing a guarantee for the applicability of the detection method; this preparation process can effectively retain the five core active ingredients, ensuring the clinical efficacy of the preparation.

[0012] In one or more embodiments, the centrifugation rate is 9000~12000 r / min, and the centrifugation time is 4~6 min.

[0013] In one or more embodiments, when the supernatant of the Er Chen Tang is collected by centrifugation, dried, and then reconstituted with a methanol-water solution, the volume fraction of methanol in the methanol-water solution is 45-55%, preferably 50%. This solvent system has good solubility for the five target components, excellent extraction effect, and can effectively reduce matrix interference and improve detection sensitivity.

[0014] In one or more embodiments, the chromatographic conditions for detecting hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol by ultra-high performance liquid chromatography are the same as those for detecting the test solution by ultra-high performance liquid chromatography. The specifications include: a chromatographic column packed with octadecylsilane-bonded silica gel; a column length of 150 mm and a diameter of 2.1 mm; a packing particle size of 5 μm; a column temperature of 40℃; an injection volume of 1.5 μL; and a flow rate of 0.3 mL / min. The mobile phase A was 20 mmol / L ammonium acetate-0.1% (volume fraction) formic acid aqueous solution, and the mobile phase B was 0.1% (volume fraction) formic acid-acetonitrile; gradient elution was used.

[0015] Preferably, the gradient elution conditions are as follows:

[0016] The chromatographic parameters are optimized to the best possible conditions, which can further improve the separation effect of each component and ensure the stability and accuracy of the detection results.

[0017] The defined gradient elution conditions enable the efficient separation of five components: uridine, glycyrrhizic acid, hesperidin, glycyrrhizin, and 6-gingerol. The peaks of each component are symmetrical, without tailing, and the resolution is greater than 1.5, which meets the requirements for quantitative analysis.

[0018] The beneficial effects of this invention are as follows: (1) This invention optimizes the mobile phase system and gradient elution program through ultra-high performance liquid chromatography, and realizes the simultaneous determination of the content of five core active ingredients in five medicinal materials in Er Chen Tang: tangerine peel (hesperidin), pinellia (uridine), licorice (glycyrrhizic acid and glycyrrhizin), and ginger (6-gingerol). The obtained chromatograms have good separation, stable baseline and symmetrical peak shape, and can accurately determine the content of each component. By establishing the detection limit and standard curve of five standards, the one-sidedness of quality control caused by single component detection is avoided, and the quality status of Er Chen Tang can be comprehensively reflected, providing a scientific and reliable technical means for its quality control and evaluation.

[0019] (2) The detection method provided by the present invention is simple and fast, with good separation effect, high sensitivity, stable results, good reproducibility and strong specificity. It is suitable for the quality detection of Er Chen Tang and is conducive to quality monitoring and industrialization promotion in industrial production. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 The standard curve for hesperidin; Figure 2 The standard curve for uridine; Figure 3 This is the standard curve for glycyrrhizic acid; Figure 4 This is the standard curve for glycyrrhizin; Figure 5 The standard curve for 6-gingerol; Figure 6 The chromatogram of the test solution; Figure 7 The chromatograms of 10 batches of Er Chen Tang are shown. Peak 1 is uridine, peak 2 is glycyrrhizin, peak 3 is hesperidin, peak 4 is glycyrrhizic acid, and peak 5 is 6-gingerol. Figure 8 The chromatogram of the test solution in Comparative Example 1 is shown below. Figure 9 The chromatogram of the test solution in Comparative Example 2; Figure 10 The chromatogram is shown for the test solution in Comparative Example 3. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0024] Currently, there are significant deficiencies in the quality control of Er Chen Tang, making it difficult to meet the multi-indicator and comprehensive quality control requirements of Er Chen Tang. This problem is specifically reflected in the following two aspects: First, the control of core components is incomplete. Existing research and related quality standards focus on controlling only a few components. For example, some studies only quantitatively analyze individual components such as hesperidin or glycyrrhizic acid, completely failing to cover the three active components closely related to the core efficacy: uridine, glycyrrhizin, and 6-gingerol. Since the efficacy of traditional Chinese medicine formulas depends on the synergistic effect of multiple components, controlling only a few components cannot fully reflect the overall quality of Er Chen Tang, easily leading to fluctuations in efficacy between different batches of the formula due to differences in the content of uncontrolled core components.

