UPLC (Ultra Performance Liquid Chromatography)-based construction method for characteristic spectrum of Xipayi gingival securing fluid and method for determining content of characteristic components of Xipayi

Abstract

The invention provides a construction method of a Xipayi gingival fixing liquid characteristic spectrum based on UPLC and a method for determining the content of characteristic components of Xipayi gingival fixing liquid. The construction method comprises the following steps: by taking gallic acid, methyl gallate, benzoic acid, 1, 2, 3, 6-tetragalloylglucose and 1, 2, 3, 4, 6-pentagalloylglucose as reference substances, respectively adding water for dissolving to obtain reference substance solutions; mixing the Xipayi gingival fixing liquid with water, and filtering to obtain a test solution; placing the test sample solution and the various reference substance solutions in an ultra-high performance liquid chromatograph, determining to obtain a test sample spectrum and a reference substance spectrum, and establishing a characteristic spectrum by adopting a traditional Chinese medicine chromatographic fingerprint spectrum similarity evaluation system. The specific chromatogram has good reproducibility and batch consistency, and can be used as a standard for overall quality evaluation. The construction method can also be used for content determination of the characteristic components, and integrated detection of characteristic spectrum construction and multi-index content determination is realized.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, and more specifically, to a method for constructing a characteristic spectrum of Sipay gingival fluid based on UPLC and a method for determining the content of its characteristic components. Background Technology

[0002] Xipay Gums Strengthening Liquid has the effects of strengthening teeth and gums, clearing blood and relieving pain, and soothing the throat and eliminating odor. Clinically, it is mainly used to treat oral diseases such as periodontitis, gingivitis and oral ulcers.

[0003] Currently, the quality standards for Xipayi gingival liquid mainly focus on the determination and identification of gallic acid content. Wu Haodong et al. established an HPLC method to simultaneously determine the content of gallic acid (GA) and ellagic acid (EA) in the residue of Xipayi gingival liquid, and found that a large number of effective components remain in the residue of gallnut, which has great utilization value. Huo Shixia et al. reported a method for simultaneously determining five gallic tannins in gallnut medicinal materials and the preparation Xipayi gingival liquid using HPLC: gallic acid, 1-gallic acid glucose (1-GG), methyl gallate (MG), 1,2,3,6-tetragalacylglucose (TeGG), and 1,2,3,4,6-pentagalloylglucose (PGG). This method solved the problem of simultaneous determination of multiple gallic tannin components in gallnut. However, in preparing the test solution, this study used methanol to treat the Sipay gingival solution, which caused the polyphenols to undergo methyl esterification during sample treatment, promoting the formation of methyl gallate and failing to accurately reflect the original quality of the preparation. Peng Yan used differential spectrophotometry to determine the gallic acid content in Sipay gingival solution. Although this method is simple, rapid, and low-cost, it only measures gallic acid as a single component, making it difficult to achieve a comprehensive characterization of its quality. Qin Yiqiang used ultraviolet spectrophotometry to determine the total tannin content in Sipay gingival solution, but this method lacks separation capabilities and has poor specificity.

[0004] Characteristic chromatograms are an important technical means for evaluating the quality of traditional Chinese medicine (TCM). Combined with cluster analysis and orthogonal partial least squares discriminant analysis, they focus on selecting representative key characteristic information from the chromatograms, serving as an important identification method for TCM quality control and fully reflecting the holistic and ambiguous characteristics of TCM. Characteristic chromatograms become a relatively ideal means of TCM quality evaluation when the material basis is unclear or reference standards are expensive. Ultra-high performance liquid chromatography (UPLC) has better separation effect, peak capacity, and sensitivity than high performance liquid chromatography (HPLC). Therefore, based on the problems of single indicators, solvation effect of sample pretreatment, and insufficient overall comprehensiveness in the above-mentioned quality control studies of Xipayi gingival solution, this study establishes an overall quality control strategy of UPLC-characteristic chromatogram and multi-index component content determination method to characterize the quality of Xipayi gingival solution from a holistic perspective. Summary of the Invention

[0005] The main objective of this invention is to provide a method for constructing a characteristic spectrum of Sipay gingival solution based on UPLC and a method for determining the content of its characteristic components, so as to solve the problem of the single quality control index of Sipay gingival solution in the prior art.

[0006] To achieve the above objectives, according to a first aspect of the present invention, a method for constructing a characteristic spectrum of Sipay gingival fixation solution is provided, the method comprising:

[0007] a) Preparation of reference solutions: Gallic acid, methyl gallate, benzoic acid, 1,2,3,6-tetragalloglucose and 1,2,3,4,6-pentagalloglucose were used as reference standards and dissolved in water respectively to obtain the above reference solutions; wherein, the above reference solutions include gallic acid solution, methyl gallate solution, benzoic acid solution, 1,2,3,6-tetragalloglucose solution and 1,2,3,4,6-pentagalloglucose;

[0008] b) Preparation of the test solution: The above-mentioned Sipay gingival solution was mixed with water and filtered to obtain the above-mentioned test solution;

[0009] c) Place the above-mentioned test solution and various above-mentioned reference solutions in an ultra-high performance liquid chromatograph, and obtain the test sample chromatogram and reference chromatogram. Use the traditional Chinese medicine chromatographic fingerprint chromatogram similarity evaluation system to establish the above-mentioned characteristic chromatograms.

[0010] Further, the concentration of the gallic acid solution is 0.8-1.0 mg / mL; preferably, the concentration of the methyl gallate solution is 0.3-0.6 mg / mL; preferably, the concentration of the benzoic acid solution is 0.3-0.6 mg / mL; preferably, the concentration of the 1,2,3,6-tetragalloglucoside solution is 0.4-0.6 mg / mL; preferably, the concentration of the 1,2,3,4,6-pentagalloglucoside solution is 0.4-0.6 mg / mL.

[0011] Furthermore, the chromatographic conditions of the above-mentioned ultra-high performance liquid chromatograph include: a Waters ACQUITYUPLC HSS T3 column; a size of 2.1×100 mm; a particle size of 1.8 μm; an inorganic acid as mobile phase A, acetonitrile as mobile phase B, a flow rate of 0.3-0.5 mL / min, a column temperature of 33-38℃, an injection volume of 1-2 μL, and gradient elution;

[0012] Preferably, the elution conditions for the gradient elution are as follows: 0-4 min, the volume fraction of mobile phase A decreases from 97% to 92%, the volume fraction of mobile phase B increases from 3% to 8%, and the detection wavelength is 240 nm; 4-6.2 min, the volume fraction of mobile phase A decreases from 92% to 90%, the volume fraction of mobile phase B increases from 8% to 10%, and the detection wavelength is 240 nm; 6.2-14.6 min, the volume fraction of mobile phase A decreases from 90% to 85%, the volume fraction of mobile phase B increases from 10% to 15%, and the detection wavelength is 240 nm; 14.6-20.8 min, the volume fraction of mobile phase A decreases from 85% to 82%, the volume fraction of mobile phase B increases from 15% to 18%, and the detection wavelength is 275 nm; 20.8-30 min, the volume fraction of mobile phase A decreases from 82% to 70%, the volume fraction of mobile phase B increases from 18% to 30%, and the detection wavelength is 275 nm.

[0013] Further, the inorganic acid includes phosphoric acid; preferably, the mass-volume concentration of the inorganic acid is 0.1%-0.3%; preferably, the volume fraction of the Sipai gingival solution in the test solution is 15%-25%.

[0014] To achieve the above objectives, according to a second aspect of the present invention, a characteristic spectrum of Sipay gingival solution constructed using the above-described construction method is provided.

[0015] Preferably, the above characteristic spectrum contains 7 characteristic peaks; wherein, peak 1 is the gallic acid peak, peak 4 is the benzoic acid peak, peak 5 is the 1,2,3,6-tetragalloylglucose peak, peak 6 is the 1,2,3,4,6-pentagalloylglucose peak, peak 7 is pentagalloylglucose (unknown isomer); peak 8 is the first unknown peak, and peak 9 is the second unknown peak;

[0016] Preferably, peak No. 4 is used as the positioning peak, with a relative retention time of 1.000. The relative retention times of the other six characteristic peaks are as follows: peak No. 1 has a relative retention time of 0.123~0.151; peak No. 5 has a relative retention time of 1.076~1.315; peak No. 6 has a relative retention time of 1.361~1.663; peak No. 7 has a relative retention time of 1.477~1.805; peak No. 8 has a relative retention time of 1.642~2.006; and peak No. 9 has a relative retention time of 1.671~2.043.

[0017] More preferably, taking peak 4 as a control, the relative peak area is 1.000; the relative peak areas of the other 6 characteristic peaks are: peak 1, relative peak area is 0.453-0.854; peak 5, relative peak area is 0.436-0.778; peak 6, relative peak area is 0.115-0.375; peak 7, relative peak area is 0.444-0.854; peak 8, relative peak area is 0.223-0.407; peak 9, relative peak area is 0.205-0.432.

