Quality evaluation method for phlegm-drinking pills based on HPLC fingerprint spectrum and multi-index component content determination

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a quality evaluation method for phlegm and fluid retention pills based on HPLC fingerprint spectrum and multi-index component content determination. The method comprises the following steps: determining the contents of nine active components in the phlegm-drinking pill and constructing an HPLC fingerprint spectrum of the phlegm-drinking pill based on a high performance liquid chromatography-high resolution mass spectrometry technology; wherein the nine active ingredients are sinapine, chlorogenic acid, cryptochlorogenic acid, 3, 6 '-dimustard acyl sucrose, rosmarinic acid, apiose isoliquiritin, isoliquiritin, glycyrrhizic acid and atractylenolide I. The invention further discloses a preparation method of the pharmaceutical composition. According to the method, the fingerprint spectrum and multi-index determination are integrated, a reproducible technical framework is provided for modern quality evaluation of the traditional Chinese medicine compound preparation with lower instrument cost and higher universality, and the industry is promoted to be transformed from'single component control 'to'overall quality management and control'.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and particularly relates to a quality evaluation method for Tanyin pills based on HPLC fingerprint and multi-index component content determination. Technical Background

[0002] Tanyin Pills, as a classic Chinese medicine prescription for warming and transforming cold phlegm, originated from traditional Chinese medicine theory. It has significant effects of warming the spleen and kidneys, and assisting yang and transforming fluid. It is widely used clinically to treat cough, shortness of breath, wheezing, coughing up white sputum, chills and cold limbs caused by spleen and kidney yang deficiency and phlegm and fluid blocking the lungs. This prescription is carefully formulated with nine medicinal materials: Radish Seed, White Mustard Seed, Radish Seed, Perilla Seed, Dried Ginger, Atractylodes, Atractylodes, Cinnamon Bark, and Licorice. Its efficacy has been verified by clinical practice for more than half a century and is deeply trusted by doctors and patients. In recent years, Tanyin Pills have been included in the authoritative guideline "Guidelines for the Clinical Application of Traditional Chinese Medicines for the Treatment of Chronic Obstructive Pulmonary Disease (COPD) (2021 Edition)", which clearly recommends its use in clinical intervention for the symptoms of spleen and kidney yang deficiency and phlegm and fluid blocking the lungs in the stable stage of COPD, further highlighting its important position in the modern medical system.

[0003] However, with the accelerated modernization and internationalization of Traditional Chinese Medicine (TCM), more stringent and scientific requirements have been placed on the quality control of TCM preparations. The quality of TCM compound preparations is influenced by numerous factors, including the origin and harvesting time of the raw medicinal materials, the processing method, and the stability of the production process. These factors directly impact the content and ratio of the active ingredients in the preparation, and thus their clinical efficacy and safety. Therefore, establishing scientific, accurate, and comprehensive quality evaluation methods is crucial to ensuring the quality stability and efficacy of TCM compound preparations such as Tanyin Pills.

[0004] Currently, research on the quality control of Tanyin Pills is relatively scarce. Existing quality control standards are primarily based on the relevant provisions of the Pharmacopoeia of the People's Republic of China, covering only thin-layer chromatography (TLC) identification and determination of a single indicator, glycyrrhizic acid, which fails to fully reflect the overall chemical characteristics and quality control requirements of Tanyin Pills. Fingerprinting technology, as an effective quality control tool, can visually reflect the consistency and stability of the chemical composition of traditional Chinese medicine preparations by identifying and comparing multiple common peaks. Multi-index component content determination further quantifies the content of key active ingredients, providing a scientific basis for precise quality control of traditional Chinese medicines. High-performance liquid chromatography (HPLC) technology, due to its advantages of high separation efficiency, rapid analysis speed, and high sensitivity, has performed well in the establishment of fingerprints for traditional Chinese medicines and the determination of multi-index component content. Furthermore, the introduction of high-resolution mass spectrometry has greatly improved the ability to identify the structures of complex traditional Chinese medicine components, providing a reliable means for accurately assigning common peaks in fingerprints. Chemometric methods, through statistical analysis, reveal the inherent relationship between chemical composition and quality differences in traditional Chinese medicine preparations, providing a more in-depth and comprehensive perspective for quality evaluation.

[0005] Therefore, the present invention aims to develop a scientific, accurate and comprehensive quality evaluation method for Tanyin Pills by establishing an HPLC fingerprint of Tanyin Pills, combining multi-index component content determination and chemometric analysis, so as to meet the needs of modern Chinese medicine quality control. Summary of the Invention

[0006] The object of the present invention is to provide a quality evaluation method for Tanyin Pills based on HPLC fingerprint and multi-index component content determination. The HPLC fingerprint and multi-index content determination method established in the present invention is accurate and reliable, can provide a scientific basis for the quality control of Tanyin Pills, and provide a reference for its standardized production and quality evaluation.

[0007] The object of the present invention is achieved through the following technical solutions:

[0008] The present invention provides a quality evaluation method for Tanyin pills based on HPLC fingerprint and multi-index component content determination, comprising determining nine active ingredients in Tanyin pills based on high performance liquid chromatography-high resolution mass spectrometry technology and constructing an HPLC fingerprint of Tanyin pills; wherein the nine active ingredients are sinapinic acid, chlorogenic acid, cryptochlorogenic acid, 3,6'-diesinapoylsucrose, rosmarinic acid, apiose isoliquiritin, isoliquiritin, glycyrrhizic acid and atractylodes lactone I.

[0009] Furthermore, the method for determining the nine active ingredients in Tanyin Pills based on high performance liquid chromatography-high resolution mass spectrometry technology specifically comprises the following steps:

[0010] (1) Grind Tanyin pills into fine powder, sieve, and prepare sample powder for use;

[0011] (2) Add methanol aqueous solution to the sample powder in step (1), extract by ultrasonication, cool to room temperature, weigh, make up the weight loss with solvent, shake well, let stand, draw supernatant, filter, and take the filtrate as the test solution;

[0012] (3) Sinapine thiocyanate, chlorogenic acid, cryptochlorogenic acid, 3,6'-diesinapoylsucrose, rosmarinic acid, apiose isoliquiritin, isoliquiritin, ammonium glycyrrhizate and atractylodes lactone I reference substances were taken to prepare reference substance solutions; fine powders of single medicinal materials such as Radix Aconiti Lateralis Preparata, Sinapis Albae Seed, Radish Seed, Perilla Seed, Dried Ginger, Atractylodes Rhizome, Atractylodes Macrocephalae, Cinnamomum Cassiae and Licorice were taken and sampled according to the corresponding proportions in the prescription of Tanyin Pills, and single medicinal material sample solutions were prepared according to the method for preparing the test solution in step (2); negative samples lacking Atractylodes Rhizome, Atractylodes Macrocephalae, Perilla Seed, Radish Seed, Sinapis Albae Seed and Licorice were prepared according to the prescription proportions and preparation process of Tanyin Pills, and negative sample solutions were prepared according to the method for preparing the test solution in step (2).