[0025] Secondly, the detection methods have inherent limitations and cannot achieve simultaneous detection of core components. Existing related studies have only achieved simultaneous determination of two components (such as hesperidin and belamcanda chinensis) in the quality control study of Ma Xing Er Chen Tang, and this detection system does not cover the core components of uridine, glycyrrhizin, and 6-gingerol identified in this application. Most detection methods are still limited to single-component determination; for example, the quality standard for Er Chen Wan stipulated in the Chinese Pharmacopoeia only targets the detection of a single component, lacking a dedicated simultaneous detection scheme for the above five core active components. From a technical perspective, traditional high-performance liquid chromatography (HPLC) suffers from drawbacks such as complex sample pretreatment, insufficient detection accuracy, and excessively long analysis time. Although ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) has been applied to the detection of Er Chen Tang components, its detection range does not match the core active components identified in this application, and therefore cannot meet the actual needs of comprehensive quality control of Er Chen Tang across multiple indicators.

[0026] To overcome the above problems, the present invention provides a method for simultaneously detecting the contents of hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol in Er Chen Tang, comprising the following steps: A standard mixture of hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol was detected by ultra-high performance liquid chromatography. Standard curves were established with the peak area of ​​each standard as the ordinate and the concentration as the abscissa. Centrifuge the Er Chen Tang to collect the supernatant, dry it, redissolve it with methanol-water solution, filter and collect the filtrate to obtain the test solution; The peak areas of each component in the test solution were detected by ultra-high performance liquid chromatography (UHPLC). Based on the standard curves, the contents of hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol in Er Chen Tang were obtained.

[0027] In this invention, by limiting the use of methanol-water solution as the solvent for the standard mixture solution, the standard can be fully dissolved to prepare the standard mixture solution, thus ensuring the accuracy of detection.

[0028] By clarifying the preparation process of Er Chen Tang, the consistency and stability of the test sample source are ensured, thus guaranteeing the applicability of the detection method. This preparation process can effectively retain the five core active ingredients, ensuring the clinical efficacy of the preparation.

[0029] In this invention, by limiting the use of methanol-water solution as the resolution solvent for the test sample solution, the five target components are well soluble and have excellent extraction effect, which can effectively reduce matrix interference and improve detection sensitivity.

[0030] By optimizing chromatographic parameters, the separation effect of each component can be further improved, ensuring the stability and accuracy of the detection results.

[0031] By optimizing the gradient elution conditions, five components—uridine, glycyrrhizic acid, hesperidin, glycyrrhizin, and 6-gingerol—can be efficiently separated. The peaks of each component are symmetrical, without tailing, and the resolution is greater than 1.5, meeting the requirements for quantitative analysis.

[0032] This invention utilizes ultra-high performance liquid chromatography (UHPLC) to optimize the mobile phase system and gradient elution program, enabling the simultaneous determination of five core active ingredients in Er Chen Tang (a traditional Chinese medicine formula): tangerine peel (hesperidin), pinellia (uridine), licorice (glycyrrhizic acid and glycyrrhizin), and ginger (6-gingerol). The resulting chromatograms exhibit good separation, stable baselines, and symmetrical peak shapes, allowing for accurate determination of each component's content. By establishing detection limits and standard curves for five standards, the invention avoids the limitations of single-component detection in quality control, comprehensively reflecting the quality status of Er Chen Tang and providing a scientific and reliable technical means for its quality control and evaluation.

[0033] The detection method provided by this invention is simple and rapid, with good separation effect, high sensitivity, stable results, good reproducibility, and strong specificity. It is suitable for the quality detection of Er Chen Tang and is beneficial to quality monitoring and industrialization promotion in industrial production.

[0034] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0035] Example 1 Instrument precision assessment and establishment of standard curves for hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol: 1.1 Instrument Precision Assessment (1) Weigh the hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol standards respectively, dissolve them in 50% methanol aqueous solution and make up to volume to prepare a series of standard mixed solutions with gradient concentrations.

[0036] (2) The same batch of standard mixed solution was detected by ultra-high performance liquid chromatography (UHPLC) six times consecutively, and the peak areas of each component were recorded. The results are as follows: Figure 1 As shown.

[0037] Ultra-high performance liquid chromatography conditions: The mobile phase A was 20 mmol / L ammonium acetate-0.1% (volume fraction) formic acid aqueous solution, and the mobile phase B was 0.1% (volume fraction) formic acid-acetonitrile; gradient elution was used.

