Determination method of glycyrrhiza index component in pediatric qixing tea and application thereof

Abstract

The application discloses a method for determining the content of licorice index components in pediatric Qixing tea and application thereof, and belongs to the technical field of detection and analysis. The technical problem to be solved is that the existing technology has the defects of single pharmacopoeia quality control index, long analysis time of the existing detection method, complex sample pretreatment, small number of simultaneously determined effective components, poor adaptability of single wavelength detection, and uneven recovery rate of conventional solvent extraction, and a method for determining the content of licorice index components in pediatric Qixing tea is provided. The technical solution points are that the HPLC-UV switching wavelength method is used to simultaneously determine the content of seven index components, i.e., isohoutuyangoside, apiosyl glycyrrhizin, neoglycyrrhizin, glycyrrhizin, isoglycyrrhizin, glycyrrhetin and glycyrrhizic acid in the pediatric Qixing tea.

Description

Technical Field

[0001] This invention relates to the field of detection and analysis technology, specifically to a method for determining the content of licorice index components in children's herbal tea and its application. Background Technology

[0002] Xiaoer Qixingcha granules are a classic pediatric formula preparation. The standard prescription consists of seven medicinal herbs: 893g of coix seed, 893g of rice sprout, 446g of hawthorn, 670g of bamboo leaf, 335g of uncaria, 112g of cicada slough, and 112g of licorice. These herbs are extracted, concentrated, granulated, and packaged. The core effects are to stimulate appetite, relieve indigestion, clear heat, and calm the nerves. Clinically, it is widely used for common pediatric symptoms such as indigestion, loss of appetite, irritability, restlessness, insomnia, constipation, and dark urine caused by food stagnation and heat in children. Each of the seven raw medicinal materials contains characteristic active substances, collectively forming the pharmacodynamic basis of the preparation: Licorice contains flavonoids and triterpenoid saponins such as glycyrrhizic acid, glycyrrhizin, neoglycyrrhizin, apigenin, and isoglycyrrhizin, which are the core active substances for strengthening the spleen, regulating balance, clearing heat, and detoxifying; Lophatherum gracile is rich in carbon glycosides such as isohexyrrhizin, which exert a calming and irritability-relieving effect; Uncaria rhynchophylla contains rhynchophylline and isorhynchophylline, which are the hallmark components for calming the liver and relieving convulsions; Hawthorn, rice sprouts, and Coix seed contain organic acids, polyphenols, and polysaccharides, respectively, which are specifically for promoting digestion and eliminating food stagnation; Cicada slough contains amino acids and sterol active components, which assist in dispelling wind and calming the mind. The synergistic effect of these multiple effective components collectively achieves the complete pharmacological efficacy of the preparation. However, the current pharmacopoeia quality standards only use high performance liquid chromatography to limit the content of glycyrrhizic acid, a single component in the preparation. Relying on a single indicator cannot characterize the authenticity of the other six medicinal materials such as bamboo leaf, uncaria, and hawthorn, nor can it fully reflect the overall content level of multiple categories of effective components such as flavonoids, triterpenes, alkaloids, and organic acids in the prescription. The quality control coverage is seriously insufficient, making it difficult to accurately and comprehensively control the overall intrinsic quality of Xiaoer Qixingcha granules, resulting in significant quality control loopholes.

[0003] Pediatric Seven-Star Tea Granules are formulated from seven Chinese medicinal herbs with significantly different properties and substance categories. Each herb contains multiple active ingredients with varying structures, polarities, and UV absorption characteristics. Existing detection methods for Pediatric Seven-Star Tea Granules mostly focus on alkaloid markers in herbs such as Uncaria rhynchophylla. There are few simultaneously detectable indicators for licorice, a key ingredient in the formula, making it difficult to fully evaluate the stability of licorice ingredient usage and accurately reflect the actual levels of licorice-related active ingredients in the preparation.

[0004] Licorice is a key ingredient in the prescription of Xiaor Qixingcha granules, and its various active components, such as triterpenoid saponins and flavonoid glycosides, are directly related to the clinical efficacy of the preparation. Licorice also contains multiple pharmacologically active substances, including glycyrrhizic acid, glycyrrhizin, and isoglycyrrhizin, with differences in their physicochemical properties and UV responses. Current conventional HPLC-UV detection methods can only determine a few indicator components in licorice, making it difficult to simultaneously quantify multiple characteristic components and fully characterize the material basis of licorice in the preparation.

[0005] While some existing literature can simultaneously detect more than twenty components of Xiaoer Qixingcha (a traditional Chinese herbal tea), they generally rely on mass spectrometry detectors, resulting in high instrument purchase and maintenance costs, which hinders its widespread application in routine laboratories. Therefore, the industry urgently needs to develop a method for detecting multiple licorice components in Xiaoer Qixingcha granules. This method should feature simple pretreatment, short analysis time, excellent chromatographic separation, high quantitative accuracy, and compatibility with general-purpose HPLC-UV equipment to meet the quality control requirements for the herbal flavor of licorice in this compound preparation.

