Establishment method of characteristic fingerprint spectrum of Jianpi antenatal granules and fingerprint spectrum thereof

Abstract

The application discloses a method for establishing characteristic fingerprint spectrum of Jianpi Antai granules and the fingerprint spectrum, and comprises the following steps: S1, preparation of a test sample solution; S2, preparation of a mixed control sample solution; S3, determination of chromatographic conditions; and S4, analysis and comparison of the test sample solution and the control sample solution according to the chromatographic conditions in step S3 to obtain the characteristic fingerprint spectrum of Jianpi Antai granules composed of common peaks. The method can be used for multi-component fingerprint spectrum research on Jianpi Antai granules, attribute research on main chromatographic peaks and similarity evaluation, and the fingerprint spectrum can comprehensively reflect the effectiveness, uniformity and stability of Jianpi Antai granules, is beneficial to the endocrine and immune microenvironment related pharmacodynamic research and safety evaluation of Jianpi Antai granules, and can increase the thin layer chromatography qualitative identification of index components and the content determination of effective components on the basis of the existing quality standard, so that a more perfect quality standard control system is established.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine granule technology, specifically relating to the method for establishing the characteristic fingerprint spectrum of Jianpi Antai granules and its fingerprint spectrum. Background Technology

[0002] Jianpi Antai Mixture is widely used in the treatment of threatened miscarriage with significant efficacy. Currently, its usage coverage among threatened miscarriage patients is approximately 95%, benefiting nearly 500,000 individuals. However, the bulky bottled form of the mixture poses disadvantages such as inconvenience in transportation and storage, significantly limiting its market application and promotion. Furthermore, the current standard for this preparation is the 2005 edition of the "Zhejiang Provincial Medical Institution Preparation Standard," which only includes thin-layer chromatography identification of paeoniflorin and astragaloside A, as well as routine tests such as pH and relative density. The thin-layer chromatography identification items are limited, lacking testing for the content of its main active ingredients and failing to detect potential components that could affect medication safety, thus failing to comprehensively and effectively control the quality of the preparation.

[0003] To address the aforementioned issues, those skilled in the art, based on the prescription of Jianpi Antai Mixture, have employed modern technology, driven by demand, and leveraging the advantages of traditional Chinese medicine to develop new formulations that are easy to store and convenient to carry and use. They have fully utilized pharmaceutical preparation processes to modify the mixture into a granule form, conducted pharmacodynamic studies and safety assessments related to the endocrine and immune microenvironment of Jianpi Antai Granules, and added thin-layer chromatography qualitative identification of its indicator components and content determination of effective ingredients to the existing quality standards. They have also determined the levels of heavy metals, harmful elements, and pesticide residues, thus establishing a more comprehensive quality standard control system.

[0004] How to conduct multi-component fingerprint spectroscopy research on Jianpi Antai granules, and to conduct attribution research and similarity evaluation of the main chromatographic peaks, are technical problems that need to be solved by those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to solve the aforementioned technical problems existing in the prior art, and to provide a method for establishing the characteristic fingerprint spectrum of Jianpi Antai Granules and its fingerprint spectrum. This method enables multi-component fingerprint spectrum studies of Jianpi Antai Granules, attribute studies and similarity evaluations of major chromatographic peaks. The fingerprint spectrum comprehensively reflects the efficacy, uniformity and stability of Jianpi Antai Granules, which is beneficial for conducting pharmacodynamic studies and safety assessments related to the endocrine and immune microenvironment of Jianpi Antai Granules. Furthermore, based on the existing quality standards, this invention adds thin-layer chromatographic qualitative identification of its indicator components and content determination of effective components, as well as determination of heavy metals, harmful elements and pesticide residues, thus establishing a more comprehensive quality standard control system.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0007] The method for establishing the characteristic fingerprint spectrum of Jianpi Antai Granules is characterized by the following steps.

[0008] S1. Preparation of the test solution: Weigh the Jianpi Antai granules, add distilled water, weigh the sample, extract with ultrasound, cool, weigh again, replenish the lost weight with distilled water, shake well, filter, and collect the filtrate to obtain the test solution.

[0009] S2. Preparation of mixed reference solution: Weigh out gallic acid, protocatechuic acid, paeoniflorin, verbascoside, hyperoside, baicalin and wedelia lactone reference standards respectively, dissolve them in 50% methanol solution to prepare a mixed reference solution.

[0010] S3. Determination of chromatographic conditions: Select a chromatographic column; Mobile phase: 0.1% formic acid aqueous solution as mobile phase A, methanol as mobile phase B, gradient elution, mobile phase flow rate 0.8-1.2 mL / min, detection wavelength 210-300 nm, column temperature 25-35 ℃, injection volume 20 μL.