[0018] To achieve the above objectives, according to a third aspect of the present invention, a method for quality testing of Sipay gingival sclerosing solution is provided. The method includes: constructing a characteristic spectrum of the Sipay gingival sclerosing solution to be tested using the above-described construction method to obtain a characteristic spectrum of the Sipay gingival sclerosing solution to be tested; and comparing the characteristic spectrum of the Sipay gingival sclerosing solution to be tested with a control characteristic spectrum obtained under the same characteristic spectrum testing conditions to achieve quality testing of the Sipay gingival sclerosing solution to be tested.

[0019] Furthermore, the characteristic peaks in the above-mentioned comparative characteristic spectrum are 7; among them, peak 1 is the gallic acid peak, peak 4 is the benzoic acid peak, peak 5 is the 1,2,3,6-tetragalloglucopyranose peak, peak 6 is the 1,2,3,4,6-pentagalloglucopyranose peak, peak 7 is pentagalloglucopyranose (unknown isomer); peak 8 is the first unknown peak, and peak 9 is the second unknown peak.

[0020] Preferably, peak No. 4 is used as the positioning peak, with a relative retention time of 1.000. The relative retention times of the other six characteristic peaks are as follows: peak No. 1 has a relative retention time of 0.123~0.151; peak No. 5 has a relative retention time of 1.076~1.315; peak No. 6 has a relative retention time of 1.361~1.663; peak No. 7 has a relative retention time of 1.477~1.805; peak No. 8 has a relative retention time of 1.642~2.006; and peak No. 9 has a relative retention time of 1.671~2.043.

[0021] More preferably, taking peak 4 as a control, the relative peak area is 1.000; the relative peak areas of the other 6 characteristic peaks are: peak 1, relative peak area is 0.453-0.854; peak 5, relative peak area is 0.436-0.778; peak 6, relative peak area is 0.115-0.375; peak 7, relative peak area is 0.444-0.854; peak 8, relative peak area is 0.223-0.407; peak 9, relative peak area is 0.205-0.432.

[0022] To achieve the above objectives, according to a fourth aspect of the present invention, a method for determining the content of characteristic components in Sipay gingival solution is provided, wherein the characteristic components are selected from any one or more of the following: gallic acid, benzoic acid, 1,2,3,6-tetragalloglucoside or 1,2,3,4,6-pentagalloglucoside.

[0023] The above determination method includes: 1) placing the reference solution and the test solution in an ultra-high performance liquid chromatograph for chromatographic detection to obtain the reference chromatogram and the test chromatogram; 2) comparing the reference chromatogram and the test chromatogram to obtain the common peak; 3) calculating the peak area of ​​the common peak in the reference chromatogram and the test chromatogram; 4) calculating the content of the above characteristic component in the test solution using the external standard method; wherein, the preparation method of the test solution is the same as the preparation method of the test solution in the above construction method; the preparation method of the reference solution includes: dissolving the above characteristic component in water to obtain the reference solution.

[0024] Furthermore, the chromatographic conditions of the ultra-high performance liquid chromatograph are the same as those in the above construction method.

[0025] To achieve the above objectives, according to a fifth aspect of the present invention, an application is provided in the quality control of Sipay gingival solution using the above-described method for constructing a characteristic spectrum of the Sipay gingival solution, or the above-described characteristic spectrum, or the above-described quality detection method, or the above-described determination method.

[0026] By applying the technical solution of this invention, water is used as a solvent to dissolve Sipay gingival syrup to prepare a test solution. Combined with five water-soluble reference standards and an optimized UPLC-gradient elution-wavelength switching program, a characteristic spectrum with no methyl esterification interference, high resolution, and high reproducibility was successfully constructed. For the first time, a holistic, non-targeted, and visualized quality characterization of thermosensitive active ingredients such as gallic acid and tetra / pentagalloglucopyranoside in Sipay gingival syrup was achieved. This method overcomes the limitations of traditional methanol extraction methods, which lead to component distortion and only measure a single indicator. A new scientific, accurate, and standardized fingerprint spectrum quality control method has been established. Attached Figure Description

[0027] The accompanying drawings, which form part of this specification, 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 undue limitation of the invention. In the drawings:

[0028] Figure 1 The UPLC chromatograms of Sipa gingival solution extracted with different extraction solvents according to embodiments of the present invention are shown; wherein, A is water; B is 20% methanol by volume; C is 40% methanol by volume; D is 60% methanol by volume; E is 20% ethanol by volume; F is 40% ethanol by volume; and G is 60% ethanol by volume. Figure 1 In the AG series, 1 represents GA; 2 represents MG; 4 represents BA; 5 represents TeGG; 6 represents PGG; 7 represents 5-GG (unknown isomer); 8 and 9 are unknown.

[0029] Figure 2 The UPLC spectra of Sipay gingival solution at different wavelengths according to embodiments of the present invention are shown; wherein, A is 240 nm; B is 275 nm; C is the switched wavelength (0~14.6 min, 240 nm; 14.6~30 min, 275 nm). Figure 2 In AC, 1 represents GA, 4 represents BA, 5 represents TeGG, and 6 represents PGG. 7 represents 5-GG (unknown isomer); 8 and 9 are unknown.

[0030] Figure 3 The UPLC spectra of Sipay gingival fixation solution at different flow rates according to embodiments of the present invention are shown; where A is 0.4 mL / min, B is 0.3 mL / min, and C is 0.2 mL / min. In the figure, 1 represents GA, 4 represents BA, 5 represents TeGG, and 6 represents PGG. 7 represents 5-GG (unknown isomer); 8 and 9 are unknown.

[0031] Figure 4The UPLC spectra of Sipay gingival stabilizing solution at different column temperatures according to embodiments of the present invention are shown; where A represents 35°C; B represents 30°C; and C represents 20°C. In the figure, 1 represents GA, 4 represents BA, 5 represents TeGG, and 6 represents PGG. 7 represents 5-GG (unknown isomer); 8 and 9 are unknown.

[0032] Figure 5 UPLC spectra of Sipa gingival solution under different mobile phases according to embodiments of the present invention are shown; wherein, A is acetonitrile-0.1% phosphoric acid solution; B is acetonitrile-0.1% formic acid solution. In the figure, 1 is GA, 4 is BA, 5 is TeGG, 6 is PGG. 7 represents 5-GG (unknown isomer); 8 and 9 are unknown.

[0033] Figure 6 The UPLC spectra of Sipa gingival solution under different gradient elution procedures according to embodiments of the present invention are shown; wherein, A represents analytical method one; B represents analytical method two; and C represents analytical method three. Figure 6 In AC, 1 represents GA, 4 represents BA, 5 represents TeGG, and 6 represents PGG. 7 represents 5-GG (unknown isomer); 8 and 9 are unknown.

[0034] Figure 7 A control spectrum of Sipay gingival solution according to an embodiment of the present invention is shown; wherein, 1 represents GA; 4 represents BA; 5 represents TeGG; 6 represents PGG; 7 represents 5-GG (unknown isomer); 8 and 9 are unknown.

[0035] Figure 8 The UPLC chromatograms of 25 batches of Sipaye gingival solution according to an embodiment of the present invention are shown; wherein, the UPLC chromatogram of batch S1 and Figure 7 The comparison charts in the charts are the same charts. Detailed Implementation

[0036] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0037] Terminology Explanation:

[0038] Characteristic chromatogram: This refers to a chromatogram obtained through chromatographic analysis (such as UPLC) of a traditional Chinese medicine preparation sample, which reflects the overall chemical composition and relative proportions of its components. This chromatogram is characterized by multiple representative, stable, and specific chromatographic peaks, reflecting the material basis characteristics of traditional Chinese medicine as "multi-component, holistic action." It does not rely solely on a single reference standard, but emphasizes the integrity, fingerprint-like nature, and batch-to-batch consistency of the chromatogram morphology.

[0039] In this invention, the characteristic chromatogram of Xipai Gingival Solution is based on measured data from 25 batches of samples. After processing with the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System," seven characteristic peaks (including four known components and three unknown tannin derivatives) were extracted. A standardized chromatogram model was formed by calculating the relative retention time and relative peak area of ​​each peak, using benzoic acid as the reference peak. This chromatogram does not rely on the content of a single component but characterizes the quality of the formulation through a multi-peak synergistic pattern, making it particularly suitable for pharmaceutical formulations with scarce reference standards, complex components, and ambiguous mechanisms of action.

[0040] Reference chromatogram: refers to the characteristic chromatogram formed by collecting and standardizing representative batches of products that meet quality standards and have been fully verified, under identical instruments, chromatographic conditions and operating procedures, and serving as a "reference benchmark" for the quality evaluation of subsequent batches.