[0013] (4) Qualitative testing: The reference solution, single herbal sample solution, and negative sample solution described in step (3) are injected into HPLC for testing, and the nine active ingredients in the test solution are determined based on the precise molecular weight and relative retention time;

[0014] (5) Quantitative detection: The test solution in step (2) and the mixed reference solution in step (3) were respectively determined by high performance liquid chromatography-high resolution mass spectrometry, and the nine active ingredients in the test solution were quantitatively detected by the external standard method.

[0015] Furthermore, in step (2), the mass volume ratio of the pharmaceutical powder to the methanol aqueous solution is 1 g: 30-100 mL; the volume fraction of the methanol aqueous solution is 30%-100%; the filtration is performed using a 0.22 μm microporous filter membrane; and the ultrasonic treatment conditions are: ultrasonic power 300-320 W, frequency 30-40 kHz, and treatment time 30-60 min.

[0016] Furthermore, in step (3), the specific preparation scheme of the reference solution is:

[0017] Using methanol-0.08 mol / L potassium dihydrogen phosphate solution with a volume ratio of 10:90 as the solvent for mustard thiocyanate and ammonium glycyrrhizate, and methanol as the solvent for other reference substances, reference substance stock solutions with a mass concentration of 0.5-1.5 mg·mL^-1 of each compound were prepared. Accurately measure an appropriate amount of each reference substance stock solution and use methanol-0.08 mol / L potassium dihydrogen phosphate solution with a volume ratio of 10:90 as the dilution solvent to prepare reference substances containing 51.00 μg·mL of mustard thiocyanate, respectively. -1 , chlorogenic acid 38.00 μg·mL -1 , cryptochlorogenic acid 7.80 μg·mL -1 3,6'-diesinapoylsucrose 44.96 μg·mL -1 , rosmarinic acid 11.60 μg·mL -1 , apiosyl isoliquiritin 2.65 μg·mL -1 , isoliquiritin 9.75 μg·mL -1 , ammonium glycyrrhizate 88.00 μg·mL -1 and Atractylodes lactone Ⅰ 12.20 μg·mL -1 mixed reference solution.

[0018] Furthermore, in step (4) or (5), the chromatographic conditions of the high performance liquid chromatography are: Agilent Poroshell 120PFP column, 4.6 mm × 150 mm, 2.7 μm; flow rate of 0.5 mL min-1; column temperature of 30°C; detection wavelengths of 345 nm (0-90 min) and 254 nm (90-105 min); mobile phase: phase A methanol-phase B 0.1% phosphoric acid aqueous solution; gradient elution.

[0019] Furthermore, the gradient elution procedure is specifically as follows: 0-10 min, 2% A→5% A; 10-30 min, 5% A→20% A; 30-90 min, 20% A→70% A; 90-105 min, 70% A→90% A.

[0020] Furthermore, the mass spectrometry conditions in step (4) or (5) are as follows: electrospray positive and negative ion modes are used, respectively; the full scan range is m / z 80-1200; the spray voltage is 2800 V for negative ions and 3500 V for positive ions; the nebulizer temperature is 400°C; the ion transfer capillary temperature is 350°C; the sheath gas flow rate is 50 arb; the auxiliary gas flow rate is 15 arb; the lens voltage is 55 V; and the collision voltage is set to step energies of 20, 40, and 60 eV, respectively.

[0021] Furthermore, the method for constructing the HPLC fingerprint of Tanyin Pills comprises the following steps:

[0022] (1) Preparation of the test solution: Grind Tanyin pills into fine powder, sieve, take 0.5 g of the sample powder, and ultrasonically extract it with 20 ml of a 75% methanol-water solution for 40 min. Let it cool to room temperature, weigh it, make up the weight loss with solvent, shake it well, let it stand, aspirate the supernatant, filter it with a 0.22 μm microporous membrane, and use the filtrate as the test solution;

[0023] (2) High performance liquid chromatography (HPLC) analysis: Agilent Poroshell 120PFP column, 4.6 mm × 150 mm, 2.7 μm; mobile phase: methanol in phase A-0.1% phosphoric acid aqueous solution in phase B; gradient elution: 0-10 min, 2% A→5% A; 10-30 min, 5% A→20% A; 30-90 min, 20% A→70% A; 90-105 min, 70% A→90% A; flow rate: 0.5 mL min-1; column temperature: 30°C; detection wavelengths: 345 nm (0-90 min) and 254 nm (90-105 min); (3) Ten batches of Tanyin pill samples were measured and analyzed and compared to obtain a standard fingerprint of Tanyin pill HPLC consisting of their common characteristic peaks.

[0024] Furthermore, the relative retention times tR of the 16 common characteristic peaks of the standard fingerprint of Tanyin Pills HPLC are: 8.52±0.2min, 27.94±0.2min, 44.03±0.2min, 47.88±0.2min, 48.85±0.2min, 60.91±0.2min, 69.61±0.2min, 70.50±0.2min, 75.60±0.2min, 81.07±0.2min, 87.96±0.2min, 89.13±0.2min, 93.64±0.2min, 98.22±0.2min, 98.66±0.2min, and 99.11±0.2min.

[0025] The present invention also provides an application of the test results obtained by utilizing the Tanyin Pills quality evaluation method in the quality control and standardized production of Tanyin Pills.

[0026] The beneficial effects of the present invention are:

[0027] The present invention utilizes an Agilent Poroshell 120PFP chromatographic column (150 mm × 4.6 mm, 2.7 μm) to increase the number of theoretical plates by approximately 40% and reduce column pressure by 40-50%, achieving separation performance close to that of UPLC on a conventional HPLC instrument. This significantly improves the separation efficiency and the number of identifiable compounds in Tanyin Pills. While conventional C18 columns are the most commonly used in chromatographic analysis of chemical components in traditional Chinese medicine, the Agilent Poroshell 120PFP column employed in the present invention, with its 2.7 μm small particle size core-shell structure, exhibits superior separation performance, significantly enhancing the separation efficiency and number of isolable compounds in Tanyin Pills.