[0011] S4. The test solution and the reference solution are analyzed and compared according to the chromatographic conditions of step S3 to obtain the characteristic fingerprint spectrum of Jianpi Antai Granules composed of common peaks.

[0012] Furthermore, a precision test was conducted to investigate the methodology of the proposed chromatographic conditions: a sample of Jianpi Antai Granules was taken, a test solution was prepared according to step S1, and then the sample was injected 6 times consecutively according to the chromatographic conditions of step S3. The characteristic fingerprint chromatogram of Jianpi Antai Granules was recorded, a reference peak was selected, and the RSD values ​​of the relative retention time and relative peak area of ​​the common peaks were calculated.

[0013] Furthermore, a stability test was conducted to investigate the methodology of the proposed chromatographic conditions: Jianpi Antai Granules samples were taken, and test solutions were prepared according to step S1. The solutions were placed at room temperature for 0, 2, 4, 8, 10, and 12 hours, respectively. Then, the samples were injected and analyzed according to the chromatographic conditions in step S3. The characteristic fingerprint chromatograms of Jianpi Antai Granules were recorded, a reference peak was selected, and the relative retention time and relative peak area of ​​the common peaks were calculated.

[0014] Furthermore, a repeatability test was conducted to investigate the methodology of the proposed chromatographic conditions: six test solutions of Jianpi Antai Granules were prepared in parallel according to step S1, and then injected and analyzed according to the chromatographic conditions of step S3. The characteristic fingerprint chromatogram of Jianpi Antai Granules was recorded, a reference peak was selected, and the RSD values ​​of the relative retention time and relative peak area of ​​the common peaks were calculated.

[0015] Furthermore, the chromatographic column used was an Agilent ZORBAX SB-C. 18 The chromatographic column has dimensions of 4.6 mm × 250 mm and a particle size of 5 μm.

[0016] Furthermore, the mobile phase flow rate was 0.8 mL / min, the detection wavelength was 254 nm, and the column temperature was 30 °C.

[0017] Furthermore, in step S3, the gradient elution procedure is as follows: .

[0018] Furthermore, 10 batches of Jianpi Antai Granules samples, batch numbers S1 to S10, were taken. Test solutions were prepared according to step S1, and then analyzed under the chromatographic conditions of step S3. Characteristic fingerprint spectra of the 10 batches of Jianpi Antai Granules were obtained. These characteristic fingerprint spectra of the 10 batches of Jianpi Antai Granules were sequentially imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012" software in AIA format for data analysis. The characteristic fingerprint spectra of batch S10 of Jianpi Antai Granules were set as the reference spectra. The characteristic fingerprint spectra of the 10 batches of Jianpi Antai Granules were automatically matched, and corrections were performed using 18 common peaks as correction points. After peak matching, a reference spectra of Jianpi Antai Granules was generated using the average method. Then, using peak number 15 as the reference peak, the components corresponding to the common peaks were identified using the reference standard method. The common peaks were identified by comparing the peak retention time and chromatographic behavior with the characteristic fingerprint spectra of the mixed reference standard solution of Jianpi Antai Granules.

[0019] Furthermore, the characteristic fingerprint data of 10 batches of Jianpi Antai Granules were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012" software. The similarity was evaluated between the characteristic fingerprint of Jianpi Antai Granules of the test solution and the control fingerprint of Jianpi Antai Granules by calculating the similarity.

[0020] The characteristic fingerprint spectrum of Jianpi Antai Granules is characterized by having 18 common peaks; among them, peak 3 is gallic acid, peak 7 is protocatechuic acid, peak 11 is paeoniflorin, peak 12 is verbascoside, peak 14 is hyperoside, peak 15 is baicalin, and peak 17 is wedelia lactone.

[0021] The present invention has the following beneficial effects due to the adoption of the above technical solution.

[0022] (1) This invention can conduct multi-component fingerprint spectroscopy studies on Jianpi Antai Granules, conduct attribution studies and similarity evaluations of the main chromatographic peaks, and the fingerprint spectroscopy comprehensively reflects the effectiveness, uniformity and stability of Jianpi Antai Granules. It is beneficial to conduct pharmacodynamic studies and safety assessments of Jianpi Antai Granules related to the endocrine and immune microenvironment. Furthermore, it adds thin-layer chromatographic qualitative identification of its indicator components and content determination of effective components to the existing quality standards, and determines the content of heavy metals, harmful elements and pesticide residues, thus establishing a more complete quality standard control system.

[0023] (2) In the chromatographic conditions of this invention, the detection wavelength of the characteristic fingerprint spectrum of Jianpi Antai Granules is selected as 254nm. The fingerprint spectrum baseline is stable, the number of fingerprint spectrum peaks is large, and the peak shape is good.