[0041] In this invention, the reference spectrum is the standardized 7-peak characteristic spectrum established by this invention. Its peak positions, relative retention times, and relative peak area ranges are all derived from the statistical mean and ±10% fluctuation range of 25 batches of qualified samples. This spectrum serves as a "benchmark" for quality control, used to compare its similarity with the spectrum of the sample to be tested, and to determine whether it is within the acceptable quality range.

[0042] As mentioned in the background section, the existing method of preparing the test solution using methanol to treat Sipay gingival solution will cause a methyl esterification reaction, which promotes the formation of methyl gallate and cannot truly reflect the original quality of the Sipay gingival solution preparation. In this invention, the inventors attempted to use water as a solvent to treat Sipay gingival solution, thereby effectively avoiding the occurrence of the methyl esterification reaction, and thus proposed the protection scheme of this invention.

[0043] In a first typical embodiment of the present invention, a method for constructing a characteristic chromatogram of Xipayi gingival solution is provided. The method includes: a) preparation of reference solutions: using gallic acid, methyl gallate, benzoic acid, 1,2,3,6-tetragalloglucoside and 1,2,3,4,6-pentagalloglucoside as references, dissolving them in water to obtain the above reference solutions; wherein the above reference solutions include gallic acid solution, methyl gallate solution, benzoic acid solution, 1,2,3,6-tetragalloglucoside solution and 1,2,3,4,6-pentagalloglucoside; b) preparation of test solution: mixing the above Xipayi gingival solution with water and filtering to obtain the above test solution; c) placing the above test solution and multiple above reference solutions in an ultra-high performance liquid chromatograph, measuring to obtain the test chromatogram and reference chromatogram, and establishing the above characteristic chromatogram using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system.

[0044] Existing methods for preparing the Sipay gingival solution test solution involve reflux treatment with methanol. However, this easily triggers gallic acid methyl esterification, leading to component distortion (such as artificially generated methyl ester peaks) and failing to accurately reflect the original formulation. This invention is the first to directly dilute Sipay gingival solution with pure water as a solvent, avoiding chemical interference from organic solvents on polyphenolic tannins and ensuring the structural integrity of heat-sensitive components such as gallic acid, tetragalloyl glucose, and pentagalloyl glucose in the sample.

[0045] This invention provides a method for constructing the characteristic spectrum of Sipay gingival solution based on direct aqueous phase injection. By establishing a multi-component reference system and a standardized UPLC detection procedure, a systematic characterization of the overall chemical composition of the formulation is achieved. The reference solution is directly prepared using water-soluble components (gallic acid, methyl gallate, benzoic acid, 1,2,3,6-tetragalloglucoside, 1,2,3,4,6-pentagalloglucoside) and is consistent with the solvent system of the test solution, ensuring the matching of peak shape, retention time, and response value.

[0046] By acquiring chromatograms using ultra-high performance liquid chromatography and using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2004A)" for peak matching and characteristic chromatogram generation, the overall quality control of multiple active ingredients in Xipai Gingival Solution was achieved.

[0047] Based on UPLC spectral analysis of 25 batches of samples, this application confirms that the method can stably obtain 7 characteristic peaks with symmetrical peak shapes and no tailing. Compared with the peak broadening and false peaks caused by methanol system in the literature, it has higher chemical fidelity and batch-to-batch reproducibility.

[0048] In a preferred embodiment of the present invention, the concentration of the gallic acid solution is 0.8-1.0 mg / mL (e.g., 0.8, 0.85, 0.9, 0.95, 0.998, or 1.0); in a preferred embodiment of the present invention, the concentration of the methyl gallate solution is 0.3-0.6 mg / mL (e.g., 0.3, 0.4, 0.5, 0.502, or 0.6 mg / mL); in a preferred embodiment of the present invention, the concentration of the benzoic acid solution is 0.3-0.6 mg / mL (e.g., 0.3, 0.4, 0.5, 0.503, or 0.6 mg / mL); in a preferred embodiment of the present invention, the concentration of the 1,2,3,6-tetragalloglucopyranoside solution is 0.4-0.6 mg / mL (e.g., 0.4, 0.5, 0.596, or 0.6 mg / mL). mg / mL); In a preferred embodiment of the present invention, the concentration of the above-mentioned 1,2,3,4,6-pentagalloglucopyranoside solution is 0.4-0.6 mg / mL (e.g., 0.4, 0.5 or 0.6 mg / mL).

[0049] This invention, validated through extensive preliminary experiments, determined the optimal concentration ranges of five reference standards in aqueous solutions. These concentrations ensured that the response values ​​of each component in UPLC detection were within the mid-range of the linear range (R² > 0.999), avoiding weak signals at low concentrations or peak tailing at high concentrations. Furthermore, PGG and TeGG, with their large molecular weights and strong polarity, require higher concentrations to obtain stable signals; however, excessive concentrations can easily lead to peak broadening. The optimized concentration achieves a balance between high sensitivity, good resolution, and good linearity. This concentration system is significantly superior to the methanol system in existing technologies, avoiding peak drift caused by differences in solvent polarity.

[0050] In a preferred embodiment of the present invention, the chromatographic conditions of the above-mentioned ultra-high performance liquid chromatograph include: a Waters ACQUITY UPLC HSS T3 column with a size of 2.1 × 100 mm and a particle size of 1.8 μm; using an inorganic acid as mobile phase A and acetonitrile as mobile phase B; a flow rate of 0.3-0.5 mL / min (e.g., 0.3 mL / min, 0.4 mL / min, or 0.5 mL / min); a column temperature of 33-38℃ (e.g., 33℃, 34℃, 35℃, 36℃, 37℃, or 38℃); an injection volume of 1-2 μL (e.g., 1 μL, 1.2 μL, 1.4 μL, 1.6 μL, 1.8 μL, or 2.0 μL); and gradient elution.

[0051] The chromatographic column used was a Waters ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm), whose hydrophilic C18 packing material has excellent retention ability for polyhydroxy tannins.

[0052] In a preferred embodiment of the present invention, the elution conditions for the gradient elution are as follows: 0-4 min, the volume fraction of mobile phase A decreases from 97% to 92%, the volume fraction of mobile phase B increases from 3% to 8%, and the detection wavelength is 240 nm; 4-6.2 min, the volume fraction of mobile phase A decreases from 92% to 90%, the volume fraction of mobile phase B increases from 8% to 10%, and the detection wavelength is 240 nm; 6.2-14.6 min, the volume fraction of mobile phase A decreases from 90% to 85%, the volume fraction of mobile phase B increases from 10% to 15%, and the detection wavelength is 240 nm; 14.6-20.8 min, the volume fraction of mobile phase A decreases from 85% to 82%, the volume fraction of mobile phase B increases from 15% to 18%, and the detection wavelength is 275 nm; 20.8-30 min, the volume fraction of mobile phase A decreases from 82% to 70%, the volume fraction of mobile phase B increases from 18% to 30%, and the detection wavelength is 275 nm.

[0053] This invention is the first to employ a UPLC detection strategy that combines gradient elution with wavelength switching to solve the problem of differences in separation and UV absorption detection response caused by the coexistence of low-polarity and high-polarity tannin components in Sipay gingival solution.

[0054] The elution program consists of three stages: For the first 14.6 min, a low organic phase ratio (3%–15% acetonitrile) is used to efficiently separate small-molecule phenolic acids (such as GA and BA) and moderately polar tannins (TeGG) at a wavelength of 240 nm. After 14.6 min, the wavelength is switched to 275 nm to enhance the UV absorption response of high-molecular-weight tannins (such as PGG) and aromatic ring structures (such as ellagic acid derivatives), while simultaneously increasing the organic phase ratio to 30%, achieving rapid elution of highly polar, large-molecule tannins. This design overcomes the problem of uneven response to multiple components in traditional fixed-wavelength methods, ensuring clear separation of all seven characteristic peaks within 30 min, without tailing or overlap, providing reliable technical support for high-resolution fingerprinting of complex traditional Chinese medicine preparations.

[0055] The aforementioned flow rate range helps promote symmetrical peak shapes without significant broadening, avoiding tailing of some characteristic peaks or decreased resolution caused by excessively fast or slow flow rates. The aforementioned column temperature range shortens analysis time and improves peak shape; within this range, the retention time fluctuations of the seven characteristic peaks are minimal, and the system pressure is stable, meeting the safety and stability requirements of UPLC instrument operation. Using the aforementioned injection volumes, no column overload or peak distortion was observed. Low-abundance unknown peak signals were detectable with 1 μL injection, and the main components were unsaturated with 2 μL injection, meeting the dual requirements of sensitivity and linear response for chromatogram construction. The above parameter combination was validated with 25 batches of samples, and without changing the elution program and detection wavelength, it can stably obtain characteristic chromatograms with consistent morphology, providing a reliable instrument response basis for subsequent similarity evaluation.