[0006] Relevant patent documents retrieved: The document, published under the country of origin China, with announcement number CN108627581B and announcement date August 31, 2021, discloses a method for determining the content of rhynchophylline and isorhynchophylline in children's seven-star tea granules. The method includes the following steps: (1) Preparation of mixed reference solution: Weigh appropriate amounts of rhynchophylline and isorhynchophylline, accurately weigh them, and add methanol to prepare a mixed reference solution of rhynchophylline and isorhynchophylline; (2) Preparation of test solution: Accurately weigh children's seven-star tea granules, add hot water to dissolve them, sonicate them, cool them, adjust the pH value with ammonia, and then extract them with ethyl acetate by shaking and recovering acetic acid. Ethyl ester solution, the residue is dissolved in methanol; (3) High performance liquid chromatography determination: use octadecylsilane bonded silica gel as the packing material; mobile phase: acetonitrile (A): 0.02-0.07% diethylamine-(7-10)mM ammonium acetate (B)=40-60:60-40V / V; flow rate: 0.8-1.2mL / min; elution time: 35-60 minutes; column temperature: 25-35℃; detection wavelength: 240-250nm; This content determination method is simple to operate, accurately reflects the true content of the product, and is suitable for routine production inspection in industrialization.

[0007] This document, published in China (CN115290769B, May 14, 2024), discloses a method for detecting the active ingredients of Xiaor Qixing Tea granules. The method comprises the following steps: First, a mixed internal standard solution, a mixed standard solution, and the sample to be tested are prepared. Then, the detection is performed as follows: The sample is injected into a Poroshel 1 120EC-C18 column with an injection volume of 1-10 μL and a column temperature of 20-40℃. Gradient elution is then performed using a mobile phase composed of phases A and B at a flow rate of 0.15-0.45 mL / min according to a prescribed procedure. Simultaneously, multiple reaction monitoring (MRM) analysis is used to obtain the spectra of the analyte and the standard. Finally, the spectra of the analyte and the standard are compared to determine the 26 active ingredients and their contents in the Xiaor Qixing Tea granules. This method can simultaneously perform qualitative and quantitative detection of 26 active ingredients in Xiaor Qixing Tea granules and has the advantages of high precision and good repeatability.

[0008] Relevant non-patent literature retrieved: The journal title is "Chinese Journal of Experimental Traditional Medical Formulae", the article title is "Fingerprint Spectrum of Xiao'er Qixingcha Granules", volume number is 13, and the publication date is July 29, 2014. This article discloses the use of high performance liquid chromatography (HPLC) with a 9-segment gradient elution program to simultaneously determine three components: glycyrrhizin, chlorogenic acid, and apigenin. The entire gradient elution process, combined with column equilibration to the initial mobile phase, has a total detection time of up to 118 minutes, resulting in a lengthy detection cycle and extremely low efficiency for large batches of samples.

[0009] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The detection cycle is lengthy and the detection efficiency for large batches of samples is extremely low. The relevant evidence is that the total detection time for a single high-performance liquid chromatography method disclosed in the literature "Fingerprint Spectrum of Pediatric Seven-Star Tea Granules" is as high as 118 min, which is low in detection efficiency.

[0010] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: Traditional Chinese medicine (TCM) has complex components. During method development, to improve peak shape, enhance chromatographic peak resolution, and strengthen detector signal response, experiments were conducted to investigate different types and concentrations of mobile phase additives, as well as parameters such as the absorption wavelength of each component. Finally, by optimizing the gradient elution program, baseline separation and quantitative analysis of the seven target components in the sample were achieved within 37 minutes. Summary of the Invention

[0011] The purpose of this invention is to provide: A method for determining the content of licorice index components in children's herbal tea, and related technologies, to solve technical problems such as improving the separation of target peaks, enhancing signal response, and improving peak shape, or combinations thereof.

[0012] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0013] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0014] Definitions of standard terms can be found in references such as the 2025 edition of the Pharmacopoeia of the People's Republic of China and Analysis of Traditional Chinese Medicine Preparations (2nd edition, edited by Cai Baochang, Higher Education Press).

[0015] Unless otherwise stated, conventional methods within the scope of the art shall be used, such as single-wavelength constant detection, programmed wavelength switching method, external standard point method, external standard curve method, specificity, precision, stability, recovery rate, linearity test, etc.

[0016] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0017] The “range” disclosed in this document takes the form of a lower limit and an upper limit. It can be one or more lower limits and one or more upper limits, respectively. A given range is defined by selecting a lower limit and an upper limit. The selected lower and upper limits define the boundaries of the particular range. All ranges that can be defined in this way are inclusive and composable; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is also expected that ranges of 60-110 and 80-120 are also expected. Furthermore, if the minimum range values ​​are listed as 1 and 2, and if the maximum ranges are listed as 3, 4, and 5, then the following ranges are all expected: 1-2, 1-4, 1-5, 2-3, 2-4, and 2-5.

[0018] In this invention, unless otherwise specified, the numerical range "ab" represents a shortened representation of any combination of real numbers from a to b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been listed in this document, and "0-5" is simply a shortened representation of these numerical combinations.

[0019] The term "Xiao'er Qixing Cha" used in this article refers to a pediatric traditional Chinese medicine preparation made from seven medicinal herbs: coix seed, rice sprout, hawthorn, light bamboo leaf, uncaria, cicada molting, and licorice. It has the effects of stimulating appetite, relieving indigestion, clearing heat, and calming the nerves.

[0020] The term "HPLC-UV" used in this article refers to High Performance Liquid Chromatography-Ultraviolet Detector, a chromatographic detection method that uses high performance liquid chromatography to separate sample components and an ultraviolet detector to perform quantitative and qualitative analysis of target substances.

[0021] The term "wavelength switching method" used in this article refers to a chromatographic detection method that automatically switches the ultraviolet detection wavelength during different retention time periods of the chromatographic program based on the differences in the maximum ultraviolet absorption wavelength of different target reference standards, thereby simultaneously determining multiple components with significant differences in polarity and ultraviolet absorption characteristics.