[0024] The mobile phases used for the characteristic fingerprint chromatogram of Jianpi Antai Granules were: 0.1% formic acid aqueous solution as mobile phase A and methanol as mobile phase B. The characteristic fingerprint chromatogram of Jianpi Antai Granules showed better peak separation, better peak shape, and a more stable baseline.

[0025] The column temperature for the characteristic fingerprint spectrum of Jianpi Antai Granules was selected at 30℃, and the peak separation of the characteristic fingerprint spectrum of Jianpi Antai Granules was good.

[0026] The flow rate for the characteristic fingerprint chromatogram of Jianpi Antai Granules was selected as 0.8 mL / min, and the peak separation of the characteristic fingerprint chromatogram of Jianpi Antai Granules was good. Attached Figure Description

[0027] The present invention will be further described below with reference to the accompanying drawings: The present invention will be further described below with reference to the accompanying drawings: Figure 1 (1) is the Agilent ZORBAX SB-C in this invention. 18 The characteristic fingerprint chromatogram of Jianpi Antai Granules corresponding to the chromatographic column; Figure 1 (2) is the Agilent Eclipse XDB-C in this invention. 18 The characteristic fingerprint chromatogram of Jianpi Antai Granules corresponding to the chromatographic column; Figure 1 (3) is Grace Alltima C in this invention. 18 The characteristic fingerprint chromatogram of Jianpi Antai Granules corresponding to the chromatographic column; Figure 2 (1) is the characteristic fingerprint spectrum of the Jianpi Antai Granules at a wavelength of 210nm in this invention; Figure 2 (2) is the characteristic fingerprint spectrum of the Jianpi Antai Granules at a wavelength of 230nm in this invention; Figure 2(3) is the characteristic fingerprint spectrum of the Jianpi Antai Granules at a wavelength of 240nm in this invention; Figure 3 (4) is the characteristic fingerprint spectrum of the Jianpi Antai Granules at a wavelength of 254nm in this invention; Figure 3 (5) is the characteristic fingerprint spectrum of the Jianpi Antai Granules at a wavelength of 260nm in this invention; Figure 3 (6) is the characteristic fingerprint spectrum of the Jianpi Antai Granules at a wavelength of 280nm in this invention; Figure 3 (7) is the characteristic fingerprint spectrum of the Jianpi Antai Granules at a wavelength of 300nm in this invention; Figure 4 (1) is the characteristic fingerprint spectrum of Jianpi Antai Granules in this invention, with 0.1% formic acid water as mobile phase A and methanol as mobile phase B; Figure 4 (2) is the characteristic fingerprint spectrum of Jianpi Antai Granules in this invention, with 0.1% formic acid water as mobile phase A and acetonitrile as mobile phase B; Figure 4 (3) is the characteristic fingerprint spectrum of Jianpi Antai Granules in this invention with 0.1% phosphoric acid water as mobile phase A and methanol as mobile phase B; Figure 4 (4) is the characteristic fingerprint spectrum of Jianpi Antai Granules in this invention with 0.1% phosphoric acid water as mobile phase A and acetonitrile as mobile phase B; Figure 5 (1) is the characteristic fingerprint spectrum of the Jianpi Antai Granules at a column temperature of 25℃ in this invention; Figure 5 (2) is the characteristic fingerprint spectrum of the Jianpi Antai Granules at a column temperature of 30℃ in this invention; Figure 5 (3) is the characteristic fingerprint spectrum of the Jianpi Antai Granules at a column temperature of 35℃ in this invention; Figure 6 (1) is the characteristic fingerprint spectrum of the Jianpi Antai Granules at a flow rate of 0.8 mL / min in this invention; Figure 6 (2) is the characteristic fingerprint spectrum of the Jianpi Antai Granules at a flow rate of 1.0 mL / min in this invention; Figure 6 (3) is the characteristic fingerprint spectrum of the Jianpi Antai Granules at a flow rate of 1.2 mL / min in this invention; Figure 7 (1) is the characteristic fingerprint spectrum of the Jianpi Antai Granules extracted by distilled water in this invention; Figure 7(2) is the characteristic fingerprint spectrum of Jianpi Antai Granules extracted by the present invention with 10% methanol as the solvent; Figure 7 (3) is the characteristic fingerprint spectrum of Jianpi Antai Granules extracted by selecting 20% ​​methanol as the solvent in this invention; Figure 7 (4) is the characteristic fingerprint spectrum of Jianpi Antai Granules extracted by the present invention with 30% methanol as the solvent; Figure 8 (5) is the characteristic fingerprint spectrum of Jianpi Antai Granules extracted by selecting 40% methanol as the solvent in this invention; Figure 8 (6) is the characteristic fingerprint spectrum of Jianpi Antai Granules extracted by selecting 50% methanol as the solvent in this invention; Figure 8 (7) is the characteristic fingerprint spectrum of Jianpi Antai Granules extracted by 50% ethanol as the solvent in this invention; Figure 9 (1) is the characteristic fingerprint spectrum of 10 batches of Jianpi Antai Granules in this invention; Figure 9 (2) is a comparative chromatogram of the Jianpi Antai Granules in this invention; Figure 9 (3) is the characteristic fingerprint spectrum of the Jianpi Antai Granules in the mixed reference solution of the present invention. Detailed Implementation

[0028] This invention takes 10 batches of Jianpi Antai Granules samples, with batch numbers 20220626, 20220811, 20220923, 20221010, 20221019, 20221107, 20221115, 20221116, 20221117, and 20221118, and provides the following technical operations.