[0056] In a preferred embodiment of the present invention, the inorganic acid includes phosphoric acid; in a preferred embodiment of the present invention, the mass-volume concentration of the inorganic acid is 0.1%-0.3% (e.g., 0.1%, 0.2% or 0.3%); in a preferred embodiment of the present invention, the volume fraction of the Sipay gingival solution in the test solution is 15%-25% (e.g., 15%, 20% or 25%) (taking a volume fraction of 20% as an example, a volume fraction of 20% means that 5 mL of the original solution is diluted to 25 mL).

[0057] The specific concentrations of inorganic acids and specific components of inorganic acids mentioned above can effectively protonate phenolic hydroxyl groups, enhancing the retention capacity of tannins on the reversed-phase column, while suppressing the silanol effect and improving peak symmetry. Furthermore, the specific volume fraction of Sipay gingival solution in the test sample helps address peak saturation issues caused by excessively high concentrations of components such as benzoic acid and gallic acid in the formulation, while ensuring the detectability of trace amounts of tannins (such as PGG). This concentration system has been validated with 25 batches of samples, maximally restoring the component ratios of the original formulation while maintaining spectral resolution and signal-to-noise ratio, avoiding information distortion caused by concentration or excessive dilution.

[0058] In a second typical embodiment of the present invention, a characteristic spectrum of Sipay gingival solution constructed using the above-described construction method is provided.

[0059] In a preferred embodiment of the present invention, the above-mentioned characteristic spectrum contains 7 characteristic peaks; wherein, peak 1 is the gallic acid peak, peak 4 is the benzoic acid peak, peak 5 is the 1,2,3,6-tetragalloglucopyranose peak, peak 6 is the 1,2,3,4,6-pentagalloglucopyranose peak, peak 7 is pentagalloglucopyranose (unknown isomer); peak 8 is the first unknown peak, and peak 9 is the second unknown peak.

[0060] This chromatogram was generated based on 25 batches of samples using a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine. It includes 4 known components, 1 isomer confirmed by mass spectrometry, and 2 stable unknown peaks. By defining the range of relative retention time and relative peak area, a non-targeted, holistic quality evaluation model was established to reflect the synergistic change pattern of tannin components in the preparation.

[0061] In a preferred embodiment of the present invention, peak No. 4 is used as the positioning peak, with a relative retention time of 1.000. The relative retention times of the other six characteristic peaks are as follows: peak No. 1 has a relative retention time of 0.123~0.151 (e.g., 0.137 or 0.138); peak No. 5 has a relative retention time of 1.076~1.315 (e.g., 1.194, 1.195 or 1.196); and peak No. 6 has a relative retention time of 1.361~1.663 (e.g., 1.511, 1.512, 1.513, 1.51). The relative retention times of peak 4 or 1.515 are 1.477 to 1.805 (e.g., 1.640, 1.641, 1.642, 1.643); the relative retention times of peak 8 are 1.642 to 2.006 (e.g., 1.821, 1.822, 1.823, 1.824, 1.825, or 1.826); and the relative retention times of peak 9 are 1.671 to 2.043 (e.g., 1.855, 1.856, 1.857, 1.858, 1.859, or 1.860).

[0062] Using benzoic acid (peak 4) as a reference peak, the relative retention time and relative peak area of ​​each peak were statistically analyzed across 25 batches of samples, all showing extremely low RSD (<1.0%), indicating that the spectrum has high batch stability.

[0063] In a more preferred embodiment of the present invention, using peak number 4 as a control, the relative peak area is 1.000; the relative peak areas of the other six characteristic peaks are:

[0064] The relative peak area of ​​peak 1 is 0.453–0.854 (e.g., 0.453, 0.504, 0.550, 0.557, 0.573, 0.625, 0.634, 0.647, 0.666, 0.669, 0.693, 0.699, 0.716, 0.724, 0.726, 0.735, 0.738, 0.754, 0.767, 0.772, 0.788, 0.793, 0.813, 0.826, or 0.854).

[0065] The relative peak area of ​​peak 5 is 0.436–0.778 (e.g., 0.436, 0.515, 0.536, 0.546, 0.560, 0.577, 0.599, 0.604, 0.617, 0.648, 0.650, 0.661, 0.662, 0.666, 0.688, 0.691, 0.696, 0.706, 0.715, 0.725, 0.726, 0.731, 0.735, 0.777, or 0.778).

[0066] The relative peak area of ​​peak 6 is 0.115–0.375 (e.g., 0.115, 0.123, 0.129, 0.132, 0.137, 0.147, 0.159, 0.204, 0.222, 0.223, 0.226, 0.230, 0.234, 0.244, 0.247, 0.253, 0.256, 0.259, 0.262, 0.263, 0.269, 0.286, 0.291, 0.304, 0.305, 0.307, 0.325, 0.326, or 0.375).

[0067] The relative peak area of ​​peak 7 is 0.444–0.854 (e.g., 0.444, 0.447, 0.450, 0.467, 0.494, 0.648, 0.667, 0.672, 0.689, 0.700, 0.716, 0.724, 0.726, 0.731, 0.735, 0.753, 0.756, 0.759, 0.774, 0.784, 0.790, 0.802, 0.826, 0.835, 0.835, or 0.854).

[0068] The relative peak area of ​​peak 8 is 0.223–0.407 (e.g., 0.223, 0.230, 0.234, 0.236, 0.247, 0.253, 0.259, 0.263, 0.269, 0.284, 0.298, 0.299, 0.305, 0.308, 0.312, 0.324, 0.334, 0.338, 0.340, ...). 0.342, 0.345, 0.347, 0.348, 0.352, 0.353, 0.354, 0.356, 0.357, 0.358, 0.363, 0.367, 0.368, 0.369, 0.370, 0.375, 0.376, 0.380, 0.388, 0.389, 0.391, 0.403 or 0.407);

[0069] The relative peak area of ​​peak 9 is 0.205 to 0.432 (e.g., 0.205, 0.223, 0.230, 0.236, 0.247, 0.269, 0.312, 0.334, 0.338, 0.342, 0.348, 0.356, 0.357, 0.363, 0.367, 0.368, 0.376, 0.380, 0.388, 0.389, 0.391, 0.399, 0.403, 0.407 or 0.432).

[0070] This invention is the first to use the retention time and peak area range of seven characteristic peaks as core parameters for quality control. This spectral system is the first to achieve simultaneous monitoring of the "known + unknown" components of Xipai gingival solution, representing a significant breakthrough in the quality control of traditional Chinese medicine from a "single indicator" to a "holistic model".

[0071] In a third typical embodiment of the present invention, a method for quality testing of Sipay gingival fixation solution is provided. This method includes: constructing a characteristic spectrum of the Sipay gingival fixation solution to be tested using the aforementioned method for constructing a characteristic spectrum of the Sipay gingival fixation solution, thereby obtaining the characteristic spectrum of the Sipay gingival fixation solution to be tested; comparing the characteristic spectrum of the Sipay gingival fixation solution to be tested with a control characteristic spectrum obtained under the same characteristic spectrum testing conditions, thereby achieving quality testing of the Sipay gingival fixation solution to be tested.

[0072] This method uses identical detection conditions and a similarity evaluation system to compare the overall morphology of sample spectra with control spectra, enabling a comprehensive judgment on batch-to-batch process fluctuations, raw material changes, or storage stability changes. It provides a systematic quality control strategy for traditional Chinese medicine preparations that goes beyond the content of a single component.

[0073] The principle behind this method is that Xipay gingival solution is a multi-component compound preparation, and its efficacy depends on the synergistic effect of multiple tannin components, rather than a single indicator. This method, through comparison of the overall spectral morphology of seven characteristic peaks, can sensitively identify batch-to-batch process deviations (e.g., abnormal abundance of peaks 8 and 9 indicates tannin degradation), achieving a comprehensive evaluation of the preparation's uniformity, batch stability, and process controllability. The technical effect is significantly superior to existing single-component detection methods.

[0074] In a more preferred embodiment of the present invention, the characteristic peaks in the above-mentioned comparative characteristic spectrum are 7; wherein, peak 1 is the gallic acid peak, peak 4 is the benzoic acid peak, peak 5 is the 1,2,3,6-tetragalloglucopyranose peak, peak 6 is the 1,2,3,4,6-pentagalloglucopyranose peak, peak 7 is pentagalloglucopyranose (unknown isomer); peak 8 is the first unknown peak, and peak 9 is the second unknown peak.

[0075] In a preferred embodiment of the present invention, peak No. 4 is used as the positioning peak, with a relative retention time of 1.000. The relative retention times of the other six characteristic peaks are as follows: peak No. 1 has a relative retention time of 0.123~0.151 (e.g., 0.137 or 0.138); peak No. 5 has a relative retention time of 1.076~1.315 (e.g., 1.194, 1.195 or 1.196); and peak No. 6 has a relative retention time of 1.361~1.663 (e.g., 1.511, 1.512, 1.513, 1.51). The relative retention times of peak 4 or 1.515 are 1.477 to 1.805 (e.g., 1.640, 1.641, 1.642, 1.643); the relative retention times of peak 8 are 1.642 to 2.006 (e.g., 1.821, 1.822, 1.823, 1.824, 1.825, or 1.826); and the relative retention times of peak 9 are 1.671 to 2.043 (e.g., 1.855, 1.856, 1.857, 1.858, 1.859, or 1.860).