[0022] The term "glycyrrhizic acid" used in this article refers to Glycyrrhizic acid, CAS: 1405-86-3, a hallmark triterpenoid saponin active ingredient in licorice, with the molecular formula C2. 42 H 62 O 16 As a medicine, it has anti-inflammatory and anti-allergic properties; as a sweetener, it is widely used in various foods.

[0023] The term "glycyrrhizin" used in this article refers to Liquiditin (CAS: 551-15-5), an active flavonoid glucosinolate found in licorice. It is a characteristic indicator for the quality control of licorice and the determination of its content, and has various pharmacological effects such as anti-oxidation and anti-HIV activity.

[0024] The term "neoliquiritin" used in this article refers to Neoliquiritin apioside, CAS: 5088-75-5, a characteristic flavonoid disaccharide glycoside of licorice. Its glycosyl substitution site differs structurally from that of apigenin glycyrrhizin, and it is the indicator to be measured in the content determination of this product.

[0025] The term “apioside” as used in this article refers to Liquiditin apioside, CAS: 74639-14-8, a flavonoid glycoside in licorice that is a apioside-glucose disaccharide substituted with apioside.

[0026] The term "isoglycyrrhizin" as used in this article refers to isoliquiritoside, also known as isoliquiritoside, a flavonoid compound with the chemical formula C. 21 H 22 O9, molecular weight 418.39, CAS: 5041-81-6; it appears as a white to yellow crystalline powder with a melting point of 185-186℃ and a boiling point of 743.5±60.0℃. It is an active isoflavone glycoside in licorice.

[0027] The term "isocarboxylic acid" as used in this article refers to luteolin-6-C-glucoside, CAS: 4261-42-1, a 6-carbon glucoside of luteolin, a herbal chemical substance with the molecular formula C63-C ... 21 H 20 O 11 Its density is 1.759 g / cm³. 3 Characteristic carbon glycosides in licorice are used for the quality evaluation of Lophatherum gracile in children's herbal tea.

[0028] In a first aspect, the present invention provides: a method for determining the content of licorice-based indicator components in children's herbal tea, comprising the following steps: (1) Preparation of reference solution: Dissolve the reference standard in a solvent to prepare a reference solution; (2) Preparation of test solution: Dissolve the test sample in a solvent, sonicate and centrifuge to obtain the test solution; (3) HPLC-UV detection: The reference solution obtained in step (1) and the test solution obtained in step (2) are subjected to HPLC-UV detection.

[0029] The solvent used in steps (1) and (2) is selected from at least one of water, methanol, ethanol, and acetonitrile; Preferably, the solvent used in steps (1) and (2) is water.

[0030] Wherein, the concentration of the reference solution in step (1) is 0.5-2 mg / mL, for example 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2 mg / mL, or any range derived thereof; Preferably, the concentration of the reference solution in step (1) is 1 mg / mL.

[0031] Wherein, the concentration of the test sample dissolved in the solvent in step (2) is 0.1-0.5 g / mL, for example 0.1 g / mL, 0.15 g / mL, 0.2 g / mL, 0.25 g / mL, 0.3 g / mL, 0.35 g / mL, 0.4 g / mL, 0.45 g / mL, 0.5 g / mL, or any range derived therefrom; Preferably, the concentration of the test sample dissolved in the solvent in step (2) is 0.2 g / mL.

[0032] Wherein, the frequency of the ultrasound in step (2) is 20-60kHz, for example 20kHz, 25kHz, 30kHz, 35kHz, 40kHz, 45kHz, 50kHz, 55kHz, 60kHz, or any range derived therefrom; Preferably, the frequency of the ultrasound in step (2) is 40 kHz.

[0033] Wherein, the power of the ultrasound in step (2) is 200-600W, for example 200W, 250W, 300W, 350W, 400W, 450W, 500W, 550W, 600W, or any range derived therefrom; Preferably, the power of the ultrasound in step (2) is 400W.

[0034] The duration of the ultrasound in step (2) is 5-20 min, for example 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, or any range derived therefrom; Preferably, the duration of ultrasound in step (2) is 10 minutes.

[0035] Wherein, the centrifugation speed in step (2) is 10000-15000 rpm, for example 10000 rpm, 10500 rpm, 11000 rpm, 11500 rpm, 12000 rpm, 12500 rpm, 13000 rpm, 13500 rpm, 14000 rpm, 14500 rpm, 15000 rpm, or any range derived therefrom; Preferably, the centrifugation speed in step (2) is 14000 rpm.

[0036] The centrifugation time in step (2) is 5-20 min, for example 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, or any range derived therefrom; Preferably, the centrifugation time in step (2) is 10 minutes.

[0037] In some specific implementations, the chromatographic column used in step (3) is an Acclaim 120 C18 (250 mm × 4.6 mm, 5 μm).

[0038] In step (3), the mobile phase A to be detected is selected from at least one of methanol and acetonitrile; Preferably, the mobile phase A detected in step (3) is acetonitrile.

[0039] Wherein, the mobile phase B detected in step (3) is selected from at least one of water, ammonium acetate aqueous solution, phosphoric acid aqueous solution, formic acid aqueous solution and acetic acid aqueous solution; Preferably, the mobile phase B detected in step (3) is an aqueous solution of acetic acid; More preferably, the acetic acid in the aqueous acetic acid solution has a weight percentage of 0.1%-1.0%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, or any range derived thereof; More preferably, the acetic acid in the aqueous acetic acid solution has a weight percentage of 0.5%.