[0029] 1) Conduct a characteristic fingerprint study of Jianpi Antai Granules, screen factors such as chromatographic column, detection wavelength, mobile phase, column temperature, preparation method of test solution, and flow rate, and determine the construction conditions of fingerprint spectrum.

[0030] 2) Conduct a methodological review of the proposed fingerprint pattern construction conditions, and explain the reliability and feasibility of the method.

[0031] 3) Establish characteristic fingerprint and reference chromatograms of Jianpi Antai Granules and identify the main chromatographic peaks.

[0032] 4) Based on the comparison chromatogram, the similarity of each batch of Jianpi Antai Granules samples was evaluated.

[0033] 1. Instruments and reagents 1.1 Instruments The instruments used in the experiment are shown in Table 2-1:

[0034] 1.2 Reference Standards and Reagents The reference standards and reagents used in the experiment are shown in Table 2-2:

[0035] 2. Method for establishing the characteristic fingerprint spectrum of Jianpi Antai Granules 2.1 Preparation of the test solution 2.0 g of Jianpi Antai Granules was accurately weighed using an electronic balance and placed in a 250 mL stoppered conical flask. 100 mL of distilled water was added through an ultrapure water system, and the weight was determined. The flask was then ultrasonically extracted (200 W, 40 kHz) for 30 min. After cooling, the flask was weighed again. The weight loss was replenished with distilled water, and the flask was shaken well. The solution was then filtered through a 0.22 μm microporous membrane, and the filtrate was collected to obtain the test solution.

[0036] 2.2 Preparation of mixed reference solution Accurately weigh appropriate amounts of gallic acid, protocatechuic acid, paeoniflorin, verbascoflavonoid glucoside, hyperoside, baicalin, and wedelia lactone reference standards, dissolve them in 50% methanol solution, and prepare a mixed reference standard solution of appropriate concentration.

[0037] 2.3 Chromatographic conditions Using Agilent ZORBAX SB-C 18 The chromatographic column (4.6 mm × 250 mm, 5 μm) was used. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was methanol. Gradient elution was performed according to Table 2-3. The mobile phase flow rate was 0.8 mL / min, the detection wavelength was 254 nm, the column temperature was 30 ℃, and the injection volume was 20 μL.

[0038]

[0039] 2.4 Methodological Examination 2.4.1 Precision Test Take a sample of Jianpi Antai Granules (batch number: 20221118), prepare the test solution according to the preparation method of the test solution in 2.1, and inject it continuously 6 times with an autosampler under the chromatographic conditions in 2.3. Record the characteristic fingerprint spectrum of Jianpi Antai Granules. Using peak 15 (baicalin) as the reference peak, calculate the RSD values ​​of the relative retention time and relative peak area of ​​the 18 common peaks.

[0040] 2.4.2 Stability Test Take a sample of Jianpi Antai Granules (batch number: 20221118), prepare the test solution according to the steps in 2.1, and after standing at room temperature for 0, 2, 4, 8, 10 and 12 hours, inject and analyze according to the chromatographic conditions in 2.3. Record the characteristic fingerprint of Jianpi Antai Granules. Using peak 15 (baicalin) as the reference peak, calculate the RSD values ​​of the relative retention time and relative peak area of ​​the 18 common peaks.

[0041] 2.4.3 Repeatability Test Take a sample of Jianpi Antai Granules (batch number: 20221118), prepare 6 test solutions in parallel according to the preparation method of test solution in 2.1, and analyze them according to the chromatographic conditions in 2.3. Record the characteristic fingerprint spectrum of Jianpi Antai Granules, and use peak 15 (baicalin) as the reference peak to calculate the RSD values ​​of the relative retention time and relative peak area of ​​the 18 common peaks.

[0042] 2.5 Fingerprint Mapping Ten batches of Jianpi Antai Granules were prepared according to the preparation method of the test solution in 2.1. They were injected and analyzed according to the chromatographic conditions in 2.3. The chromatograms were recorded for 65 min, and the characteristic fingerprint chromatogram of Jianpi Antai Granules was established.