[0028] At the same time, to address the issue of chromatographic peak splitting in mustard, the present invention clarified the stability of mustard in acidic environments and compared the effects of various solvents on its stability. This led to the development of a methanol-0.08 mol / L potassium dihydrogen phosphate (10:90) mixed solvent system for the first time. This mixed solvent system effectively resolves the chromatographic peak splitting issue caused by conventional solvent dissolution in mustard, ensuring the accuracy and reliability of the analytical results and providing a solid foundation for the precise analysis of key components.

[0029] Relying on the Q-Exactive Orbitrap high-resolution mass spectrometer (resolution 70,000, mass error <3ppm), the present invention successfully identified 14 of the 16 common peaks under positive and negative ion dual-mode scanning, and identified new components such as trisinapoylsucrose for the first time. This mass spectrometer has a resolution of up to 70,000, which is significantly better than existing commonly used technologies (such as Q-TOF with a resolution of approximately 30,000), and the accurate mass error of the obtained data is controlled at <3ppm. With this high-precision advantage, the problem of accurate identification of isomers such as 1,2-diesinapoylgentiobioside and 3,6'-diesinapoylsucrose has been successfully solved. For the identification of the primary parent ion m / z 753.2247[MH]-, the present invention integrates multidimensional high-precision information, including molecular formula fitting based on the accurate mass number of the primary mass spectrometry, systematic deduction of the secondary fragmentation pathway, and cross-comparison of the spectra of single medicinal materials and Tanyin Pill preparations. Finally, it verifies the precise match with the reference substance in terms of chromatographic retention time and secondary fragmentation spectrum, providing a high-precision technical paradigm for the analysis of the components of complex traditional Chinese medicine systems.

[0030] The present invention constructs a quality evaluation method for multi-index coordinated control, and simultaneously determines the contents of 9 active ingredients such as sinapinic acid and chlorogenic acid, covering the basis of multiple pharmacodynamic substances. It also screens out 8 key quality difference components through chemometric analysis, and combines the fingerprint similarity evaluation (S≥0.956) to form a three-dimensional evaluation system of "ingredient group-process stability-pharmacodynamic association", which significantly improves the quality control level of traditional Chinese medicine preparations. The batch quality differentiation pattern and the influence of raw medicinal materials revealed by the study provide a scientific basis for production enterprises to optimize procurement and process parameters. By integrating fingerprints and multi-index determination, this method provides a replicable technical framework for the modern quality evaluation of traditional Chinese medicine compound preparations with lower instrument costs (compared to mass spectrometry fingerprints) and higher popularity, and promotes the transformation of the industry from "single ingredient control" to "overall quality control". BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 HPLC fingerprints of 10 batches of Tanyin Pills in Example 1 of the present invention;

[0033] Figure 2 HPLC charts of the Tanyin Pill samples and individual medicinal materials in the prescription in Example 1 of the present invention;

[0034] Figure 3This is a mass spectrometry fragmentation pathway diagram of 3,6'-diesinapoylsucrose in Example 1 of the present invention;

[0035] Figure 4 Figure 1 shows the HCA results (A) and PCA scores (B) of 10 batches of Tanyin Pill samples in Example 1 of the present invention;

[0036] Figure 5 Graphs showing the PLS-DA scores (A) and VIP values ​​(B) of 10 batches of Tanyin Pill samples in Example 1 of the present invention;

[0037] Figure 6 This is a chromatogram in Example 1 of the present invention, wherein A is the test solution of Tanyin Pills, B is the mixed reference solution, and C is the negative sample. The specific numbers in the figure are 2. sinapinic acid thiocyanate; 4. chlorogenic acid; 5. cryptochlorogenic acid; 7. 3,6'-diesinapoylsucrose; 9. rosmarinic acid; 11. apiose isoliquiritigenin; 12. isoliquiritigenin; 13. ammonium glycyrrhizate; and 14. Atractylodes lactone I. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] Example 1

[0044] This example uses high performance liquid chromatography and high-resolution mass spectrometry to establish a fingerprint of Tanyin Pills and conduct a similarity evaluation. Simultaneously, the contents of nine active ingredients in Tanyin Pills, including sinapinic acid, chlorogenic acid, cryptochlorogenic acid, 3,6'-diesinapoylsucrose, rosmarinic acid, apiosyl isoliquiritin, isoliquiritin, glycyrrhizic acid, and atractylodes lactone I, were determined. The goal was to establish a scientific and comprehensive quality evaluation method. The specific research methods and results are as follows:

[0045] 1. Instruments and Materials

[0046] Instruments: Agilent 1260 high-performance liquid chromatograph, Thermo Fisher Q Exactive Focus mass spectrometer, and Dionex U3000 liquid chromatograph coupled with the mass spectrometer; Kunshan KQ-400 ultrasonic cleaning machine; Beijing Sartorius BS210S 1 / 10,000 and BT25S 1 / 100,000 electronic balances.

[0047] Materials: Ten batches of Tanyin pills were provided by Xi'an Zhengda Pharmaceutical Co., Ltd. (specification: 0.18 g per pill; batch numbers: 230105k (S1), 230301 (S2), 230302 (S3), 230503 (S4), 230704 (S5), 240502k (S6), 240601 (S7), 240702 (S8), 241003 (S9), and 241204 (S10). Reference substances, sinapinic thiocyanate (batch number 24032702) and cryptochlorogenic acid (batch number 24041504), were purchased from Glip Biotechnology Co., Ltd.; ammonium glycyrrhizate (batch number CHB2) was purchased from Glip Biotechnology Co., Ltd. 31005), chlorogenic acid (batch number CHB201114), and rosmarinic acid (batch number CHB230702) were purchased from Chengdu Kroma Biotechnology Co., Ltd., 3,6'-diesinapoylsucrose (batch number 19031208) was purchased from Shanghai Qiming Biotechnology Co., Ltd., and apigenin isoliquiritin (batch number HR111W7), isoliquiritin (batch number HR5423S1), and atractylodes lactone I (batch number HS14914B1) were purchased from Baoji Chenguang Biotechnology Co., Ltd. The purity of all these reference substances was ≥98%. Methanol and phosphoric acid were of chromatographic grade, formic acid was of mass spectrometry grade, and all other reagents used were of analytical grade.