[0076] In a more preferred embodiment of the present invention, using peak number 4 as a control, the relative peak area is 1.000; the relative peak areas of the other six characteristic peaks are:

[0077] The relative peak area of ​​peak 1 is 0.453–0.854 (e.g., 0.453, 0.504, 0.550, 0.557, 0.573, 0.625, 0.634, 0.647, 0.666, 0.669, 0.693, 0.699, 0.716, 0.724, 0.726, 0.735, 0.738, 0.754, 0.767, 0.772, 0.788, 0.793, 0.813, 0.826, or 0.854).

[0078] The relative peak area of ​​peak 5 is 0.436–0.778 (e.g., 0.436, 0.515, 0.536, 0.546, 0.560, 0.577, 0.599, 0.604, 0.617, 0.648, 0.650, 0.661, 0.662, 0.666, 0.688, 0.691, 0.696, 0.706, 0.715, 0.725, 0.726, 0.731, 0.735, 0.777, or 0.778).

[0079] The relative peak area of ​​peak 6 is 0.115–0.375 (e.g., 0.115, 0.123, 0.129, 0.132, 0.137, 0.147, 0.159, 0.204, 0.222, 0.223, 0.226, 0.230, 0.234, 0.244, 0.247, 0.253, 0.256, 0.259, 0.262, 0.263, 0.269, 0.286, 0.291, 0.304, 0.305, 0.307, 0.325, 0.326, or 0.375).

[0080] The relative peak area of ​​peak 7 is 0.444–0.854 (e.g., 0.444, 0.447, 0.450, 0.467, 0.494, 0.648, 0.667, 0.672, 0.689, 0.700, 0.716, 0.724, 0.726, 0.731, 0.735, 0.753, 0.756, 0.759, 0.774, 0.784, 0.790, 0.802, 0.826, 0.835, 0.835, or 0.854).

[0081] The relative peak area of ​​peak 8 is 0.223–0.407 (e.g., 0.223, 0.230, 0.234, 0.236, 0.247, 0.253, 0.259, 0.263, 0.269, 0.284, 0.298, 0.299, 0.305, 0.308, 0.312, 0.324, 0.334, 0.338, 0.340, ...). 0.342, 0.345, 0.347, 0.348, 0.352, 0.353, 0.354, 0.356, 0.357, 0.358, 0.363, 0.367, 0.368, 0.369, 0.370, 0.375, 0.376, 0.380, 0.388, 0.389, 0.391, 0.403 or 0.407);

[0082] The relative peak area of ​​peak 9 is 0.205 to 0.432 (e.g., 0.205, 0.223, 0.230, 0.236, 0.247, 0.269, 0.312, 0.334, 0.338, 0.342, 0.348, 0.356, 0.357, 0.363, 0.367, 0.368, 0.376, 0.380, 0.388, 0.389, 0.391, 0.399, 0.403, 0.407 or 0.432).

[0083] The relative retention times and relative peak areas of the seven characteristic peaks in the comparative chromatograms of this application were obtained based on statistical data from 25 batches of products (n=25), and were confirmed to be statistically representative by analysis of variance (ANOVA). Setting the above relative retention time range to the mean ± 10% complies with the recommended principle of the "acceptable range" for characteristic chromatograms in the General Principles of the Chinese Pharmacopoeia.

[0084] In a fourth typical embodiment of the present invention, a method for determining the content of characteristic components of Sipay gingival solution is provided, wherein the characteristic components are selected from any one of the following: gallic acid, benzoic acid, 1,2,3,6-tetragalloglucoside or 1,2,3,4,6-pentagalloglucoside.

[0085] The above determination method includes: 1) placing the reference solution and the test solution in an ultra-high performance liquid chromatograph for chromatographic detection to obtain the reference chromatogram and the test chromatogram; 2) comparing the reference chromatogram and the test chromatogram to obtain the common peak; 3) calculating the peak area of ​​the common peak in the reference chromatogram and the test chromatogram; 4) calculating the content of the above characteristic component in the test solution using the external standard method; wherein, the preparation method of the test solution is the same as the preparation method of the test solution in the above-mentioned method for constructing the characteristic chromatogram of Xipai gingival solution; the preparation method of the reference solution includes: dissolving the above characteristic component in water to obtain the reference solution.

[0086] In a preferred embodiment of the present invention, the chromatographic conditions of the ultra-high performance liquid chromatograph are the same as those in the method for constructing the characteristic spectrum of the Sipay gingival solution.

[0087] Within the linear range, the peak area of ​​the above-mentioned characteristic components exhibits a good linear relationship with their content. Calculations and analysis show that R... 2 ≥0.999. Among them, the linear range of GA is 2.993~998.0 μg / mL; the linear range of BA is 1.508~502.8 μg / mL; the linear range of TeGG is 1.788~596.0 μg / mL; and the linear range of PGG is 1.812~604.0 μg / mL.

[0088] By using a unified chromatographic system for both qualitative and quantitative detection—that is, the same UPLC procedure—it can be used for both characteristic chromatogram similarity evaluation and content determination of four key components, greatly improving detection efficiency and reducing laboratory equipment and labor costs. This "one method for two purposes" design is significantly superior to the traditional "chromatogram + content" separation test mode, and is an important manifestation of the integration and intelligence of traditional Chinese medicine quality control methods.

[0089] In a fifth typical embodiment of the present invention, an application is provided in the quality control of Sipay gingival solution using the above-described method for constructing a characteristic spectrum of the Sipay gingival solution, or the above-described characteristic spectrum, or the above-described quality detection method, or the above-described determination method.

[0090] The method for constructing characteristic spectra, the characteristic spectra themselves, the quality detection method, and the method for determining component content provided by this invention can be systematically applied to the raw material input control, production process monitoring, intermediate inspection, finished product release, and stability studies of Xipay gingival liquid. For example, at the raw material end, it can verify the consistency of tannin composition in gallic acid medicinal material; at the production end, it can monitor whether key process parameters such as extraction, concentration, and dispensing lead to an imbalance in component ratios; at the quality release end, it can replace the traditional low-level standard of only measuring "gallic acid" and achieve multi-indicator monitoring. This technical solution has universality and can be extended to other traditional Chinese medicine preparations such as mouthwashes, eye drops, and patches with tannins as the main active ingredient, and has significant industrialization value.

[0091] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0092] Example 1

[0093] 1. Instruments and reagents

[0094] 1.1 Instruments

[0095] Waters UPLC liquid chromatograph; SCIEX Zeno TOF™ 7600 quadrupole tandem time-of-flight high-resolution mass spectrometer; KQ 250DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); AE200 electronic balance (Mettler, Switzerland) Toledo (DHG) Model 9070A Electric Heating Constant Temperature Drying Oven (Shanghai Jinghong Experimental Equipment Co., Ltd.)

[0096] 1.2 Drug Testing

[0097] A total of 25 batches of Xipayi Gingival Solution were produced, all from Xinqikang Pharmaceutical Co., Ltd. (batch numbers are 240813, 240815, 240816, 240902, 240906, 240908, 240909, 241003, 241007, 241009, 241010, 241011, 241012, 241101, 241104, 241105, 241107, 241111, 241115, 241117, 241123, 241211, 241212, 241213, and 240350, numbered S1 to S25 respectively). Reference standards included gallic acid (GA), methyl gallate (MG), benzoic acid (BA), 1,2,3,6-tetragalloglucopyranoside (TeGG), and 1,2,3,4,6-pentagalloglucopyranoside (PGG) (batch numbers N2304195025, M24012209531, M240319093713, N2427891153, and N2504962510, respectively). All reference standards were purchased from Sichuan Hengcheng Zhiyuan Biotechnology Co., Ltd., and the mass fraction of all reference standards was ≥98%. Acetonitrile was of chromatographic grade; all other reagents were of analytical grade; and ultrapure water was used in the experiments.

[0098] 2. Methods and Results

[0099] 2.1 Preparation of the test sample:

[0100] 2.1.1 Method 1: Reference Methods (Wu Haodong, Li Yan, Zhang Fengxue, et al. Simultaneous determination of gallic acid and ellagic acid content in Xipayi gingival liquid residue by HPLC [J]. Journal of Xinjiang Medical University, 2017, 40(3):366-368+71; Huo Shixia, Chen Liangmian, Zulipikar Wusman, et al. Simultaneous determination of 5 gallic tannins in gallic medicinal materials and preparations Xipayi gingival liquid by one-test-multiple-evaluation method [J]. Journal of Pharmaceutical Analysis, 2023, 43(5): 780-92; Qin Yiqiang, Cai Xiaoling, Gong Xun. Determination of total tannins and gallic acid content in Xipayi gingival liquid [J]. Chinese Journal of Pharmaceutical Science, 2012, 2(5): 97-8.).