[0040] The gradient elution conditions for detection in step (3) are as follows: Table 1 Gradient elution conditions

[0041] Wherein, the column temperature detected in step (3) is selected from 30-45℃, such as 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, or any range derived therefrom; Preferably, the column temperature detected in step (3) is 35°C.

[0042] Wherein, the injection volume for detection in step (3) is selected from 2-40 μL, for example 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, or any range derived therefrom; Preferably, the injection volume for the detection in step (3) is 15 μL.

[0043] The flow rate detected in step (3) is selected from 0.5-2 mL / min, for example 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, 1.6 mL / min, 1.7 mL / min, 1.8 mL / min, 1.9 mL / min, 2 mL / min, or any range derived therefrom; Preferably, the flow rate detected in step (3) is 1.0 mL / min.

[0044] The wavelength for detection in step (3) is selected from 250-380nm; Furthermore, the wavelength detected in step (3) is detected by switching wavelengths; Preferably, the detection wavelength in step (3) is 288 nm, 252 nm, or 380 nm; Specifically, the correspondence between the detection wavelength, the target compound, and the detection time in step (3) is as follows: Table 2 Wavelength selection at different times

[0045] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred embodiment: the mobile phase B detected in step (3) is selected from at least one of water, ammonium acetate aqueous solution, phosphoric acid aqueous solution, formic acid aqueous solution, and acetic acid aqueous solution; preferably, the mobile phase B detected in step (3) is acetic acid aqueous solution; more preferably, the weight percentage of acetic acid in the acetic acid aqueous solution is 0.1%-1.0%; even more preferably, the weight percentage of acetic acid in the acetic acid aqueous solution is 0.5%. This technical solution, based on solving the technical problem of "more chromatographic peaks and better peak shapes in the obtained spectrum", further solves the technical problem of "improving the resolution of the target peak and enhancing the signal response".

[0046] The second preferred option is that the column temperature detected in step (3) is selected from 30-45℃; preferably, the column temperature detected in step (3) is 35℃. This technical solution, based on solving the technical problem of "improving the resolution of the target peak and enhancing the signal response", further solves the technical problem of "improving the resolution of chromatographic peaks, separation efficiency and chromatographic column life".

[0047] The third preferred option: the injection volume of the detection in step (3) is selected from 2-40 μL; preferably, the injection volume of the detection in step (3) is 15 μL. This technical solution solves the technical problem of "improving the separation of the target peak and enhancing the signal response" and further solves the technical problem of "further improving the separation of the target peak and enhancing the signal response".

[0048] Secondly, the present invention provides the application of the determination method in the comprehensive quality control of Xiaoer Qixingcha granules.

[0049] The phrase "the solvent used in steps (1) and (2) is selected from at least one of water, methanol, ethanol, and acetonitrile" is summarized from the foregoing explanation and / or the corresponding technical feature in Example 2, "a total of five solvent systems were selected and compared, namely water, 30%, 50%, 70%, and 100% methanol." Therefore, those skilled in the art can reasonably infer that "the solvent used in steps (1) and (2) is selected from at least one of water, methanol, ethanol, and acetonitrile," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of this invention.

[0050] The phrase "the duration of ultrasound in step (2) is 5-20 min" is a generalization derived from the aforementioned explanation and / or the corresponding technical feature in Example 2, "the time for ultrasonic extraction of the sample is examined, including 5 min, 10 min, and 20 min." Therefore, those skilled in the art can reasonably infer that "the duration of ultrasound in step (2) is 5-20 min," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing technical level, should all fall within the protection scope of this invention.

[0051] The statement that "the weight percentage of acetic acid in the acetic acid aqueous solution is 0.1%-1.0%" is a generalization derived from the foregoing explanation and / or the corresponding technical features in Example 2 such as "acetonitrile-0.1% acetic acid aqueous solution, acetonitrile-0.2% acetic acid aqueous solution, acetonitrile-0.5% acetic acid aqueous solution, and acetonitrile-1.0% acetic acid aqueous solution." Therefore, those skilled in the art can reasonably infer that "the weight percentage of acetic acid in the acetic acid aqueous solution is 0.1%-1.0%", its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on the existing technical level, should all fall within the protection scope of this invention.

[0052] The phrase "column temperature selected from 30-45℃" is a generalization derived from the foregoing explanation and / or the corresponding technical feature "different chromatographic column temperatures (30℃, 35℃, 40℃, 45℃)" in Example 2. Therefore, those skilled in the art can reasonably infer that "column temperature selected from 30-45℃," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of this invention.

[0053] The phrase "injection volume selected from 2-40 μL" is a generalization derived from the foregoing explanation and / or the corresponding technical feature "different injection volumes (2 μL, 5 μL, 10 μL, 15 μL, 20 μL, 30 μL, 40 μL)" in Example 2. Therefore, those skilled in the art can reasonably infer that "injection volume selected from 2-40 μL," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional and common knowledge based on the existing level of technology should all fall within the protection scope of this invention.

[0054] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: (1) The target peaks in the spectrum obtained by the determination method of the present invention have the best separation and the signal response is strong; (2) The determination method of the present invention is for different target components. In this experiment, the wavelength switching function is used to select the maximum absorption wavelength of each component for quantitative detection, so as to improve the detection sensitivity of the method. (3) The determination method of the present invention has high precision, good repeatability, good stability, and is simple and efficient, providing a reliable reference for the comprehensive quality control of children's Qixing Tea granules. Attached Figure Description

[0055] Figure 1 The spectrum obtained in Example 1 is shown.