[0048] 2 Methods and Results

[0049] 2.1 Chromatographic conditions

[0050] An Agilent Poroshel 1120PFP column (4.6 mm × 150 mm, 2.7 μm) was used. The mobile phase consisted of methanol-0.1% phosphoric acid gradient elution (0–10 min, 2%–5% methanol; 10–30 min, 5%–20% methanol; 30–90 min, 20%–70% methanol; 90–105 min, 70%–90% methanol). The detection wavelengths were 345 nm (0–90 min) and 254 nm (90–105 min). The column temperature was 30°C and the flow rate was 0.5 ml min. -1 The injection volume was 10 μL. When the compounds were identified by LC-MS, phosphoric acid was replaced with volatile formic acid, and other chromatographic conditions were the same.

[0051] 2.2 Mass spectrometry conditions

[0052] Positive and negative ion detection was performed simultaneously under HESI ion source conditions; spray voltage: negative ion 2800V, positive ion 3500V; nebulizer temperature 400℃, ion transfer capillary temperature 350℃, sheath gas flow rate 50arb, auxiliary gas flow rate 15arb, lens voltage 55V, collision voltage set to step energy of 20, 40, and 60eV respectively. Scan mode was full scan / data-dependent secondary mass spectrometry scan (FullMS / dd-MS 2 ), FullMS resolution was 70000, dd-MS2 resolution was 17500, and the scanning range was m / z 80-1200.

[0053] 2.3 Preparation of sample solution

[0054] 2.3.1 Preparation of reference solution

[0055] Weigh an appropriate amount of each reference substance accurately, add solvent to prepare a solution containing 1.02 mg·mL of sinapinic acid thiocyanate. -1 , chlorogenic acid 0.76 mg·mL -1 , cryptochlorogenic acid 0.78 mg·mL -1 , 3,6'-diesinapoylsucrose 1.12 mg·mL -1 , rosmarinic acid 1.16 mg·mL -1 , apiose isoliquiritigenin 0.53 mg·mL -1 , isoliquiritin 0.78 mg·mL -1 , ammonium glycyrrhizate 0.88 mg·mL -1 and Atractylodes lactone Ⅰ 0.61 mg·mL -1The reference substance stock solutions were prepared. The solvent for sinapine thiocyanate and ammonium glycyrrhizate was methanol-0.08 mol / L potassium dihydrogen phosphate solution (volume ratio of 10:90). The solvent for other reference substances was methanol. Accurately measure an appropriate amount of each reference substance stock solution and dilute it with methanol-0.08 mol / L potassium dihydrogen phosphate solution (volume ratio of 10:90) to prepare 51.00 μg mL of sinapine thiocyanate, respectively. -1 , chlorogenic acid 38.00 μg·mL -1 , cryptochlorogenic acid 7.80 μg·mL -1 3,6'-diesinapoylsucrose 44.96 μg·mL -1 , rosmarinic acid 11.60 μg·mL -1 , apiosyl isoliquiritin 2.65 μg·mL -1 , isoliquiritin 9.75 μg·mL -1 , ammonium glycyrrhizate 88.00 μg·mL -1 and Atractylodes lactone Ⅰ 12.20 μg·mL -1 mixed reference solution.

[0056] 2.3.2 Preparation of test solution

[0057] Tanyin pills were crushed into fine powder, passed through No. 2 sieve, and about 0.5 g of sample powder was taken, accurately weighed, and ultrasonically extracted with 20 ml of 75% methanol aqueous solution for 40 min. The powder was filtered through a 0.22 μm microporous membrane, and the filtrate was taken for analysis.

[0058] 2.3.3 Preparation of single herbal sample solutions

[0059] Take the fine powder of each single medicinal material (pass through No. 2 sieve), sample it according to the corresponding proportion in the prescription, and prepare the single medicinal material sample solution according to the method under "2.3.2".

[0060] 2.3.4 Negative sample solution

[0061] According to the prescription ratio and preparation process of Tanyin Pills, prepare a negative sample that is missing Atractylodes lancea, Atractylodes macrocephala, Perilla seed, Radish seed, White mustard seed, and Licorice root. Prepare the negative sample solution according to the method under "2.3.2".

[0062] 2.4 Study on HPLC fingerprint of Tanyin Pills

[0063] 2.4.1 Fingerprint Methodology Review

[0064] (1) Precision test

[0065] Tanyin Pills sample solution, batch number 230301, was used for the study, with six consecutive injections. Rosmarinic acid, the ninth common peak, was selected as a reference, and the relative standard deviations (RSDs) of the relative retention times and relative peak areas of the 16 common peaks were calculated. The results showed that the RSDs for the relative retention times were all less than 0.38%, and the RSDs for the relative peak areas were all less than 1.48%, indicating good precision of the method.

[0066] (2) Repeatability test

[0067] Weigh 0.5 g of Tanyin Pills batch number 230301 and prepare six sample solutions in parallel according to the method in "2.3.2" for chromatographic analysis. Using rosmarinic acid, the common peak No. 9, as a reference, the relative standard deviations (RSDs) of the relative retention times and relative peak areas of the 16 common peaks were calculated. The results showed that the RSDs for the relative retention times did not exceed 0.81%, and the RSDs for the relative peak areas did not exceed 2.68%, demonstrating good reproducibility of the method.

[0068] (3) Stability test

[0069] Weigh 0.5 g of Tanyin Pills sample, batch number 230301, and prepare the test solution according to the method under "2.3.2." The solution was placed at room temperature for 0, 2, 4, 6, 8, and 12 hours, and then subjected to chromatographic analysis. Using common peak No. 9, rosmarinic acid, as a reference, the RSDs of the relative retention times and relative peak areas of the 16 common peaks were calculated. The results showed that the RSD values ​​for the relative retention times were all less than 0.22%, and the RSD values ​​for the relative peak areas were all less than 2.02%, indicating that after the sample was prepared into the test solution, it maintained good stability at room temperature for 12 hours.

[0070] 2.4.2 Fingerprint establishment and similarity evaluation

[0071] Take the sample powder of each batch of Tanyin Pills, prepare the test solution according to the method under "2.3.2", and perform chromatographic analysis according to the experimental conditions under "2.1". Use Agilent Open Lab CDSData Analysis offline workstation to export the data file to "AIA" format, and then use the 2012 version of Chinese medicine chromatographic fingerprint similarity evaluation software to analyze the data. The median method is used to generate the reference fingerprint (R). A total of 16 common peaks in the fingerprint are identified, such as Figure 1 The similarities between the fingerprints of the 10 batches of samples (S1-S10) and the control were 0.989, 0.970, 0.993, 0.995, 0.997, 0.993, 0.991, 0.956, 0.975 and 0.967, respectively. The similarity values ​​were all high, indicating that the quality of Tanyin Pills was well controlled during the production process and the quality between batches was highly consistent.