[0038] The conditions for gradient elution are:

[0039] Table 1 Results of Instrument Precision Testing

[0040] The results in Table 1 show that the ultra-high performance liquid chromatography (UHPLC) instrument has good precision, and minor fluctuations in the instrument and operating environment have no significant impact on the measurement results. 1.2 Establishment of standard curves for hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol A standard mixture of hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol was detected by ultra-high performance liquid chromatography. Standard curves were established with the peak area of ​​each standard as the ordinate and the concentration as the abscissa.

[0041] Ultra-high performance liquid chromatography conditions: Column: Thermo Scientific Accucore™ C18; column length 150 mm, diameter 2.1 mm, packing particle size 5 μm, column temperature 40℃; injection volume 1.5 μL; flow rate 0.3 mL / min; The mobile phase A was 20 mmol / L ammonium acetate-0.1% (volume fraction) formic acid aqueous solution, and the mobile phase B was 0.1% (volume fraction) formic acid-acetonitrile; gradient elution was used.

[0042] The conditions for gradient elution are:

[0043] The peak area and concentration correspondence of the hesperidin series of graded concentration standard mixed solutions are shown in Table 2 and... Figure 1 As shown, the linear regression equation is y = 2571.6x + 19613, and the correlation coefficient R0 is... 2 =0.9995, retention time is 7.9 min. According to the limit of quantitation requirements under the Validation Guidelines for Analytical Methods in General Chapter 9101 of the Chinese Pharmacopoeia, the minimum quantitation concentration is calculated to be 0.699 μg / mL.

[0044] Table 2. Relationship between peak area and concentration of hesperidin standard

[0045] The peak area and concentration correspondence of the mixed solutions of uridine series standard concentration gradients are shown in Table 3 and... Figure 2 As shown, the linear regression equation is y = 535.01x + 3055.9, and the correlation coefficient R0 is... 2 =0.9998, retention time is 1.0 min. According to the limit of quantitation requirements under the Validation Guidelines for Analytical Methods in General Chapter 9101 of the Chinese Pharmacopoeia, the minimum quantitation concentration is calculated to be 2.770 μg / mL.

[0046] Table 3. Relationship between peak area and concentration of uridine standard

[0047] The peak area and concentration correspondence of glycyrrhizic acid series gradient concentration standard mixture solutions are shown in Table 4 and Figure 3 As shown, the linear regression equation is y = 1238.4x + 4050, and the correlation coefficient R0 is... 2 =0.9993, retention time is 9.4 min. According to the limit of quantitation requirements under the Validation Guidelines for Analytical Methods in General Chapter 9101 of the Chinese Pharmacopoeia, the minimum quantitation concentration is calculated to be 1.373 μg / mL.

[0048] Table 4. Correspondence between peak area and concentration of glycyrrhizic acid standard

[0049] The peak area and concentration correspondence of the glycyrrhizin series of graded concentration standard mixed solutions are shown in Table 5. Figure 4 As shown, the linear regression equation is y = 2621.1x + 20367, and the correlation coefficient R0 is... 2 =0.9992, retention time is 7.0 min. According to the analytical method verification principle in General Chapter 9101 of the Chinese Pharmacopoeia, the minimum quantitative concentration is calculated to be 0.763 μg / mL.

[0050] Table 5. Correspondence between peak area and concentration of glycyrrhizin standard

[0051] The peak area and concentration correspondence of the 6-gingerol series of graded concentration standard mixed solutions are shown in Table 6 and . Figure 5 As shown, the linear regression equation is y = 823.48x - 489.12, and the correlation coefficient R0 is... 2 =0.9993, retention time is 11.9 min. According to the limit of quantitation requirements under the Validation Guidelines for Analytical Methods in General Chapter 9101 of the Chinese Pharmacopoeia, the minimum quantitation concentration is calculated to be 3.643 μg / mL.

[0052] Table 6. Relationship between peak area and concentration of 6-gingerol standard

[0053] Example 2 Preparation of Er Chen Tang: Derived from the standard formula and dosage of Er Chen Tang in "Taiping Huimin Heji Jufang": 15 g dried tangerine peel, 15 g ginger-processed pinellia, 9 g poria cocos, 4.5 g licorice root, 1.5 g fresh ginger, and 3 g dried plum; The above-mentioned tangerine peel, ginger, pinellia, poria, licorice, ginger and dried plum are pulverized and mixed. Add water equal to 8 times the total weight of the Chinese medicine powder, soak for 30 minutes, decoct for 1 hour, and filter. Add water equal to 8 times the weight of the dregs, decoct for 1 hour, filter, and combine the two decoctions to obtain Er Chen Tang.