[0043] 2.6 Establishment of reference maps The characteristic fingerprint chromatograms of 10 batches of Jianpi Antai Granules were sequentially imported into the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" (2012 version) software in AIA format. The relevant parameters such as the number of peaks, peak area, and retention time of the test solutions of the 10 batches of Jianpi Antai Granules were analyzed. Compared with the other 9 batches of Jianpi Antai Granules, the characteristic fingerprint chromatogram of Jianpi Antai Granules (S10) with batch number 20221118 showed higher chromatographic peak separation and better peak shape of the main components. Therefore, the characteristic fingerprint chromatogram of Jianpi Antai Granules with batch number 20221118 was used as the reference chromatogram. The time window width was set to 0.1 s, and multi-point correction was performed. After peak matching, the control chromatogram was generated by the average method.

[0044] 2.7 Similarity Evaluation The similarity between the characteristic fingerprint of Jianpi Antai Granules and the control fingerprint of Jianpi Antai Granules was evaluated using the "Calculate Similarity" function in the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version) software. Specific Implementation

[0045] 1. Screening of chromatographic conditions 1.1 Selection of Chromatographic Column This invention examines the Agilent ZORBAX SB-C 18Chromatographic column (4.6 mm × 250 mm, 5 μm), Agilent Eclipse XDB-C 18 Chromatographic column (4.6 mm × 250 mm, 5 μm) and Grace Alltima C 18 Separation performance of the chromatographic column (4.6 mm × 250 mm, 5 μm).

[0046] Specifically, Agilent ZORBAX SB-C 18 The characteristic fingerprint of Jianpi Antai Granules corresponding to the chromatographic column is as follows: Figure 1 As shown in (1); Agilent Eclipse XDB-C 18 The characteristic fingerprint of Jianpi Antai Granules corresponding to the chromatographic column is as follows: Figure 1 As shown in (2); Grace Alltima C 18 The characteristic fingerprint of Jianpi Antai Granules corresponding to the chromatographic column is as follows: Figure 1 As shown in (3), the small image in the figure is a partial magnified view of the fingerprint spectrum.

[0047] The results showed that among the three different column types, the Agilent ZORBAX SB-C... 18 The chromatographic column produces sharp peaks with a large total amount of peak information and good resolution. Therefore, this type of chromatographic column was selected to construct the characteristic fingerprint spectrum of Jianpi Antai Granules.

[0048] 1.2 Selection of detection wavelength The detection wavelength is 210-300nm. This invention collects characteristic fingerprint spectra of Jianpi Antai Granules at different wavelengths of 210nm, 230nm, 240nm, 254nm, 260nm, 280nm and 300nm respectively.

[0049] Specifically, Figure 2 (1) is the characteristic fingerprint spectrum of Jianpi Antai Granules at a wavelength of 210 nm; Figure 2 (2) is the characteristic fingerprint spectrum of Jianpi Antai Granules at a wavelength of 230nm; Figure 2 (3) is the characteristic fingerprint spectrum of Jianpi Antai Granules at a wavelength of 240nm; Figure 3 (4) is the characteristic fingerprint spectrum of Jianpi Antai Granules at a wavelength of 254nm; Figure 3 (5) is the characteristic fingerprint spectrum of Jianpi Antai Granules at a wavelength of 260nm; Figure 3 (6) is the characteristic fingerprint spectrum of Jianpi Antai Granules at a wavelength of 280nm; Figure 3 (7) is the characteristic fingerprint spectrum of Jianpi Antai Granules at a wavelength of 300nm.

[0050] The results showed that the baseline of the chromatograms at 210nm, 230nm, and 240nm was less stable, and the number of peaks at 260nm, 280nm, and 300nm was less, and the peak shape was not as good as that at 254nm. Therefore, 254nm was selected as the detection wavelength of the characteristic fingerprint spectrum of Jianpi Antai Granules.

[0051] 1.3 Selection of mobile phase This invention uses Agilent ZORBAX SB-C 18 The chromatographic column (4.6 mm × 250 mm, 5 μm) was used with a detection wavelength of 254 nm.

[0052] This invention examines the characteristic fingerprint spectrum of Jianpi Antai Granules under the following mobile phase: (1) 0.1% formic acid solution is used as mobile phase A, and methanol is used as mobile phase B. Figure 4 Middle (1); (2) 0.1% formic acid solution is used as mobile phase A, and acetonitrile is used as mobile phase B. Figure 4 (2) (3) 0.1% phosphoric acid solution was used as mobile phase A, and methanol was used as mobile phase B. Figure 4 (3) (4) 0.1% phosphoric acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B. Figure 4 (4) The inset image is a magnified view of the fingerprint spectrum, showing gradient elution performed using the aforementioned mobile phase.