[0072] 2.4.3 Attribution of common peaks in fingerprints

[0073] Based on the compound chromatographic retention time and UV spectrum information, the chromatograms of Tanyin Pills and its single medicinal materials were compared to determine the origin of the common peaks in the fingerprints. Sample solutions of the 9 medicinal materials in the preparation were taken separately and injected and measured according to the chromatographic conditions under "2.1". The chromatograms are shown in Figure 2 The results showed that peak 2 in the fingerprint of Tanyin Pills was shared by white mustard seeds and radish seeds; peaks 3, 7, and 10 originated from radish seeds; peaks 4, 5, 14, 15, and 16 were jointly derived from Atractylodes macrocephala and Atractylodes lancea; peak 6 originated from cinnamon bark; peaks 8 and 9 originated from Perilla seeds; and peaks 11, 12, and 13 originated from Licorice root. Peak 1 had no corresponding medicinal source and was likely an unknown component produced during the preparation production process.

[0074] 2.4.4 Identification of common peaks in fingerprints

[0075] The chromatographic and mass spectrometric conditions under "2.1" and "2.2" were used to analyze the sample solution of Tanyin Pills by liquid chromatography-mass spectrometry. Based on the high-resolution mass spectrometric data of the common peaks, including the primary quasi-molecular ion peak and the secondary fragment ion, a total of 12 common peaks were identified in the fingerprint of Tanyin Pills, of which 9 common peaks were further compared with the reference substances to identify sinapines (common peak No. 2), chlorogenic acid (common peak No. 4), cryptochlorogenic acid (common peak No. 5), 3,6'-diesinapoylsucrose (common peak No. 7), rosmarinic acid (common peak No. 9), apiosyl isoliquiritin (common peak No. 11), isoliquiritin (common peak No. 12), glycyrrhizic acid (common peak No. 13), and Atractylodes lactone I (common peak No. 14). The results are shown in Table 1. The mass spectrometric identification process of the common peaks is described as follows using 3,6'-diesinapoylsucrose as an example ( Figure 3 The primary quasi-molecular ion of common peak No. 7 in negative ion mode is m / z 753.2242 [MH] - The fitting molecular formula is C 34 H 42 O 19 The primary quasi-molecular ion undergoes ester bond cleavage to produce sinapinic acid ions m / z 223.0609 and m / z 547.1673 [MH-disinapoyl] - (disinapoyl is sinapinoyl), m / z 547.1673 [MH-disinapoyl] - Loss of one molecule of H2O produces a secondary fragment m / z 529.1574 [MH-disinapoyl-H2O] -, and at the same time loses a molecule of glucose (Glc) to produce a fragment ion m / z 367.1028 [MH-disinapoyl-Glc] - The sinapinic acid ion m / z 223.0609 lost H2O, CH3·, and CH3· in sequence to produce characteristic fragment ions m / z 205.0502, m / z 190.0266, and m / z 175.0029, respectively. Finally, by comparing the secondary fragments and retention times with those of the reference substance, the common peak No. 7 was identified as 3,6'-diesinapoylsucrose.

[0076] Table 1 High-resolution mass spectrometry identification results of common peaks of Tanyin Pills

[0077]

[0078]

[0079] *Compared with reference substances; / No secondary fragment ions were detected

[0080] 2.4.5 Chemometric analysis

[0081] Chemometric analysis was performed using SIMCA-P 14.0 software. HCA analysis was performed using the peak areas of 16 common peaks of 10 batches of Tanyin Pill samples (S1 to S10) as variables. The results are shown in Figure 4 A. The samples were divided into two categories: Category I was S1 to S5, and Category II was S6 to S10. The 16 common peak areas of 10 batches of Tanyin Pill samples were subjected to PCA analysis. The cumulative variance of principal components 1 and 2 was greater than 87%, indicating that these two principal components played a leading role in reflecting the common peak information of the fingerprint of Tanyin Pill samples. PCA analysis results are shown in Figure 4 B. From the score graph, we can see that the 10 batches of Tanyin pill samples can be divided into two categories, which is consistent with the HCA classification results. Based on PCA, we further selected the supervised PLS-DA model to analyze the 10 batches of Tanyin pill samples and screen the components that contribute most to the differences between groups. The PLS-DA model score graph is shown in Figure 5 A. The results show that the cumulative explanatory power parameter R 2 X and R 2 Y were 0.812 and 0.870 respectively, and the prediction ability parameter Q 2 The values ​​of VIP and VIP were 0.649, which were all greater than 0.5, indicating that the established model was stable and reliable with strong predictive ability. The differential chemical components of 10 batches of samples were screened according to the variable importance in projection (VIP). The larger the VIP value, the greater its contribution to the quality difference. The VIP values ​​of 16 peak area variables in the model were extracted within the confidence interval of 0.95. Figure 5 B shows that there are 8 components with VIP>1 (marked in red), namely, No. 10 common peak trisinapoylsucrose (VIP=1.2913), No. 8 common peak isosmarinic acid glycoside (VIP=1.1525), No. 3 common peak sinapinoylsucrose (VIP=1.1399), No. 7 common peak 3,6'-diesinapoylsucrose (VIP=1.1356), No. 11 common peak apiosyl isoliquiritigenin (VIP=1.0729), No. 6 unknown common peak (VIP=1.0687), No. 2 common peak sinapinic acid (VIP=1.0659), No. 9 common peak rosmarinic acid (VIP=1.0439), suggesting that these 8 components may be important components affecting the quality differences of Tanyin Pills.

[0082] 2.5 Determination of content of multiple index components

[0083] 2.5.1 Methodological Investigation

[0084] (1) Specificity test

[0085] Take appropriate amount of the mixed reference solution, test solution and negative sample solution prepared under "2.3" respectively, and analyze them according to the chromatographic conditions under "2.1". Record the chromatogram. Figure 6 The results showed that chromatographic peaks with the same retention time could be observed in the chromatogram of the test solution at the corresponding positions of the sinapinic thiocyanate, chlorogenic acid, cryptochlorogenic acid, 3,6'-diesinapoylsucrose, rosmarinic acid, apiose isoliquiritin, isoliquiritin, ammonium glycyrrhizate, and Atractylodes lactone I reference substances. The UV absorption spectra of these chromatographic peaks were highly consistent with the UV spectra of the reference substances. In contrast, no chromatographic peaks appeared at the corresponding positions of the negative chromatogram, indicating that there was no interference from the negative sample, thus confirming the good specificity of the method.