[0054] Example 3 3.1 Preparation of the test solution: Take 5 mL of the Er Chen Tang prepared in Example 2, prepare 23 portions in parallel, centrifuge at 10000 r / min for 5 min, take the supernatant, dry in a water bath, reconstitute with 5 mL of 50% methanol aqueous solution, weigh, cool to room temperature, make up the weight loss with 50% methanol aqueous solution, shake well, filter through a 0.45 μm organic phase filter membrane, and take the filtrate as the test solution.

[0055] 3.2 Method repeatability and 24-hour stability study: Ultra-high performance liquid chromatography conditions: Column: Thermo Scientific Accucore™ C18; column length 150 mm, diameter 2.1 mm, packing particle size 5 μm, column temperature 40℃; injection volume 1.5 μL; flow rate 0.3 mL / min; The mobile phase A was 20 mmol / L ammonium acetate-0.1% (volume fraction) formic acid aqueous solution, and the mobile phase B was 0.1% (volume fraction) formic acid-acetonitrile; gradient elution was used.

[0056] The conditions for gradient elution are:

[0057] The repeatability test results are shown in Table 7. As can be seen from Table 7, the RSD of each analyte in the 6 parallel test solutions is ≤2.0%, indicating that the method has good repeatability and the consistency of the test sample pretreatment and determination process is high.

[0058] Table 7 Results of Repeatability Testing

[0059] Take the same batch of test solution and analyze it at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after preparation, respectively, according to the aforementioned limited ultra-high performance liquid chromatography conditions. Record the peak area of ​​each analyte and conduct a 24-h short-term stability study. The results are shown in Table 8. The results show that the recovery rate of the test solution sample is distributed from 92.0% to 105.0% within 24 h. The results show that the sample is stable and it is recommended to complete the detection within 24 h.

[0060] Table 8 Stability test results

[0061] 3.3 Determination of the contents of hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol: Take the test solution and perform ultra-high performance liquid chromatography (UHPLC) detection. The UHPLC conditions are the same as specified in 3.2. Record the chromatogram. The results are as follows: Figure 6 As shown.

[0062] The results of the determination of hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol in 10 batches of Er Chen Tang are shown in Table 9 and 10. Figure 7 As shown. During the content determination process, the content was qualitatively determined based on the peak elution time, and the content of each component was calculated based on the peak area and the standard curve obtained in Example 1.

[0063] Table 9. Results of determination of hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol content in 10 batches of Er Chen Tang.

[0064] The results show that the method established in this invention can accurately determine the content of five components in Er Chen Tang. The determination results of 10 batches of samples are stable and reproducible, and can be used for the quality control of Er Chen Tang.

[0065] Comparative Example 1 Change of mobile phase: Replace mobile phase A as specified in 3.2 with 0.1% (v / v) formic acid aqueous solution. Keep all other UHPLC conditions the same as specified in 3.2. Perform UHPLC analysis on the test solution and record the chromatogram. The results are as follows: Figure 8 As shown.

[0066] Figure 6 and Figure 8 The comparison revealed that, Figure 8 The mid-component peaks 1 and 2 overlapped, exhibited peak tailing and broadening, and showed severe baseline drift. Peak 5 also showed severe tailing and low separation from peak 6. This was because the aqueous phase containing only formic acid lacked buffer salts, failing to provide stable ionic strength and buffering capacity. This resulted in disordered retention behavior of the target component, intensified secondary interactions with the stationary phase, and fluctuations in ionization efficiency. The 20 mmol / L ammonium acetate added in this invention optimized the interaction between the component and the stationary phase, suppressed secondary adsorption, stabilized the background signal and ionization efficiency, and significantly improved the separation of multiple components, peak symmetry, baseline stability, and quantitative reliability.

[0067] Replace the elution conditions specified in 3.2 with:

[0068] Other ultra-high performance liquid chromatography (UHPLC) conditions are the same as those specified in 3.2. The test solution is then subjected to UHPLC detection, and the chromatogram is recorded. The results are as follows: Figure 9 As shown.

[0069] Figure 6 and Figure 9 The comparison revealed that, Figure 6 The peaks (peaks 1 to 5) of each target component have a reasonable peak time distribution, sharp and symmetrical peak shapes, and baseline separation of each component is achieved without cross interference. The baseline is stable throughout the entire process without obvious noise or drift. Figure 9 The overall elution time of the target component was significantly delayed, the peak shape was broadened, and peaks 5 and 6 showed severe tailing, resulting in insufficient separation.