[0053] The results showed that the number of peaks in the chromatograms obtained by gradient elution under methanol and acetonitrile conditions was similar. Considering the price factor of both, methanol, which is more affordable, was selected. Comparing the two mobile phase systems, 0.1% formic acid water (mobile phase A)-methanol (mobile phase B) and 0.1% phosphoric acid water (mobile phase A)-methanol (mobile phase B), the former showed better peak resolution, better peak shape, and a more stable baseline than the latter. Therefore, 0.1% formic acid water was selected as mobile phase A and methanol as mobile phase B as the mobile phase for the characteristic fingerprint chromatogram of Jianpi Antai Granules.

[0054] 1.4 Column Temperature Selection The column temperature is 25–35°C. This invention uses an Agilent ZORBAX SB-C. 18 A chromatographic column (4.6 mm × 250 mm, 5 μm) was used with a detection wavelength of 254 nm. The column temperature was controlled by the column oven of the high performance liquid chromatograph, and the effects of different column temperatures (25℃, 30℃, and 35℃) on the separation of chromatographic peaks were compared.

[0055] Specifically, Figure 5 (1) is the characteristic fingerprint spectrum of Jianpi Antai Granules at a column temperature of 25℃; Figure 5(2) is the characteristic fingerprint spectrum of Jianpi Antai Granules at a column temperature of 30℃; Figure 5 (3) is the characteristic fingerprint spectrum of Jianpi Antai Granules at a column temperature of 35℃. In the figure, the small figure is a partial magnified view of the fingerprint spectrum.

[0056] The results showed that column temperature had little effect on the fingerprint spectrum, but 30℃ was slightly better. Therefore, 30℃ was selected as the column temperature for constructing the characteristic fingerprint spectrum of Jianpi Antai Granules.

[0057] 1.5 Flow rate selection The mobile phase flow rate is 0.8–1.2 mL / min. This invention uses Agilent ZORBAX SB-C. 18 A chromatographic column (4.6 mm × 250 mm, 5 μm) was used with a detection wavelength of 254 nm. The flow rate was controlled by a quaternary pump of the high-performance liquid chromatograph, and the effects of three different flow rates (0.8 mL / min, 1.0 mL / min, and 1.2 mL / min) on chromatographic separation were compared.

[0058] Specifically, Figure 6 (1) is the characteristic fingerprint spectrum of Jianpi Antai Granules at a flow rate of 0.8 mL / min; Figure 6 (2) is the characteristic fingerprint spectrum of Jianpi Antai Granules at a flow rate of 1.0 mL / min; Figure 6 In the middle (3), the fingerprint spectrum of Jianpi Antai Granules at a flow rate of 1.2 mL / min is shown. In the figure, the small figure is a partial magnified view of the fingerprint spectrum.

[0059] The results showed that the chromatographic peaks were well separated at 0.8 mL / min, so 0.8 mL / min was selected as the flow rate used to construct the characteristic fingerprint of Jianpi Antai Granules.

[0060] By optimizing the above influencing factors, the chromatographic conditions for constructing the characteristic fingerprint of Jianpi Antai Granules were determined as follows: Column: Agilent ZORBAX SB-C 18 Chromatographic column; Mobile phase: 0.1% formic acid aqueous solution as mobile phase A, methanol as mobile phase B; Detection wavelength: 254 nm; column temperature: 30 ℃; flow rate: 0.8 mL / min; injection volume: 20 μL.

[0061] 2. Preparation of the test solution 2.1 Selection of extraction solvent Take approximately 2.0 g of Jianpi Antai Granules sample (batch number: 20220626), weigh 7 portions in parallel, and place them in 250 mL stoppered conical flasks. Add 100 mL each of distilled water, 10% methanol, 20% methanol, 30% methanol, 40% methanol, 50% methanol, and 50% ethanol respectively, weigh them, and extract by ultrasonication (200 W, 40 KHz) for 30 min. After cooling, weigh them again, replenish the lost weight with the extraction solvent, shake well, filter through a 0.22 μm microporous membrane, and collect the filtrate to obtain the final product.

[0062] The sample was analyzed by high performance liquid chromatography under the chromatographic conditions described above for constructing the characteristic fingerprint of Jianpi Antai Granules. The concentration and relative content of each component in the sample were accurately measured by a DAD detector.

[0063] Specifically, Figure 7 (1) The characteristic fingerprint spectrum of Jianpi Antai Granules under distilled water as the extraction solvent; Figure 7 (2) The characteristic fingerprint spectrum of Jianpi Antai Granules was selected as the extraction solvent with 10% methanol. Figure 7 (3) The characteristic fingerprint spectrum of Jianpi Antai Granules was selected as the extraction solvent under 20% methanol. Figure 7 (4) The characteristic fingerprint spectrum of Jianpi Antai Granules was selected as the extraction solvent under 30% methanol. Figure 8 (5) The characteristic fingerprint spectrum of Jianpi Antai Granules was selected as the extraction solvent under 40% methanol. Figure 8 (6) The characteristic fingerprint spectrum of Jianpi Antai Granules was selected as the extraction solvent under 50% methanol. Figure 8 (7) is the characteristic fingerprint spectrum of Jianpi Antai Granules under 50% ethanol as the extraction solvent; in the figure, the small figure is a partial magnified view of the fingerprint spectrum.