[0086] (2) Linear relationship

[0087] Methanol-0.08 mol·L -1 Potassium dihydrogen phosphate solution (volume ratio of 10:90) was used as the dilution solvent. Accurately measure an appropriate amount of the mixed reference solution under "2.3.1" and dilute it into a series of mixed reference solutions with different concentrations according to the dilution multiples of 1, 2, 5, 10, 25, and 50 times, respectively. Inject the solution and analyze. The mass concentration of each reference solution (X, μg·mL -1 ) as the abscissa and the peak area value (Y) as the ordinate to draw the standard curve, and calculate the linear equation and correlation coefficient. The results are shown in Table 2. The results show that the reference substances have a good linear relationship within the corresponding concentration range.

[0088] Table 2 Standard curves, correlation coefficients, and linear ranges of the nine components

[0089]

[0090] (3) Precision test

[0091] 10 μL of the same mixed reference solution was accurately aspirated and injected 6 times. The peak areas were measured and the RSDs of the peak areas of sinapinic thiocyanate, chlorogenic acid, cryptochlorogenic acid, 3,6'-diesinapoylsucrose, rosmarinic acid, apiose isoliquiritin, isoliquiritin, ammonium glycyrrhizate and atractylodes lactone I were calculated to be 0.93%, 0.37%, 0.94%, 0.63%, 0.50%, 1.61%, 0.24%, 0.72% and 0.23%, respectively, indicating that the instrument had good precision.

[0092] (4) Stability test

[0093] The same test solution (batch number 230301) was placed at room temperature and injected at 0, 2, 6, 4, 8, and 12 h. The calculated peak area RSDs for sinapinic thiocyanate, chlorogenic acid, cryptochlorogenic acid, 3,6'-diesinapoylsucrose, rosmarinic acid, apiose isoliquiritin, isoliquiritin, ammonium glycyrrhizate, and atractylodes lactone I were 0.53%, 0.47%, 1.20%, 1.10%, 1.28%, 0.65%, 0.92%, 1.34%, and 1.14%, respectively, indicating good stability of the test solution within 12 h.

[0094] (5) Repeatability test

[0095] Six samples (batch number 230301) were accurately weighed and the test solutions were prepared according to the method in "2.3.2". The samples were injected and determined according to the chromatographic conditions in "2.1". The average contents of sinapine thiocyanate, chlorogenic acid, cryptochlorogenic acid, 3,6'-diesinapoylsucrose, rosmarinic acid, apiose isoliquiritin, isoliquiritin, ammonium glycyrrhizate, and atractylodes lactone I were calculated to be 0.9696, 0.3627, 0.0654, 0.5247, 0.2379, 0.0645, 0.1824, 1.0905, and 0.2726 mg·g, respectively. -1 , RSDs were 0.60%, 1.27%, 1.47%, 0.77%, 0.98%, 1.42%, 1.25%, 0.70% and 0.17%, respectively. The results showed that the method had good repeatability.

[0096] (6) Sample recovery test

[0097] Accurately weigh 0.25 g of sample powder (batch number 230301) with known content of each component. Six aliquots were weighed and placed in stoppered Erlenmeyer flasks. Each aliquot was precisely spiked with the reference substances (sinapine thiocyanate, chlorogenic acid, cryptochlorogenic acid, 3,6'-diesinapoylsucrose, rosmarinic acid, apiose isoliquiritin, isoliquiritin, ammonium glycyrrhizate, and atractylodes lactone I) at a 1:1 ratio. Six test sample solutions were prepared in parallel according to the method in "2.3.2." Samples were then injected and analyzed under the chromatographic conditions in "2.1" to determine their content and calculate their recoveries. The results showed that the average recoveries of the nine components ranged from 98.0% to 101.8%, with RSDs of no more than 1.9%, demonstrating the good accuracy of this method.

[0098] 2.5.2 Sample content determination

[0099] About 0.5 g of powder from each of 10 batches of Tanyin Pills samples was taken and accurately weighed. The test solution was prepared according to the method in "2.3.2". The samples were injected and analyzed under the chromatographic conditions in "2.1". The contents of sinapinic acid thiocyanate, chlorogenic acid, cryptochlorogenic acid, 3,6'-diesinapoylsucrose, rosmarinic acid, apiose isoliquiritin, isoliquiritin, glycyrrhizic acid (ammonium glycyrrhizate content / 1.0207) and atractylodes lactone I were calculated, as well as the total content of the nine indicator components in different batches of samples. The results are shown in Table 3.

[0100] Table 3 Sample content determination (mg g -1 ,n=3)

[0101]

[0102]

[0103] 3. Results Analysis

[0104] 3.1 Selection of experimental conditions

[0105] The present invention investigates the preparation method of the test solution, including extraction methods (ultrasonic extraction, heating reflux extraction), extraction solvents (30%, 50%, 75%, 100% methanol), and extraction times (30, 45, and 60 minutes). Based on factors such as the number of peaks, peak response intensity, and resolution, the extraction condition is selected as 75% methanol ultrasonic 45 minutes. Chromatographic conditions are investigated, including detection wavelengths (230, 254, 280, 300, and 345 nm) and mobile phases (acetonitrile-water, methanol-water, and methanol-0.1% phosphoric acid aqueous solution). Based on factors such as the number of chromatographic peaks, resolution, and peak response intensity, methanol-0.1% phosphoric acid aqueous solution is selected as the mobile phase. A wavelength switching method is also employed, i.e., the wavelength is 345 nm from 0 to 90 minutes and is switched to 254 nm from 90 to 105 minutes to facilitate the detection of components such as glycyrrhizic acid and atractylodes lactone I in the second half of the chromatogram. In addition, the present invention finds that the sinapine thiocyanate reference substance is prone to chromatographic peak splitting when directly dissolved with methanol. Since sinapine is a quaternary ammonium base, it is relatively stable in an acidic environment. Therefore, the present invention refers to the preparation method of the sinapine thiocyanate reference substance under the mustard item of the 2020 edition of the Chinese Pharmacopoeia, and selects methanol-0.08mol / L potassium dihydrogen phosphate solution (volume ratio of 10:90) as the dissolving solvent of sinapine thiocyanate and the diluting solvent of the mixed reference solution. The sinapine chromatographic peak under this condition has a good peak shape, thereby meeting the requirements of the determination.