[0101] Accurately pipette 5.0 mL of Sipaye gingival solution into a round-bottom flask, accurately add 20 mL of 40% methanol solution, weigh, reflux in a water bath for 4 h, cool, replenish the lost weight with 40% methanol solution, shake well, filter, accurately take 1.0 mL of the filtrate into a 10 mL volumetric flask, dilute to the mark with 40% methanol solution, shake well, filter through a 0.45 μm microporous membrane, and collect the filtrate to obtain the Sipaye gingival solution test solution.

[0102] 2.1.2 Method 2: Accurately pipette 5.0 mL of Sipaye gingival solution into a 25 mL volumetric flask, dilute with pure water to the mark, shake well, filter through a 0.45 μm microporous membrane, and collect the filtrate as the test solution for Sipaye gingival solution.

[0103] 2.1.3 Method 3:

[0104] Three batches of Sipaye gingival solution were extracted with 40% methanol and water, and the MG content was found to be increased in all of them. This may be related to the transformation of components such as GA, ellagic acid, and polygalloyl glucose naturally present in gallic acid during the extraction process. In addition, after alcohol extraction, the content of some components in Sipaye gingival solution decreased or even disappeared.

[0105] Literature reports that gallic acid readily esterifies with methanol under acidic conditions to form MG; while β-glucosylgallic acid or polygalloylglucose in gallium may be hydrolyzed to form gallic acid, which is then esterified to methyl gallate. However, the specific reaction substrates and mechanisms of the esterification reaction in Sipay gingival solution require further investigation.

[0106] In addition to the two sample preparation methods of water dilution and 40% methanol reflux extraction, this experiment also investigated the effects of five extraction solvents—20% methanol, 60% methanol, 20% ethanol, 40% ethanol, and 60% ethanol—on Sipay gingival sclerosing solution. The test solution was prepared according to the method described in section "2.1.1" and injected under the conditions described in section "2.3".

[0107] The results showed that after reflux extraction with methanol and ethanol at different ratios, several original peaks of Xipayi gingival slurry showed varying degrees of decrease in response values, or even disappeared. During reflux extraction with methanol at different ratios, MG (peak 2) appeared in all cases of Xipayi gingival slurry, and as the proportion of methanol increased, the gallic acid content decreased, while peak 2 (MG) increased. During reflux extraction with ethanol at different ratios, ethyl gallate (EG, peak 3) appeared in all cases of Xipayi gingival slurry, and as the proportion of ethanol increased, the gallic acid content decreased, while peak 3 (EG) increased.

[0108] Therefore, this application ultimately chose water as the extraction solvent to prepare the test sample.

[0109] 2.2 Preparation of reference solutions: Accurately weigh appropriate amounts of GA, MG, BA, TeGG, and PGG reference standards, dissolve them in water, and prepare single reference standard stock solutions of a certain concentration. Accurately pipette appropriate amounts of the above reference standard stock solutions, dilute them with water, and prepare a mixed reference standard solution containing GA 0.998 mg / mL, MG 0.502 mg / mL, BA 0.503 mg / mL, TeGG 0.596 mg / mL, and PGG 0.600 mg / mL, and store at 4℃ for later use.

[0110] 2.3 Chromatographic conditions: The column was a Waters ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm); the flow rate was 0.4 mL / min, the column temperature was 35℃, the mobile phase A was 0.1% phosphoric acid water, and the mobile phase B was acetonitrile. Gradient elution: 0-4 min, 3~8% B; 4-6.2 min, 8~10% B; 6.2-14.6 min, 10~15% B; 14.6-20.8 min, 15~18% B; 20.8-25 min, 18~30% B; wavelength switching program: 0~14.6 min, 240 nm; 14.6~30 min, 275 nm; injection volume was 1 μL.

[0111] UPLC chromatograms of the reference standard and sample solutions prepared by different methods are shown below. Figure 1 .

[0112] The above chromatographic conditions were obtained after the inventors made many attempts and optimizations. The specific optimization process is shown below.

[0113] 2.3.1 Optimization of chromatographic wavelength

[0114] Take an appropriate amount of this product (batch number: 240813), prepare the test solution according to the method in section "2.1.2", and inject it under the conditions in section "2.3". By comparing the results at 240 nm and 275 nm, it was found that there are significant differences in the UV absorption characteristics of different components. Among them, GA, TeGG, and PGG have strong absorption near 275 nm, while BA has strong absorption at 240 nm and weak absorption at 275 nm (experimental results are shown in [link to results]). Figure 2 Based on the aforementioned differences in absorption characteristics, a single detection wavelength is insufficient to ensure sensitive detection of all components; therefore, a wavelength switching method was employed. Thus, a detection wavelength of 240 nm was set from 0 to 14.6 min, and 275 nm from 14.6 to 30 min. After wavelength switching, the separation between the chromatographic peaks of each component was good, with symmetrical and sharp peak shapes, meeting the analytical requirements. Subsequent characteristic chromatogram construction and content determination studies were conducted using this wavelength switching method.

[0115] 2.3.2 Examination of Flow Velocity

[0116] Take an appropriate amount of this product (batch number: 240813), prepare the test solution according to the method in section "2.1.2", and conduct the test at flow rates of 0.2 mL / min, 0.3 mL / min and 0.4 mL / min respectively, based on the chromatographic conditions in section "2.3". Inject once for each test and record the chromatogram (see...). Figure 3 ).

[0117] When investigating the effects of three different flow rates (0.4 mL / min, 0.3 mL / min, and 0.2 mL / min) on the chromatographic peak separation, it was found that as the mobile phase flow rate decreased, the retention time of each component generally shifted later, indicating a longer analysis cycle. Under the condition of 0.4 mL / min, the main chromatographic peaks showed good shape and high resolution, and the analysis time was short. Therefore, 0.4 mL / min was ultimately selected as the optimal flow rate.

[0118] 2.3.3 Investigation of column temperature

[0119] Take an appropriate amount of this product (batch number: 240813), prepare the test solution according to the method in section "2.1.2", and conduct the test at column temperatures of 25℃, 30℃ and 35℃ respectively, based on the chromatographic conditions in section "2.3". Inject the sample once for each temperature and record the chromatogram (see...). Figure 4 ).

[0120] When investigating the effects of three different column temperatures (25℃, 30℃, and 35℃) on the separation of chromatographic peaks, it was found that as the column temperature increased, the retention time of the target components gradually shortened. Simultaneously, the peak shape became sharper, the peak symmetry significantly improved, and the tailing phenomenon was significantly reduced. Furthermore, it was observed that the lower the column temperature, the higher the system pressure. Therefore, 35℃ was ultimately determined to be the optimal column temperature for this liquid chromatography analysis.

[0121] 2.3.4 Selection of Mobile Phase

[0122] This experiment investigated the effects of acetonitrile-0.1% formic acid solution and acetonitrile-0.1% phosphoric acid solution. An appropriate amount of this product (batch number: 240813) was taken, and the test solution was prepared according to the method described in section "2.1.2". The sample was then injected under the conditions described in "2.3". The chromatogram obtained using the acetonitrile-0.1% phosphoric acid solution system as the mobile phase showed a stable baseline and good compound separation; therefore, acetonitrile-0.1% phosphoric acid solution was selected as the mobile phase system. The experimental results are shown below. Figure 5 .

[0123] 2.3.5 Selection of Gradient Elution Program

[0124] Take an appropriate amount of this product (batch number: 240813) and prepare the test solution according to the method in section “2.1.2”. Analytical conditions: chromatographic column: Waters ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm); flow rate: 0.4 mL / min; column temperature: 35℃; mobile phase A: 0.1% phosphoric acid water; mobile phase B: acetonitrile; wavelength switching program: 0~14.6 min, 240 nm; 14.6~30 min, 275 nm; injection volume: 1 μL. First, based on the gradient condition analysis of the references (Huo Shixia, Chen Liangmian, Zulipikar Wusman, et al. Simultaneous determination of five gallic tannins in gallnut medicinal material and preparation Xipai gingival liquid by one-test-multiple-evaluation method [J]. Journal of Pharmaceutical Analysis, 2023, 43(5): 780-92), and with appropriate adjustments, analytical methods one through three were obtained. Finally, analytical method three was determined as the final condition; the experimental results are shown in […]. Figure 6 , Figure 6 In AC, 1 stands for GA, 4 for BA, 5 for TeGG, and 6 for PGG.