[0064] The results showed that using 50% ethanol as the extraction solvent resulted in fewer peaks and an unstable baseline in the chromatogram. As the proportion of methanol in the extraction solvent decreased, the peak shape gradually improved, the number of peaks gradually increased, and the sample information became richer. Therefore, distilled water was chosen as the extraction solvent.

[0065] 3. Methodological Examination 3.1 Precision Test The relative retention times results of the precision test are shown in Table 3-1:

[0066] The relative peak area results are shown in Table 3-2:

[0067] The relative retention times (RSD) of the 18 common peaks in the test solution were between 0.003% and 0.033%, and the relative peak areas (RSD) were between 0.106% and 2.728%, indicating that the instrument was stable and met the requirements of the precision test.

[0068] 3.2 Stability Test The relative retention times from the stability tests are shown in Table 3-3:

[0069] The relative peak area results are shown in Table 3-4:

[0070] The relative retention time RSDs of the 18 common peaks in the test solution ranged from 0.003% to 0.160%, and the relative peak area RSDs ranged from 0.716% to 5.458%. Among them, peak 14 (hyperoside) had a relatively small peak area, resulting in a relative peak area RSD of 5.458%. The RSDs of the relative peak areas of the remaining common peaks were all less than 3%, indicating that the sample solution was relatively stable within 12 hours.

[0071] 3.3 Repeatability Test The relative retention times of the repeatability tests are shown in Table 3-5:

[0072] The relative peak area results are shown in Table 3-6:

[0073] The relative retention times (RSDs) of the 18 common peaks in the test solution ranged from 0.006% to 0.499%, and the relative peak areas (RSDs) ranged from 0.115% to 2.641%, indicating that the method has good repeatability and meets the requirements for fingerprint pattern construction.

[0074] 4 Sample Analysis 4.1 Establishment of the characteristic fingerprint spectrum of Jianpi Antai Granules Ten batches of Jianpi Antai Granules samples were collected. Test solutions were prepared according to the above-described method, and analyzed under the above chromatographic conditions. Characteristic fingerprint chromatograms of the ten batches of Jianpi Antai Granules were obtained. Data analysis was performed using the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine" (2012 version). The characteristic fingerprint chromatogram of Jianpi Antai Granules with batch number 20221118 (S10) was set as the reference chromatogram. The fingerprint chromatograms of the ten batches were automatically matched. After multi-point correction, the results are as follows: Figure 9 As shown in (1).

[0075] 4.2 Establishment of a comparative chromatogram for Jianpi Antai Granules The characteristic fingerprint chromatograms of 10 batches of Jianpi Antai Granules were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) software. Using 18 common peaks as correction points, a control chromatogram for Jianpi Antai Granules was established. The results are as follows: Figure 9 As shown in (2).

[0076] 4.3 Common Peak Identification Using peak 15 (baicalin) as a reference peak, the components corresponding to the common peaks were identified using the reference standard method. By comparing the retention times and chromatographic behavior of the characteristic fingerprint chromatograms of Jianpi Antai Granules with those of the mixed reference standard solution, a total of 7 chromatographic peaks were identified: peak 3 was gallic acid, peak 7 was protocatechuic acid, peak 11 was paeoniflorin, peak 12 was verbascoside glucoside, peak 14 was hyperoside, peak 15 was baicalin, and peak 17 was wedelia lactone. The characteristic fingerprint chromatogram of the mixed reference standard solution of Jianpi Antai Granules is shown below. Figure 9 As shown in (3).

[0077] 4.4 Similarity Evaluation Similarity was calculated using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 edition) issued by the Chinese Pharmacopoeia Commission. The HPLC chromatogram data of 10 batches of Jianpi Antai Granules were imported, and the similarity was calculated (see Table 3-7). The similarity of all 10 batches of samples reached above 0.998, indicating that the batches of Jianpi Antai Granules were homogeneous in texture and had good similarity.

[0078]

[0079] This invention enables multi-component fingerprint spectroscopy studies of Jianpi Antai granules, attribute analysis and similarity evaluation of major chromatographic peaks. The fingerprint spectroscopy comprehensively reflects the efficacy, uniformity and stability of Jianpi Antai granules, which is beneficial for endocrine and immune microenvironment-related pharmacodynamic studies and safety assessments. Furthermore, it adds thin-layer chromatographic qualitative identification of indicator components and content determination of effective components to the existing quality standards, and determines heavy metals, harmful elements and pesticide residues, thus establishing a more comprehensive quality standard control system.