[0056] Figure 2 The figure shows the total peak area of ​​the seven components obtained by optimizing the extraction conditions in Example 2. In the figure, A represents the total peak area of ​​the seven components obtained by different extraction solvents, and B represents the total peak area of ​​the seven components obtained by different extraction solvents.

[0057] Figure 3 The chromatograms are of samples separated by different mobile phase systems in Example 2.

[0058] Figure 4 The chromatograms of samples separated by different mobile phase additives in Example 2 are shown below. The correspondence between the numbers and the components in the figure is as follows: 1: isopropargyl glycoside, 2: apigenin, 3: neoglycyrrhizin, 4: glycyrrhizin, 5: isoglycyrrhizin, 6: glycyrrhizin, 7: glycyrrhizic acid.

[0059] Figure 5 The chromatograms of the acetic acid additive samples separated at different concentrations in Example 2 are shown below. The correspondence between the numbers and the components in the figure is as follows: 1: isopropargyl glycoside, 2: apigenin, 3: neoglycyrrhizin, 4: glycyrrhizin, 5: isoglycyrrhizin, 6: glycyrrhizin, 7: glycyrrhizic acid.

[0060] Figure 6 The figures shown are chromatograms of samples separated at different column temperatures in Example 2. The correspondence between the numbers and the components in the figures is as follows: 1: isopropargyl glycoside, 2: apigenin, 3: neoglycyrrhizin, 4: glycyrrhizin, 5: isoglycyrrhizin, 6: glycyrrhizin, 7: glycyrrhizic acid.

[0061] Figure 7 The chromatograms of the separated samples under different injection volumes in Example 2 are shown below. The correspondence between the numbers and the components in the figure is as follows: 1: isopropargyl glycoside, 2: apigenin, 3: neoglycyrrhizin, 4: glycyrrhizin, 5: isoglycyrrhizin, 6: glycyrrhizin, 7: glycyrrhizic acid.

[0062] Figure 8 The figures represent the maximum absorption peaks of each component in Example 2. In the figure, A represents isoglobin, B represents apigenin, C represents neoglycyrrhizin, D represents glycyrrhizin, E represents isoglycyrrhizin, F represents glycyrrhizin, and G represents glycyrrhizic acid.

[0063] Figure 9 This is the chromatogram of the blank solvent in Example 2.

[0064] Figure 10 The chromatogram of the mixed reference solution in Example 2 is shown below. The correspondence between the numbers and the components in the figure is as follows: 1: isopropargyl glycoside, 2: apigenin, 3: neoglycyrrhizin, 4: glycyrrhizin, 5: isoglycyrrhizin, 6: glycyrrhizin, 7: glycyrrhizic acid.

[0065] Figure 11 The chromatogram of the test solution in Example 2 is shown below. The correspondence between the numbers and the components in the figure is as follows: 1: isopropargyl glycoside, 2: apigenin, 3: neoglycyrrhizin, 4: glycyrrhizin, 5: isoglycyrrhizin, 6: glycyrrhizin, 7: glycyrrhizic acid.

[0066] Figure 12 The figure shows the standard curves of the seven target components in Example 2. In the figure, A is isopropargyl glycoside, B is apigenin, C is neoglycyrrhizin, D is glycyrrhizin, E is isoglycyrrhizin, F is glycyrrhizin, and G is glycyrrhizic acid.

[0067] Figure 13 The spectrum obtained in Comparative Example 1 is shown.

[0068] Figure 14 The spectrum is obtained from Comparative Example 2-4. Detailed Implementation

[0069] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0070] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0071] Example 1 (1) Instruments and reagents Instruments: Thermo Scientific Vanquish high-performance liquid chromatograph (Thermo Fisher Scientific) including VC-P32-A dual ternary high-pressure gradient pump, VC-A13-A autosampler, VC-D11-A variable wavelength ultraviolet detector (DAD), VC-C10-A column oven and Chameleon chromatography management software (Chromeleon 7); Milli-Q ultrapure water treatment system (Millipore, USA); CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 5425R high-speed refrigerated centrifuge (Eppendorf).

[0072] Reagents: Acetonitrile, methanol, and glacial acetic acid were all chromatographically pure reagents. The ultrapure water (18.2 Mohm.cm) used in the experiment was prepared by the Milli-Q ultrapure water system.

[0073] Reference standards: Glycyrrhizic acid (CAS: 1405-86-3), Liquiritin (CAS: 551-15-5), Liquiritigenin (CAS: 578-86-9), Neo-glycyrrhizin (CAS: 5088-75-5), Liquiritin apioside (CAS: 74639-14-8), Isoliquiritoside (CAS: 5041-81-6), and Luteolin-6-C-glucoside (CAS: 4261-42-1). All chemical reference standards were purchased from Chengdu Manster Biotechnology Co., Ltd. and had a purity of ≥97%. All reference materials were confirmed by UV absorption spectroscopy, high-resolution mass spectrometry, and multi-stage fragmentation data before use.

[0074] Experimental Samples: All batches of finished Xiaoqixingcha granules samples in the experiment were provided by Guangzhou Wanglaoji Pharmaceutical Co., Ltd. (Table 3).

[0075] (2) Preparation of solution Preparation of reference solutions: Take appropriate amounts of each reference standard, weigh them accurately, place them in brown volumetric flasks, add pure water to dissolve them to prepare standard stock solutions (1 mg / mL), and store them in a refrigerator at 4℃ for later use.