[0125] Analysis Method 1 ( Figure 6 (A in the literature method): 0-4 min, 3%-8% B; 4-7 min, 8%~10% B; 7-16 min, 10%~15% B; 16-22 min, 15%-18% B; 22-30 min, 18%-30% B;

[0126] Analysis Method Two ( Figure 6 B in B): 0-4 min, 3%-8%B; 14-5 min, 8%~10%B1; 5-10 min, 10%~15% B; 10-14 min, 15%-18%B; 14-18 min, 18%B; 18-25 min, 18%~30%B;

[0127] Analysis Method 3 ( Figure 6 C in): 0-4 min, 3%~8%B; 4-6.2 min, 8%~10%B; 6.2-14.6 min, 10%~15%B; 14.6-20.8 min, 15%~18%B; 20.8~30 min, 18%~30%B.

[0128] Method A, as described in the literature, can achieve basic separation of the target components. Method B, based on this, compresses and rearranges the gradient program. Although this shortens the analysis time to some extent, the separation degree of some components decreases due to the relatively concentrated changes in the intermediate gradients, and the TeGG (peak 5) is lost, suggesting that the system is quite sensitive to the rate of gradient change.

[0129] Method C makes minor, fine-tuning adjustments to Method A, primarily by providing a more balanced time allocation within the critical elution range of 10%–18%B and refining the gradient inflection point. This optimization does not alter the overall elution trend but helps improve the separation and peak shape of some adjacent peaks, enhancing the method's stability and reproducibility. In summary, Method C, while maintaining a similar analytical framework to the literature methods, achieves optimization of chromatographic separation details and is therefore selected as the final analytical method.

[0130] 3. Establishment of Feature Maps

[0131] Although the quality evaluation methods for Xipai Gingival Solution, as a marketed formulation, already include content determination and qualitative identification, many components in the formulation are still not under control. Characteristic chromatograms are currently one of the commonly used methods for overall evaluation of formulations and can provide a basis for quality consistency evaluation in terms of process stability, etc.

[0132] 3.1 Chromatographic conditions: Same as "2.3"

[0133] 3.2 Preparation of test solution

[0134] Accurately measure 5.0 ml of each of the 25 batches of Sipaye gingival solution and add it to a 25 ml volumetric flask. Make up to volume with pure water, filter through a 0.45 μm microporous membrane, and collect the filtrate to obtain the Sipaye test solution.

[0135] 3.3 Methodology

[0136] Precision test: Take an appropriate amount of this product (batch number: 240813), prepare the test solution according to the method in section "2.1.2", inject the sample under the conditions in section "2.3", and measure 6 times. With BA as the reference chromatographic peak, the relative retention time and relative peak area RSD of each characteristic peak were all less than 3%, indicating that the instrument precision is good.

[0137] Repeatability test: Take an appropriate amount of this product (batch number: 240813) and prepare 6 test solutions in parallel according to the method in section "2.1.2". Inject the samples under the conditions in section "2.3". Using BA as the reference chromatographic peak, the relative retention time and relative peak area RSD of each characteristic peak were all less than 3%, indicating that the method has good repeatability.

[0138] Stability study: Take an appropriate amount of this product (batch number: 240813), prepare the test solution according to the method in section "2.1.2", and inject it at 0, 2, 4, 8, 12, 18 and 24 h under the conditions in section "2.3" for determination. With BA as the reference chromatographic peak, the relative retention time and relative peak area RSD of each characteristic peak were all less than 3%, indicating that the test solution has good stability within 24 h.

[0139] 3.4 Atlas Generation

[0140] Take 25 batches of samples, prepare test solutions according to the method in section "2.1.2", inject the samples under the conditions in section "2.3", import the relevant data into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software (2004A version), and record the chromatograms. Figure 7 and Figure 8 As a result, a total of 7 characteristic peaks were identified. With peak 2 (BA) as a reference, the relative retention time RSD of each characteristic peak was 0.05%~0.55% (see Table 1). The relative peak area of ​​each characteristic peak is shown in Table 2.

[0141] Table 1. Relative retention times of characteristic peaks in 25 batches of Sipaye gingival solution

[0142]

[0143] Table 2. Relative peak areas of characteristic peaks in 25 batches of Sipaye gingival solution

[0144]

[0145] 3.3 Chemometric Analysis

[0146] Similarity evaluation involved importing relevant data into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software (version 2004A), using the chromatogram of S1 (batch number 240813) as a reference. The median method was employed, with a time window width of 0.1 min, to determine the similarity. The similarity between different batches of samples was greater than 0.95, indicating stable intrinsic quality and good consistency (see Table 3).

[0147] Table 3. Similarity evaluation of 25 batches of Xipai gingival solution

[0148]

[0149] 4. Content determination

[0150] GA, BA, TeGG, and PGG showed good separation and high response values. Therefore, these components were selected for content determination in this experiment.

[0151] 4.1 Chromatographic conditions

[0152] The chromatographic conditions are the same as in "2.3".

[0153] 4.2 Preparation of reference solution

[0154] The preparation of the reference solution is the same as in "2.2".

[0155] 4.3 Preparation of the test solution

[0156] The preparation of the test solution is the same as in "2.1.2".

[0157] 4.4 Linearity and Linear Range Assessment: Take an appropriate amount of the reference solution, gradually dilute it, and inject it for determination under the conditions described in section "2.3". Plot a standard curve with peak area (Y) as the ordinate and mass concentration (X, μg / ml) as the abscissa, as shown in Table 4. The results show that each component has a good linear relationship within its respective range.

[0158] Table 4 Examination of Linear Relationships

[0159]

[0160] 4.5 Limit of Quantitation (LOQ) and Limit of Detection (LOD): The lowest concentration of the standard curve was used for injection analysis. The LQ was calculated with a signal-to-noise ratio (S / N) of 10 and the LOD was calculated with a S / N of 3. The LQs for GA, BA, TeGG, and PGG were 2.993, 1.508, 1.788, and 1.812 μg / mL, respectively, and the LODs were 1.496, 0.754, 0.894, and 0.906 μg / mL, respectively.

[0161] 4.6 Precision Test: High, medium, and low concentration reference solutions were taken and injected and measured 6 times on the same day under the conditions described in section "2.3". If the RSD of the peak area of ​​each reference solution was less than 3%, it indicates good intra-day precision of the instrument. After three consecutive days of testing, the inter-day precision was calculated. If the RSD was less than 3%, it indicates good inter-day precision.

[0162] 4.7 Repeatability test: Take an appropriate amount of this product (batch number: 240813) and prepare 6 test solutions in parallel according to the method in section “2.1.2”. Inject the samples under the conditions in section “2.3”. The RSD of the contents of GA, BA, TeGG and PGG is less than 3%, indicating that the method has good repeatability.

[0163] 4.8 Stability test: Take an appropriate amount of this product (batch number: 240813), prepare the test solution according to the method in section “2.1.2”, and inject and determine the sample at 0, 1, 3, 7, 12, 18, 20 and 24 h under the conditions in section “2.3”. The peak area RSD of GA, BA, TeGG and PGG was less than 3%, indicating that the solution has good stability within 24 h.

[0164] 4.9 Spiking Recovery Experiment: Accurately pipette 0.5 mL of this product (batch number: 240813) into a 5 mL volumetric flask. Accurately add 0.5 mL of the mixed reference solution at 50%, 100%, and 150% of the test sample content, respectively. Dilute with water to the mark, mix well, and perform three aliquots for each level. Inject the samples under the conditions described in section “2.3”, determine the recovery rate, and calculate the average recovery rate. The results showed that the average recovery rates of GA, BA, TeGG, and PGG were 99.67%, 101.62%, 99.13%, and 99.57%, respectively, with RSDs of 1.29%, 1.51%, 1.89%, and 2.01%, respectively. The experimental results are shown in Table 5.

[0165] Table 5. Results of sample recovery

[0166]

[0167] 4.10 Sample Determination: 25 batches of samples were taken, and test solutions were prepared according to the method described in section “2.1.2”. The samples were injected under the conditions described in section “2.3”, and the contents of the four components were determined and calculated (all units are mg / mL). The results are shown in Table 6. Specifically, the contents of GA (gallic acid) ranged from 1.425 mg / mL to 2.557 mg / mL, BA (benzoic acid) ranged from 0.4339 mg / mL to 0.4831 mg / mL, TeGG (1,2,3,6-tetragalloglucopyranoside) ranged from 0.3946 mg / mL to 0.6739 mg / mL, and PGG (1,2,3,4,6-pentagalloglucopyranoside) ranged from 0.1290 mg / mL to 0.3478 mg / mL.

[0168] Table 6 Content determination results

[0169]

[0170] As can be seen from the above description, this application has for the first time established a whole-process quality control system for Xipai gingival solution, which takes direct aqueous phase injection as the core, UPLC gradient elution and wavelength switching as the means, and multi-index characteristic peak characterization and multi-index component content determination as the standard. It systematically solves the three major technical problems in traditional methods: "solvent interference causing component distortion, single index cannot characterize the whole, and lack of quantifiable standards".