[0106] 3.2 Fingerprint analysis

[0107] The present invention established HPLC fingerprints of 10 batches of Tanyin Pill samples and identified 16 common peaks. The attribution and identification of the common peaks clarified the sources of the main ingredients in the preparation, such as sinapines (white mustard seeds / radish seeds), rosmarinic acid (perilla seeds), atractylodes lactone I (atractylodes macrocephala), glycyrrhizic acid (licorice), etc., which are consistent with the pharmacological material basis of the prescription composition medicinal materials. Q-Exactive Orbitrap high-resolution mass spectrometry technology further improves the reliability of component identification, especially the analysis of the cleavage pathway of structurally complex compounds (such as 3,6'-diesinapoylsucrose), which can provide an important reference for subsequent research. It is worth noting that the corresponding medicinal material source was not found for common peak No. 1, and it is speculated that it may be a newly generated component in the preparation process. Since its response signal was not seen during the mass spectrometry data acquisition process, the present invention did not identify the common peak, and it can be further analyzed in the future through phytochemical separation combined with nuclear magnetic resonance technology.

[0108] 3.3 Multi-index content determination

[0109] Modern pharmacological studies have shown that the combined effects of multiple components in Tanyin Pills form the material basis for their clinical efficacy. Sinapine can improve inflammatory responses and mucus hypersecretion in asthmatic lung tissue, thereby mediating the prescription's expectorant and dampness-removing effects. Chlorogenic acid, cryptochlorogenic acid, and rosmarinic acid possess significant antioxidant and anti-inflammatory properties, potentially enhancing the overall efficacy of Tanyin Pills by inhibiting inflammation and regulating immune function. 3,6'-diesinapoylsucrose significantly alleviates oxidative stress and exhibits anti-apoptotic effects. Glycyrrhizic acid, isoliquiritin, and apiolactone isoliquiritin, derived from licorice, exert antitussive and expectorant effects through a multi-target mechanism. Atractylodes lactone I plays an important role in regulating intestinal flora and immunity, contributing to the spleen-tonifying and qi-invigorating effects of Tanyin Pills and improving lung damage caused by recurrent respiratory infections. Therefore, considering the correlation between the main active ingredients of each medicinal flavor in the preparation and the clinical efficacy, as well as the identification and separation of the common peaks in the fingerprint, sinapinic acid, chlorogenic acid, cryptochlorogenic acid, 3,6'-diesinapoylsucrose, rosmarinic acid, apiosyl isoliquiritin, isoliquiritin, glycyrrhizic acid and Atractylodes lactone I were selected as content determination indicators, so as to achieve a more comprehensive quality evaluation of Tanyin Pills.

[0110] 3.4 Quality Evaluation and Analysis of Tanyin Pills

[0111] The present invention evaluates the quality consistency of Tanyin Pills preparation by combining HPLC fingerprint with chemometric method. The results of the similarity (S≥0.956) between the fingerprints of 10 batches of samples and the control indicate that the quality stability of the preparation is generally good. The results of HCA and PCA analysis show that the quality characteristics of the 10 batches of samples show significant clustering characteristics (such as Figure 4 As shown). Among them, the 2023 production batches (S1-S5) were classified as Class I, and the 2024 production batches (S6-S10) were clustered into Class II. This significant quality differentiation pattern based on the production year suggests that batch differences in raw medicinal materials may be the key influencing factor. The common peak areas of the 10 batches of samples were further analyzed by PLS-DA. Among them, 3 of the 8 common peaks with VIP values ​​greater than 1 were derived from radish seeds (trisinapoylsucrose, sinapoylsucrose, 3,6'-diesinapoylsucrose), 2 were derived from perilla seeds (isorosmarinic acid glycosides, rosmarinic acid), 1 was derived from licorice (apiose isoliquiritigenin), 1 was shared by radish seeds and white mustard seeds (sinapine), and 1 was unknown. On the other hand, the results of multi-index quantitative analysis showed that the dispersion of 3,6'-diesinapoylsucrose from radish seeds was the most prominent (the content range was 0.1197~0.5247mg·g -1The above overall data characteristics suggest that the quality stability control of raw medicinal materials such as radish seeds, perilla seeds, white mustard seeds, and liquorice should be strengthened, especially the quality control of radish seeds. It is recommended to establish a traceability system for the origin and a standardized processing technology to ensure the stability of the chemical composition content between batches of preparations.

[0112] In summary, this study systematically evaluated the chemical characteristics and quality consistency of Tanyin Pills by integrating fingerprint analysis with multi-index content determination, providing a scientific basis for quality control of Tanyin Pills and setting a precedent for establishing a modern evaluation system for compound Chinese medicine preparations. Future work could further expand the sample size and integrate pharmacodynamic experiments to explore the correlation between chemical characteristics and clinical efficacy, thereby promoting the shift in quality control of Chinese medicine from a "composition-oriented" to a "efficacy-oriented" approach.

[0113] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for evaluating the quality of Tanyin pills based on HPLC fingerprint and multi-index component content determination, characterized in that: It includes the determination of nine active ingredients in Tanyin Pills based on high performance liquid chromatography-high resolution mass spectrometry technology and the construction of an HPLC fingerprint of Tanyin Pills; wherein, the nine active ingredients are sinapinic acid, chlorogenic acid, cryptochlorogenic acid, 3,6'-diesinapoylsucrose, rosmarinic acid, apiosyl isoliquiritin, isoliquiritin, glycyrrhizic acid and Atractylodes lactone I.