[0076] Preparation of the test solution: Take an appropriate amount of Xiaoqixingcha granules, place them in a mortar and grind them finely. Take about 2 g, weigh it accurately, place it in a 50 mL centrifuge tube, add 10 mL of pure water accurately, shake well, sonicate (KQ-400DE; power / frequency, 400 W / 40kHz) for 10 min, let it stand, cool it, take 2 mL of the supernatant in a centrifuge tube, centrifuge at high speed (14,000 rpm) for 10 min, and take the supernatant to obtain the test solution.

[0077] Table 3. Batch sizes of Xiaoqixingcha granules used in the experiment

[0078] (3) Chromatographic conditions The chromatographic column used was an Acclaim 120 C18 (250 mm × 4.6 mm, 5 μm). Mobile phase A was 0.5% aqueous acetic acid, and mobile phase B was acetonitrile. Gradient elution was used: 0–20 min, 20%–30% B; 20–32 min, 30%–50% B; 32–33 min, 50%–100% B; 33–37 min, 100% B. Flow rate: 1.0 mL / min; column temperature: 35℃; injection volume: 15 μL. Diode array detector (DAD) detection wavelengths: 288 nm, 252 nm, 380 nm; sampling frequency: 5 Hz.

[0079] Table 1. Gradient elution conditions

[0080] The obtained spectrum is shown in Figure 1 .

[0081] Example 2: Optimization of process parameters (1) Optimization of extraction process This experiment investigated the sample extraction solvent systems, comparing the ultrasonic extraction efficiencies of five solvent systems: water, 30%, 50%, 70%, and 100% methanol. The results are shown below. Figure 2 In section A, when water, 30% methanol, 50% methanol, and 70% methanol were used as extraction solvents, the seven target chromatographic peaks were integrated. The results showed that when pure water was used as the extraction solvent, the peak area of ​​each target peak and the total peak area were the largest. Therefore, pure water was selected as the extraction solvent for the sample.

[0082] (2) Optimization of extraction time After selecting pure water as the extraction solvent, to obtain higher extraction efficiency, the ultrasonic extraction time of the samples was investigated, including 5 min, 10 min, 20 min, 30 min, 60 min, and 90 min. After HPLC analysis, the peak values ​​of each target sample were integrated, and the results are shown below. Figure 2 In section B, as the extraction time increases, the total peak area of ​​the target substance gradually increases, indicating that the extraction efficiency is also improving; after 10 min, the total peak area tends to stabilize. Therefore, based on the principle of ensuring extraction efficiency while saving time, the ultrasonic extraction time was selected as 10 min in subsequent studies.

[0083] (3) Optimization of chromatographic conditions HPLC was used to optimize sample separation conditions, including mobile phase composition, mobile phase additives, column temperature, injection volume, gradient program, and detection wavelength, so that the seven target components in the analyzed sample could be baseline separated in the shortest possible time and quantitatively detected.

[0084] ① Selection of mobile phase The experiment first investigated methanol-water (MeOH-Water) and acetonitrile-water (ACN-Water) as mobile phases. The results showed that using acetonitrile-water as the mobile phase for gradient elution yielded more chromatographic peaks with better peak shapes. Therefore, the acetonitrile-water system was chosen for further research. Figure 3 ).

[0085] Meanwhile, to improve peak shape, enhance chromatographic peak resolution, and strengthen the detector signal response, the experiment investigated different types and concentrations of mobile phase additives, including ammonium acetate (ACN-0.2M NH4OAc), phosphoric acid (ACN-0.1% PA), formic acid (ACN-0.1% FA), and acetic acid (ACN-0.5% HAc). Figure 4 It is known that acetic acid has the best target peak separation and the strongest signal response when used as a mobile phase additive. Therefore, acetic acid was selected as the mobile phase additive.

[0086] The experiment further investigated different concentrations of acetic acid additives: acetonitrile-0.1% acetic acid solution (ACN-0.1% HAc, pH=3.52), acetonitrile-0.2% acetic acid solution (ACN-0.2% HAc, pH=3.42), acetonitrile-0.5% acetic acid solution (ACN-0.5% HAc, pH=3.01), and acetonitrile-1.0% acetic acid solution (ACN-1.0% HAc, pH=2.92). Figure 5 The results showed that the separation of chromatographic peaks was better within 5-10 min under the conditions of acetonitrile-0.5% acetic acid. Considering the pH tolerance and repeatability of the chromatographic column, acetonitrile-0.5% acetic acid water was used as the mobile phase for gradient elution analysis in this experiment.

[0087] ② Selection of chromatographic column temperature The temperature of the chromatographic column has a significant impact on the separation of compounds with similar structures. This experiment investigated the effect of different column temperatures (30℃, 35℃, 40℃, and 45℃) on sample separation. The results are as follows: Figure 6 As the column temperature increases, the sample separation time gradually shortens and the separation efficiency improves, but the resolution of the target peaks decreases, especially for apigenin and neoglycyrrhizin. Therefore, in order to ensure the resolution of chromatographic peaks, separation efficiency and column life, the column temperature was set at 35℃ in this experiment.

[0088] ③Selection of injection volume The injection volume in liquid chromatography directly affects peak shape, resolution, and signal intensity. This experiment investigated the effects of different injection volumes (2 μL, 5 μL, 10 μL, 15 μL, 20 μL, 30 μL, and 40 μL) on the separation of the target peak in the sample. Figure 7 It can be seen that as the injection volume increases, the target chromatographic peaks broaden and the response intensity increases, but the resolution decreases, especially for apigenin and neoglycyrrhizin. Considering both resolution and response intensity, 15 μL was selected as the injection volume for this experiment.