[0070] Comparative Example 3 Change of mobile phase: Replace mobile phase A as specified in 3.2 with 0.1% (v / v) formic acid ammonia solution. Keep all other UHPLC conditions the same as specified in 3.2. Perform UHPLC analysis on the test solution and record the chromatogram. The results are as follows: Figure 10 As shown.

[0071] Figure 6 and Figure 10 The comparison revealed that, Figure 10 The mid-component elution time was generally delayed, the peak shape was severely broadened and tailed, some peaks showed abnormal responses and the baseline was significantly raised. This was because the formic acid ammonia solution lacked sufficient ionic strength and buffering capacity, and the pH was poorly compatible with the target component, resulting in abnormal retention behavior of the target component, intensified secondary interactions with the stationary phase, and fluctuations in ionization efficiency. The 20 mmol / L ammonium acetate added in this invention provides a stable ionic strength and buffering system, optimizes the interaction between the component and the stationary phase, inhibits secondary adsorption, stabilizes the background signal and ionization efficiency, and significantly improves the separation of multi-components, peak symmetry, baseline stability and quantitative reliability.

[0072] By optimizing chromatographic parameters, the separation effect of each component can be further improved, ensuring the stability and accuracy of the detection results.

[0073] By optimizing the gradient elution conditions, five components—uridine, glycyrrhizic acid, hesperidin, glycyrrhizin, and 6-gingerol—can be efficiently separated. The peaks of each component are symmetrical, without tailing, and the resolution is greater than 1.5, meeting the requirements for quantitative analysis.

[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for simultaneously detecting the contents of hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol in Er Chen Tang, characterized in that, Includes the following steps: A standard mixture of hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol was detected by ultra-high performance liquid chromatography. Standard curves were established with the peak area of ​​each standard as the ordinate and the concentration as the abscissa. Centrifuge the Er Chen Tang to collect the supernatant, dry it, redissolve it with methanol-water solution, filter and collect the filtrate to obtain the test solution; The peak areas of each component in the test solution were detected by ultra-high performance liquid chromatography (UHPLC). Based on the standard curves, the contents of hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol in Er Chen Tang were obtained.

2. The method as described in claim 1, characterized in that, The solvent in the mixed solution of standard hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol is an aqueous methanol solution.

3. The method as described in claim 2, characterized in that, The volume fraction of methanol in the methanol-water solution is 45-55%, preferably 50%.

4. The method as described in claim 1, characterized in that, The preparation method of Er Chen Tang includes the following steps: According to the formula, mix dried tangerine peel powder, pinellia powder, poria powder, licorice powder, ginger powder and dried plum powder, add water, soak, and then decoct twice and combine the two filtrates to obtain Er Chen Tang.

5. The method as described in claim 4, characterized in that, The total mass ratio of tangerine peel powder, pinellia powder, poria powder, licorice powder, ginger powder, and dried plum powder to water is 1:(7~9), preferably 1:

8.

6. The method as described in claim 4, characterized in that, The soaking time is 20 to 40 minutes, preferably 30 minutes.

7. The method as described in claim 4, characterized in that, The cooking time for both decoction processes was 0.5 to 1 hour.

8. The method as described in claim 1, characterized in that, The centrifugation rate is 9000~12000 r / min, and the centrifugation time is 4~6 min.

9. The method as described in claim 1, characterized in that, When collecting the supernatant by centrifuging the Er Chen Tang, drying it, and then resolving it with a methanol aqueous solution, the volume fraction of methanol in the methanol aqueous solution is 45-55%, preferably 50%.

10. The method as described in claim 1, characterized in that, The chromatographic conditions for the detection of hesperidin, uridine, glycyrrhizic acid, glycyrrhizin and 6-gingerol by ultra-high performance liquid chromatography are the same as those for the detection of the test solution by ultra-high performance liquid chromatography. The specifications include: a chromatographic column packed with octadecylsilane-bonded silica gel; a column length of 150 mm and a diameter of 2.1 mm; a packing particle size of 5 μm; a column temperature of 40 °C; an injection volume of 1.5 μL; and a flow rate of 0.3 mL / min. The mobile phase A was 20 mmol / L ammonium acetate-0.1% (volume fraction) formic acid aqueous solution, and the mobile phase B was 0.1% (volume fraction) formic acid-acetonitrile; gradient elution was used. Preferably, the gradient elution conditions are as follows: 。

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