2. The method for evaluating the quality of Tanyin Pills according to claim 1, wherein: The method for determining the nine active ingredients in Tanyin Pills based on high performance liquid chromatography-high resolution mass spectrometry specifically comprises the following steps: (1) Grind Tanyin pills into fine powder, sieve, and prepare sample powder for use; (2) Add methanol aqueous solution to the sample powder in step (1), extract by ultrasonication, cool to room temperature, weigh, make up the weight loss with solvent, shake well, let stand, draw supernatant, filter, and take the filtrate as the test solution; (3) Sinapine thiocyanate, chlorogenic acid, cryptochlorogenic acid, 3,6'-diesinapoylsucrose, rosmarinic acid, apiose isoliquiritin, isoliquiritin, ammonium glycyrrhizate and atractylodes lactone I reference substances were taken to prepare reference substance solutions; fine powders of single medicinal materials such as Radix Aconiti Lateralis Preparata, Sinapis Albae Seed, Radish Seed, Perilla Seed, Dried Ginger, Atractylodes Rhizome, Atractylodes Macrocephalae, Cinnamomum Cassiae and Licorice were taken and sampled according to the corresponding proportions in the prescription of Tanyin Pills, and single medicinal material sample solutions were prepared according to the method for preparing the test solution in step (2); negative samples lacking Atractylodes Rhizome, Atractylodes Macrocephalae, Perilla Seed, Radish Seed, Sinapis Albae Seed and Licorice were prepared according to the prescription proportions and preparation process of Tanyin Pills, and negative sample solutions were prepared according to the method for preparing the test solution in step (2). (4) Qualitative testing: The reference solution, single herbal sample solution, and negative sample solution described in step (3) are injected into HPLC for testing, and the nine active ingredients in the test solution are determined based on the precise molecular weight and relative retention time; (5) Quantitative detection: The test solution in step (2) and the mixed reference solution in step (3) were respectively determined by high performance liquid chromatography-high resolution mass spectrometry, and the nine active ingredients in the test solution were quantitatively detected by the external standard method.

3. The method for evaluating the quality of Tanyin Pills according to claim 2, wherein: In step (2), the mass volume ratio of the drug powder to the methanol aqueous solution is 1 g: 30-100 mL; the volume fraction of the methanol aqueous solution is 30%-100%; the filtration is performed using a 0.22 μm microporous filter membrane; and the ultrasonic treatment conditions are: ultrasonic power 300-320 W, frequency 30-40 kHz, and treatment time 30-60 min.

4. The method for evaluating the quality of Tanyin Pills according to claim 2, wherein: In step (3), the specific preparation scheme of the reference solution is: Using methanol-0.08 mol / L potassium dihydrogen phosphate solution with a volume ratio of 10:90 as the solvent for mustard thiocyanate and ammonium glycyrrhizate, and methanol as the solvent for other reference substances, reference substance stock solutions with a mass concentration of 0.5-1.5 mg·mL^-1 of each compound were prepared. Accurately measure an appropriate amount of each reference substance stock solution and use methanol-0.08 mol / L potassium dihydrogen phosphate solution with a volume ratio of 10:90 as the dilution solvent to prepare reference substances containing 51.00 μg·mL of mustard thiocyanate, respectively. -1 , chlorogenic acid 38.00 μg·mL -1 , cryptochlorogenic acid 7.80 μg·mL -1 3,6'-diesinapoylsucrose 44.96 μg·mL -1 , rosmarinic acid 11.60 μg·mL -1 , apiosyl isoliquiritin 2.65 μg·mL -1 , isoliquiritin 9.75 μg·mL -1 , ammonium glycyrrhizate 88.00 μg·mL -1 and Atractylodes lactone Ⅰ 12.20 μg·mL -1 mixed reference solution.

5. The method for evaluating the quality of Tanyin Pills according to claim 2, wherein: In step (4) or (5), the chromatographic conditions of the high performance liquid chromatography are: Agilent Poroshell 120PFP column, 4.6 mm × 150 mm, 2.7 μm; flow rate of 0.5 mL min-1; column temperature of 30°C; detection wavelengths of 345 nm (0-90 min) and 254 nm (90-105 min); mobile phase: phase A methanol-phase B 0.1% phosphoric acid aqueous solution; gradient elution.

6. The method for evaluating the quality of Tanyin Pills according to claim 5, wherein: The gradient elution procedure is specifically as follows: 0-10 min, 2% A→5% A; 10-30 min, 5% A→20% A; 30-90 min, 20% A→70% A; 90-105 min, 70% A→90% A.

7. The method for evaluating the quality of Tanyin Pills according to claim 2, wherein: The mass spectrometry conditions in step (4) or (5) are as follows: electrospray positive and negative ion modes are used, respectively; the full scan range is m / z 80-1200; the spray voltage is 2800V for negative ions and 3500V for positive ions; the nebulizer temperature is 400°C; the ion transfer capillary temperature is 350°C; the sheath gas flow rate is 50arb; the auxiliary gas flow rate is 15arb; the lens voltage is 55V; and the collision voltage is set to step energies of 20, 40, and 60eV, respectively.

8. The method for evaluating the quality of Tanyin Pills according to claim 1, wherein: The method for constructing the HPLC fingerprint of Tanyin Pills comprises the following steps: (1) Preparation of the test solution: Grind Tanyin pills into fine powder, sieve, take 0.5 g of the sample powder, and ultrasonically extract it with 20 ml of a 75% methanol-water solution for 40 min. Let it cool to room temperature, weigh it, make up the weight loss with solvent, shake it well, let it stand, aspirate the supernatant, filter it with a 0.22 μm microporous membrane, and use the filtrate as the test solution; (2) HPLC analysis: Agilent Poroshell 120PFP column, 4.6 mm × 150 mm, 2.7 μm; Mobile phase: methanol in phase A-0.1% phosphoric acid in water in phase B; gradient elution: 0-10 min, 2% A→5% A; 10-30 min, 5% A→20% A; 30-90 min, 20% A→70% A; 90-105 min, 70% A→90% A; flow rate, 0.5 mL min-1; column temperature, 30°C; detection wavelengths, 345 nm (0-90 min) and 254 nm (90-105 min); (3) Ten batches of Tanyin Pill samples were measured and analyzed and compared to obtain a standard fingerprint of Tanyin Pill HPLC consisting of their common characteristic peaks.

9. The method for evaluating the quality of Tanyin Pills according to claim 8, wherein: The relative retention times tR of the 16 common characteristic peaks of the standard fingerprint of the Tanyin Pills HPLC are: 8.52±0.2min, 27.94±0.2min, 44.03±0.2min, 47.88±0.2min, 48.85±0.2min, 60.91±0.2min, 69.61±0.2min, 70.50±0.2min, 75.60±0.2min, 81.07±0.2min, 87.96±0.2min, 89.13±0.2min, 93.64±0.2min, 98.22±0.2min, 98.66±0.2min, 99.11±0.2min.

10. Use of the test results obtained by the quality evaluation method of Tanyin Pills according to any one of claims 1 to 9 in the quality control and standardized production of Tanyin Pills.

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