[0089] ④ Gradient elution procedure and detection wavelength optimization The Xiaoer Qixingcha granules are composed of seven Chinese medicinal herbs, which contain a large number and variety of chemical components. Therefore, this study used HPLC-UV technology and a 250 mm high-performance liquid chromatography column to separate and detect the seven target components in the sample.

[0090] To achieve high sensitivity, this experiment first measured the ultraviolet absorption spectra of each target component to determine its maximum absorption wavelength. The results are as follows: Figure 8 As shown, different compounds have different maximum absorption wavelengths, and the differences are quite large. For example, the maximum absorption wavelength of isopropionol is 349 nm, while that of apigenin is 276 nm. Therefore, for different target components, this experiment uses the wavelength switching function to select the maximum absorption wavelength of each for quantitative detection, so as to improve the detection sensitivity of the method.

[0091] Furthermore, during the subsequent gradient optimization process, it was discovered that due to the extremely complex sample matrix, some compounds experienced interference from other components at their maximum absorption wavelength, making it impossible to achieve the required quantification. For example, at the maximum absorption wavelength of 349 nm, the separation degree between the isopropanol peak and adjacent peaks was less than 1, exhibiting a severe "bifurcation peak." Therefore, considering both the matrix and sensitivity, the target component detection wavelengths were determined as shown in Table 3.

[0092] Finally, by optimizing the gradient elution procedure, the seven target components in the sample can be baseline separated and quantitatively analyzed within 37 minutes.

[0093] Table 2. Wavelength selection at different times

[0094] Example 3 Methodological Investigation For the established sample quantitative detection method, this experiment conducted a methodological investigation in accordance with the relevant provisions under General Chapter 9101 of the Chinese Pharmacopoeia (2020 edition).

[0095] (1) Specificity examination To investigate the specificity of the method, blank solvent (methanol), mixed reference solution, and test solution were injected into a high-performance liquid chromatograph (HPLC) for determination and chromatogram recording. The results showed that no interfering peaks were observed in the blank solvent (methanol) chromatogram. Figure 9 ).

[0096] Meanwhile, the reference standard ( Figure 10 ) and sample chromatogram ( Figure 11 The seven target analytes showed good peak shapes, high separation, and no interference from other matrix components, indicating that the method has good specificity.

[0097] (2) Examination of linear range Take appropriate amounts of each reference standard stock solution and dilute with methanol to obtain a series of standard curve solutions of different concentrations. Perform the determination according to the chromatographic conditions of Example 1. Plot the reference standard concentration (μg / mL) as the abscissa (c) and the peak area as the ordinate (A). Fit the data using the least squares method to draw the standard curve, and calculate the linear regression equation and correlation coefficient.

[0098] The standard curve of the compound is shown in Figure 12 The regression equations, ranges, and correlation coefficients are shown in the table below. The results show that the correlation coefficients of isoglobin, neoglycyrrhizin, apigenin, glycyrrhizin, isoglycyrrhizin, glycyrrhizin, and glycyrrhizic acid are all greater than 0.9990 within their respective concentration ranges, indicating good linearity among the compounds.

[0099] Table 4. Results of linearity analysis of component content determination, as well as limits of detection and quantitation.

[0100] (3) Precision test A mixed reference solution was prepared and injected six times consecutively within the same day to determine intra-day precision. Inter-day precision was then determined by injecting once daily for six consecutive days. Precision was evaluated using the relative standard deviation (RSD) of the peak area of ​​the target components (%). In the intra-day precision study, the RSD of the peak area of ​​each target component was less than 2% (Table 5).

[0101] Table 5. Intraday Precision

[0102] In the daytime precision study, the RSD values ​​of the peak areas of each target component were all less than 4% (Table 6), indicating that the method has good precision.

[0103] Table 6. Daytime Precision

[0104] (4) Accuracy assessment The accuracy of this method was evaluated by the determination of recovery rates. 1.0 g of sample powder (batch: 2409012) was accurately weighed, and appropriate amounts of isoharmonic acid, neoglycyrrhizin, apigenin, glycyrrhizin, isoglycyrrhizin, glycyrrhizin, and glycyrrhizic acid reference standards were added to it, ensuring a ratio of reference standard to analyte in the sample of 1:1. Six parallel sample solutions were prepared and injected into the liquid chromatograph for determination, and the percentage recovery rate was calculated. The results are shown in the table below. The recoveries of isoharmonic acid, apigenin, neoglycyrrhizin, glycyrrhizin, isoglycyrrhizin, glycyrrhizin, and glycyrrhizic acid were 102.3%, 99.0%, 97.4%, 102.6%, 102.6%, 99.9%, and 103.9%, respectively, with RSD values ​​less than or equal to 5%, indicating that the accuracy of this method meets the requirements.

[0105] Table 7. Recovery rate analysis

[0106] (5) Stability test The test solution (batch: 2408017) was prepared according to the sample preparation method, and the chromatographic peak areas were recorded and RSD values ​​were calculated at 0, 2, 4, 8, 12, 16, and 24 h to assess the stability of the method. The results showed that the RSD values ​​of the peak areas of the seven target components were all less than 4% within 24 h, indicating that the test solution had good stability within 24 h (Table 8).