[0171] Specifically, the method of diluting the test sample with pure water instead of the traditional methanol reflux method effectively solves the problem of false positive peaks and misjudgment of content caused by gallic acid methyl esterification reaction; by establishing a standardized characteristic chromatogram containing 7 characteristic peaks (including 2 unidentified but stable unknown peaks), and for the first time specifying the acceptable range of relative retention time and relative peak area for each peak, the method achieves the transformation from qualitative to quantitative analysis; by unifying chromatographic conditions, the characteristic chromatogram and content determination (GA, BA, TeGG, PGG) share the same system, realizing dual detection of qualitative and quantitative results, and significantly improving detection efficiency and data consistency.

[0172] The technical solution of this application significantly improves the batch-to-batch quality stability of Xipai Gingival Solution (similarity ≥ 0.95), truly reflects the material basis of its multi-component synergistic effect, fills the gap in the lack of characteristic chromatographic control in the Chinese Pharmacopoeia and local standards, and provides a replicable and scalable scientific paradigm for the modernization, standardization and internationalization of ethnic minority medicines. It has significant technological innovation, industrial applicability and standard-leading value.

[0173] 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 constructing a characteristic spectrum of a Sipay gingival sclerosing solution, characterized in that, The construction method includes: a) Preparation of reference solutions: Gallic acid, methyl gallate, benzoic acid, 1,2,3,6-tetragalloglucoside and 1,2,3,4,6-pentagalloglucoside were used as reference standards and dissolved in water respectively to obtain the reference solutions. The reference solution includes gallic acid solution, methyl gallate solution, benzoic acid solution, 1,2,3,6-tetragalloglucoside solution and 1,2,3,4,6-pentagalloglucoside solution. b) Preparation of the test solution: The Sipay gingival solution was mixed with water and filtered to obtain the test solution; c) The test solution and various reference solutions are placed in an ultra-high performance liquid chromatograph to obtain the chromatograms of the test solution and the reference solutions. The characteristic chromatograms are established using a traditional Chinese medicine chromatographic fingerprint chromatogram similarity evaluation system.

2. The construction method according to claim 1, characterized in that, The concentration of the gallic acid solution is 0.8-1.0 mg / mL; Preferably, the concentration of the methyl gallate solution is 0.3-0.6 mg / mL; Preferably, the concentration of the benzoic acid solution is 0.3-0.6 mg / mL; Preferably, the concentration of the 1,2,3,6-tetragalloglucoside solution is 0.4-0.6 mg / mL; Preferably, the concentration of the 1,2,3,4,6-pentagalloglucoside solution is 0.4-0.6 mg / mL.

3. The construction method according to claim 1, characterized in that, The chromatographic conditions of the ultra-high performance liquid chromatograph include: The chromatographic column was a Waters ACQUITY UPLC HSS T3 column; the size was 2.1 × 100 mm; the particle size was 1.8 μm; the mobile phase was inorganic acid A and acetonitrile B; the flow rate was 0.3-0.5 mL / min; the column temperature was 33-38℃; the injection volume was 1-2 μL; and gradient elution was used. Preferably, the elution conditions for the gradient elution are as follows: From 0 to 4 min, the volume fraction of mobile phase A decreased from 97% to 92%, while the volume fraction of mobile phase B increased from 3% to 8%, with a detection wavelength of 240 nm. From 4 to 6.2 minutes, the volume fraction of mobile phase A decreased from 92% to 90%, while the volume fraction of mobile phase B increased from 8% to 10%, with a detection wavelength of 240 nm. From 6.2 to 14.6 min, the volume fraction of mobile phase A decreased from 90% to 85%, while the volume fraction of mobile phase B increased from 10% to 15%, with a detection wavelength of 240 nm. From 14.6 to 20.8 min, the volume fraction of mobile phase A decreased from 85% to 82%, while the volume fraction of mobile phase B increased from 15% to 18%. The detection wavelength was 275 nm. Over 20.8–30 min, the volume fraction of mobile phase A decreased from 82% to 70%, while the volume fraction of mobile phase B increased from 18% to 30%, with a detection wavelength of 275 nm.

4. The construction method according to claim 3, characterized in that, The inorganic acid includes phosphoric acid; Preferably, the mass-volume concentration of the inorganic acid is 0.1%-0.3%; Preferably, the volume fraction of the Sipay gingival solution in the test solution is 15%-25%.

5. A characteristic spectrum of Sipay gingival sclerotherapy solution constructed using the construction method according to any one of claims 1-4; Preferably, the feature spectrum contains 7 characteristic peaks; wherein, Peak 1 is the gallic acid peak, peak 4 is the benzoic acid peak, peak 5 is the 1,2,3,6-tetragalloglucoside peak, peak 6 is the 1,2,3,4,6-pentagalloglucoside peak, peak 7 is pentagalloglucoside; peak 8 is the first unknown peak, and peak 9 is the second unknown peak. Preferably, peak number 4 is used as the positioning peak, with a relative retention time of 1.000; the relative retention times of the other six characteristic peaks are: The relative retention time of peak 1 is 0.123–0.

151. The relative retention time of peak 5 is 1.076–1.

315. The relative retention time of peak 6 is 1.361–1.

663. The relative retention time of peak 7 is 1.477–1.

805. The relative retention time of peak 8 is 1.642–2.

006. The relative retention time of peak 9 is 1.671–2.

043. More preferably, taking peak number 4 as a control, the relative peak area is 1.000; the relative peak areas of the other 6 characteristic peaks are: The relative peak area of ​​peak 1 is 0.453–0.854; The relative peak area of ​​peak 5 is 0.436–0.778; The relative peak area of ​​peak 6 is 0.115–0.375; The relative peak area of ​​peak 7 is 0.444–0.854; The relative peak area of ​​peak 8 is 0.223–0.407; The relative peak area of ​​peak 9 is 0.205–0.

432.

6. A method for quality testing of Sipay gingival fixation solution, characterized in that, The quality testing method includes: Using the construction method of any one of claims 1-4, a characteristic spectrum is constructed for the test Sipay gingival solution to obtain the characteristic spectrum of the test Sipay gingival solution; the characteristic spectrum of the test Sipay gingival solution is compared with the control characteristic spectrum obtained under the same characteristic spectrum detection conditions to achieve the quality detection of the test Sipay gingival solution.

7. The quality inspection method according to claim 6, characterized in that, The characteristic peaks in the reference feature spectrum are 7; Among them, peak 1 is the gallic acid peak, peak 4 is the benzoic acid peak, peak 5 is the 1,2,3,6-tetragalloglucopyranose peak, peak 6 is the 1,2,3,4,6-pentagalloglucopyranose peak, peak 7 is pentagalloglucopyranose; peak 8 is the first unknown peak, and peak 9 is the second unknown peak. Preferably, peak number 4 is used as the positioning peak, with a relative retention time of 1.000; the relative retention times of the other six characteristic peaks are: The relative retention time of peak 1 is 0.123–0.

151. The relative retention time of peak 5 is 1.076–1.

315. The relative retention time of peak 6 is 1.361–1.

663. The relative retention time of peak 7 is 1.477–1.

805. The relative retention time of peak 8 is 1.642–2.

006. The relative retention time of peak 9 is 1.671–2.

043. More preferably, taking peak number 4 as a control, the relative peak area is 1.000; the relative peak areas of the other 6 characteristic peaks are: The relative peak area of ​​peak 1 is 0.453–0.854; The relative peak area of ​​peak 5 is 0.436–0.778; The relative peak area of ​​peak 6 is 0.115–0.375; The relative peak area of ​​peak 7 is 0.444–0.854; The relative peak area of ​​peak 8 is 0.223–0.407; The relative peak area of ​​peak 9 is 0.205–0.

432.

8. A method for determining the content of characteristic components in a Sipay gingival sclerosing solution, characterized in that, The characteristic component is selected from any one or more of the following: gallic acid, benzoic acid, 1,2,3,6-tetragalloglucoside or 1,2,3,4,6-pentagalloglucoside. The determination method includes: 1) Place the reference solution and the test solution in an ultra-high performance liquid chromatograph for chromatographic detection to obtain the reference solution chromatogram and the test solution chromatogram; 2) Compare the chromatograms of the reference standard and the test sample to obtain common peaks; 3) Calculate the peak area of ​​the common peak in the reference sample chromatogram and the test sample chromatogram; 4) The content of the characteristic component in the test solution was calculated using the external standard method; The method for preparing the test solution is the same as the method for preparing the test solution in any one of claims 1-4. The method for preparing the reference solution includes: dissolving the characteristic component in water to obtain the reference solution.

9. The determination method according to claim 8, characterized in that, The chromatographic conditions of the ultra-high performance liquid chromatograph are the chromatographic conditions described in the construction method of claim 3 or 4.

10. The application of the method for constructing the characteristic spectrum of Xipay gingival solution according to any one of claims 1-4, or the characteristic spectrum according to claim 5, or the quality detection method according to claim 6 or 7, or the determination method according to claim 8 or 9 in the quality control of Xipay gingival solution.

Images