[0080] The above are merely specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Any simple changes, equivalent substitutions, or modifications made based on the present invention to solve essentially the same technical problems and achieve essentially the same technical effects are all covered within the protection scope of the present invention.

Claims

1. A method for establishing the characteristic fingerprint spectrum of Jianpi Antai Granules, characterized in that, Includes the following steps: S1. Preparation of the test solution: Weigh out the Jianpi Antai granules, add distilled water, weigh them, extract with ultrasound, cool, weigh them again, replenish the lost weight with distilled water, shake well, filter, and take the filtrate to obtain the test solution. S2. Preparation of mixed reference solution: Weigh out gallic acid, protocatechuic acid, paeoniflorin, verbascoflavonoid glucoside, hyperoside, baicalin and wedelia lactone reference standards respectively, dissolve them in 50% methanol solution to prepare a mixed reference standard solution; S3. Determination of chromatographic conditions: Column selection: The column used is an Agilent ZORBAX SB-C. 18 The chromatographic column has dimensions of 4.6 mm × 250 mm and a particle size of 5 μm. Mobile phase: 0.1% formic acid aqueous solution as mobile phase A, methanol as mobile phase B, gradient elution, the gradient elution procedure is as follows: ； The mobile phase flow rate was 0.8 mL / min, the detection wavelength was 254 nm, the column temperature was 30 °C, and the injection volume was 20 μL. S4. The test solution and the reference solution are analyzed and compared according to the chromatographic conditions of step S3. Peak 15 is used as the reference peak, which is the baicalin peak. The characteristic fingerprint spectrum of Jianpi Antai Granules composed of common peaks is obtained.

2. The method for establishing the characteristic fingerprint spectrum of Jianpi Antai Granules according to claim 1, characterized in that: The proposed chromatographic conditions were investigated using a precision test: Jianpi Antai Granules sample was taken, and the test solution was prepared according to step S1. Then, the sample was injected 6 times consecutively according to the chromatographic conditions of step S3. The characteristic fingerprint chromatogram of Jianpi Antai Granules was recorded. Peak 15 was used as the reference peak, and the relative retention time and relative peak area of ​​the common peaks were calculated.

3. The method for establishing the characteristic fingerprint spectrum of Jianpi Antai Granules according to claim 1, characterized in that: Stability tests were conducted to investigate the methodology of the proposed chromatographic conditions: Jianpi Antai Granules samples were taken, and test solutions were prepared according to step S1. The test solutions were placed at room temperature, and samples were taken at 0, 2, 4, 8, 10, and 12 hours. The samples were then injected and analyzed according to the chromatographic conditions of step S3. The characteristic fingerprint chromatogram of Jianpi Antai Granules was recorded. Peak 15 was used as the reference peak, and the relative retention time and relative peak area of ​​the common peaks were calculated.

4. The method for establishing the characteristic fingerprint spectrum of Jianpi Antai Granules according to claim 1, characterized in that: The proposed chromatographic conditions were investigated using repeatability tests: six test solutions of Jianpi Antai Granules were prepared in parallel according to step S1, and then injected and analyzed according to the chromatographic conditions of step S3. The characteristic fingerprint chromatogram of Jianpi Antai Granules was recorded. Peak 15 was used as the reference peak, and the relative retention time and relative peak area of ​​the common peaks were calculated.

5. The method for establishing the characteristic fingerprint spectrum of Jianpi Antai Granules according to claim 1, characterized in that: Ten batches of Jianpi Antai Granules samples, batch numbers S1 to S10, were collected. Test solutions were prepared according to step S1, and the samples were analyzed under the chromatographic conditions specified in step S3. Characteristic fingerprint chromatograms of the ten batches of Jianpi Antai Granules were obtained. These chromatograms were then sequentially imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012" software in AIA format for data analysis. The characteristic fingerprint chromatogram of batch S10 was set as the reference chromatogram. The characteristic fingerprint chromatograms of the ten batches of Jianpi Antai Granules were automatically matched, and corrections were performed using 18 common peaks as correction points. After peak matching, a reference chromatogram of Jianpi Antai Granules was generated using the average method. Peak 15 was used as the reference peak, and the components corresponding to the common peaks were identified using the reference standard method. The common peaks were identified by comparing the peak retention time and chromatographic behavior with the characteristic fingerprint chromatogram of the mixed reference standard solution of Jianpi Antai Granules.

6. The method for establishing the characteristic fingerprint spectrum of Jianpi Antai Granules according to claim 5, characterized in that: Data from the characteristic fingerprint chromatograms of 10 batches of Jianpi Antai Granules were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012" software. The similarity was evaluated between the characteristic fingerprint chromatogram of the test solution of Jianpi Antai Granules and the control chromatogram of Jianpi Antai Granules by calculating the similarity.

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