[0107] Table 8. Stability Study

[0108] (6) Repeated examination Six test solutions (batch: 2408017) were prepared in parallel according to the method in Example 1, injected into a liquid chromatograph for analysis, and the peak areas of the target components were recorded. The RSD values ​​were calculated to evaluate the repeatability of the method. The results showed that the RSD values ​​of isoglobin, neoglycyrrhizin, apigenin, glycyrrhizin, isoglycyrrhizin, glycyrrhizin, and glycyrrhizic acid were all less than 4%, indicating that the method had good repeatability (Table 9).

[0109] Table 9. Repeatability Tests

[0110] (7) Durability test The test solution (batch: 2409020) was prepared in parallel according to the method in Example 1. The robustness of the method was investigated by changing the flow rate, column temperature, and different brands of chromatographic columns. The results showed that at different column temperatures (30 ℃, 35 ℃, 40 ℃, 45 ℃), flow rates (0.8 mL / min, 1.0 mL / min, 1.2 mL / min), and chromatographic columns, the theoretical plate numbers of the seven target components were all above 5000, the resolution was all greater than 1.5, and the tailing factor was all between 1.0 and 1.3, indicating that the method has good robustness and meets the requirements.

[0111] (8) Sample determination Twenty-three batches of Xiaoqixingcha granule test solutions were prepared according to the method described in Example 1, injected into a high-performance liquid chromatograph for analysis, peak areas were recorded, and the content of each component in the test samples was calculated. The content calculation results are shown in the table below.

[0112] Table 10. Results of content determination of each compound in different batches (Mean±SD, μg / g)

[0113] Comparative Example 1 The corresponding spectrum was obtained by testing according to the method described in the journal article "Fingerprint Spectrum of Pediatric Seven-Star Tea Granules".

[0114] The obtained spectrum is shown in Figure 13 .

[0115] Comparative Example 2 Compared with Example 1, the only difference is that the wavelength is not switched and the detection wavelength is 252nm. All other conditions are basically the same as in Example 1.

[0116] Comparative Example 3 Compared with Example 1, the only difference is that the wavelength is not switched and the detection wavelength is 288nm. All other conditions are basically the same as in Example 1.

[0117] Comparative Example 4 Compared with Example 1, the only difference is that the wavelength is not switched and the detection wavelength is 380nm. All other conditions are basically the same as in Example 1.

[0118] The spectra obtained from Comparative Examples 2-4 are shown below. Figure 14 .

[0119] Verification of technical effectiveness and / or analysis of technical problem solving This application employs HPLC-UV wavelength switching to simultaneously determine the content of seven indicator components in licorice from Xiaoer Qixing Tea, including isopropargylin, apigenin, neoglycyrrhizin, glycyrrhizin, isoglycyrrhizin, glycyrrhizin, and glycyrrhizic acid. By optimizing the extraction solvent, extraction time, detection wavelength, mobile phase, mobile phase additives, chromatographic column, column temperature, injection volume, and gradient elution program, a quality control method for licorice in Xiaoer Qixing Tea was established. This method is highly precise, reproducible, stable, simple, and efficient, providing a reliable reference for the comprehensive quality control of Xiaoer Qixing Tea granules.

[0120] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for determining the content of licorice-based indicator components in children's herbal tea, characterized in that, Including the following steps: (1) Preparation of reference solution: Dissolve the reference standard in a solvent to prepare a reference solution; (2) Preparation of test solution: Dissolve the test sample in a solvent, sonicate and centrifuge to obtain the test solution; (3) HPLC-UV detection: The reference solution obtained in step (1) and the test solution obtained in step (2) were subjected to HPLC-UV detection; The gradient elution conditions for detection in step (3) are as follows: 。 2. The determination method according to claim 1, characterized in that, The solvents used in steps (1) and (2) are selected from at least one of water, methanol, ethanol, and acetonitrile; The concentration of the reference solution in step (1) is 0.5-2 mg / mL; The concentration of the test sample dissolved in the solvent in step (2) is 0.1-0.5 g / mL.

3. The determination method according to claim 2, characterized in that, The solvent used in steps (1) and (2) is water; The concentration of the reference solution in step (1) is 1 mg / mL; the concentration of the test sample dissolved in the solvent in step (2) is 0.2 g / mL.

4. The determination method according to claim 1, characterized in that, The frequency of the ultrasound in step (2) is 20-60kHz, the power is 200-600W, and the duration is 5-20min; In step (2), the centrifugation speed is 10000-15000 rpm and the duration is 5-20 min.

5. The determination method according to claim 1, characterized in that, The mobile phase A detected in step (3) is selected from at least one of methanol and acetonitrile; The mobile phase B used in step (3) is selected from at least one of water, ammonium acetate aqueous solution, phosphoric acid aqueous solution, formic acid aqueous solution and acetic acid aqueous solution.

6. The determination method according to claim 5, characterized in that, The acetic acid aqueous solution contains 0.1%-1.0% acetic acid by weight.

7. The determination method according to claim 1, characterized in that, In step (3), the column temperature for detection is selected from 30-45℃, the sample volume is selected from 2-40μL, the flow rate is selected from 0.5-2mL / min, and the detection wavelength is selected from 250-380nm.

8. The determination method according to claim 7, characterized in that, In step (3), the column temperature for detection is 35℃, the injection volume is 15μL, the flow rate is 1.0 mL / min, and the detection wavelength is obtained by switching wavelengths.

9. The determination method according to claim 8, characterized in that, The correspondence between the detection wavelength, target compound, and detection time in step (3) is shown below: 。 10. The application of the determination method according to any one of claims 1-9 in the comprehensive quality control of Xiaoer Qixingcha granules.

Images