Jiangtangning granule fingerprint spectrum and multi-component quantitative quality control method

Abstract

The invention discloses a Jiangtangning granule fingerprint spectrum and a multi-component quantitative quality control method, and belongs to the technical field of pharmaceutical analysis. According to the method, high performance liquid chromatography is adopted, and fingerprint spectrum determination and quantitative analysis of 10 components are synchronously realized under the same chromatographic condition. The method comprises the following steps: preparing a test solution and a reference solution, carrying out gradient elution with an acetonitrile-phosphoric acid aqueous solution, and detecting under optimized chromatographic conditions. The fingerprint spectrum determines common peaks based on multiple batches of samples, and the similarity is greater than or equal to 0.9; 10 components such as calycosin-7-glucoside are measured at the same time, the separation degree is larger than or equal to 1.5, and the linear relation is good. The method solves the problems of few control indexes and low efficiency in the prior art, can comprehensively characterize the product quality, improve the detection efficiency and reduce the cost, and is suitable for quality control of the Huangtangning granules.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and specifically proposes a method for quality control of Jiangtangning granules fingerprint and multi-component quantification. Background Art

[0002] Jiangtangning Granules (National Medicine Approval No. Z20205004) are a traditional Chinese medicine compound preparation composed of seven herbs: astragalus root, rehmannia root, radix trichosanthis, schisandra chinensis, pseudoginseng, licorice, and pumpkin powder. They are said to invigorate qi and nourish yin, promote fluid production and quench thirst. Jinpai Zhizhengtang Pharmaceutical Co., Ltd. is the exclusive marketing authorization holder for this product. The active ingredients in Jiangtangning Granules are complex, including astragaloside, calycosin glucoside, calycosin, schisandrin A, schisandrin B, liquiritin, apigenin, glycyrrhizic acid, liquiritigenin, formononetin, and formononetin, making quality control challenging.

[0003] Chinese invention patent application CN1698705A discloses a method for preparing Jiangtangning tablets, describing the tablet preparation process but lacking multi-component quality control methods. Liu Yuan et al., in "Simultaneous Determination of Five Active Ingredients in Jiangtangning Capsules by HPLC and Cluster Analysis," describe a method for quantitative analysis of five components in Jiangtangning capsules. However, this method suffers from poor resolution of highly polar components, resulting in no product fingerprint being constructed. Furthermore, the number of quantitative components is relatively small, failing to capture key components such as schisandrin A, schisandrin B, glycyrrhizin, and glycyrrhizic acid.

[0004] The complex composition of the seven medicinal ingredients in the Jiangtangning preparation often makes it difficult to simultaneously characterize the fingerprint of the traditional Chinese medicine compound and perform multi-index content detection using a single chromatographic condition. This typically requires the development of multiple detection methods, which also leads to problems such as high workload and low efficiency. Furthermore, research on quality evaluation and control methods for Jiangtangning granules is relatively weak, with a limited number of controlled indicators, hindering comprehensive quality evaluation of the product.

[0005] Therefore, it is urgent to conduct research on the intrinsic quality of Jiangtangning Granules and to construct a quality control method that can simultaneously realize the fingerprint of Jiangtangning Granules and multi-component quantification. Summary of the Invention

[0006] In light of this, the present invention proposes an analytical method that can simultaneously perform fingerprint analysis and multi-index quantification under the same chromatographic conditions, thereby improving the quality control of Jiangtangning Granules and addressing the lack of comprehensive quality control methods for Jiangtangning Granules in the prior art. The established method is stable and efficient, requiring only a single injection, and can quantitatively analyze all ten index components of Jiangtangning Granules. It also allows for control of the overall quality attributes of the chemical composition of Jiangtangning Granules through fingerprint analysis, which is of great significance for improving the quality control of the Jiangtangning Granules described herein.

[0007] The technical scheme of the present application is implemented as follows: the present application provides a method for quality control of Zangneng granules fingerprint and multi-component quantification, comprising the following steps: (1) Preparation of test sample solution: take the Zangneng granules to be tested, add an extraction solvent to extract, filter, and take the filtrate to obtain the test sample solution; (2) Preparation of control sample solution: take calycosin-7-glucoside, formononetin, ononin, glycyrrhizin, glycyrrhizic acid, calycosin, formononetin, glycyrrhizin, schizandrin A, and schizandrin B to prepare a mixed control sample solution; (3) Chromatographic analysis conditions: use a chromatographic column filled with octadecylsilane-bonded silica gel, gradient elution with acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B, and sample injection detection by a UV detector; (4) Fingerprint development: respectively take the test sample solution and the control sample solution, inject them into the HPLC chromatograph, and integrate the minimum peak area ≥ 0.5‰ total peak area, to obtain the fingerprint according to the common peaks in the spectra measured from multiple batches of test samples, wherein the number of the multiple batches of test samples is ≥ 10 batches, and the multiple batches of test samples cover typical variations in raw material sources and production processes; (5) Multi-component quantitative analysis: select 10 components with a separation degree (Ri) ≥ 1.5, i.e. calycosin-7-glucoside, formononetin, ononin, glycyrrhizin, glycyrrhizic acid, calycosin, formononetin, glycyrrhizin, schizandrin A, and schizandrin B, for content determination, and perform product quality control through the content of each component.

[0008] In some embodiments, the extraction solvent is at least one of water, methanol, and ethanol.

[0009] In some embodiments, the extraction solvent is methanol.

[0010] In some embodiments, the ratio of the mass of the test sample to the volume of the extraction solvent used is (1-4):(12.5-50), and the ratio unit is g / ml.

[0011] In some embodiments, the extraction step uses heated reflux extraction, the reflux temperature is 75-85°C, and the extraction time is 30-60 min.

[0012] In some embodiments, in step (2), the control sample solution contains: calycosin-7-glucoside 150-250 μg per 1 ml of the control sample solution; and / or, calycosin 100-200 μg per 1 ml of the control sample solution; and / or, Each 1 ml of the reference solution contains 500-1500 μg of liquiritin; and / or Each 1 ml of the reference solution contains 50-150 μg of liquiritin; and / or, Each 1 ml of the reference solution contains 50-150 μg of formononetin; and / or Each 1 ml of the reference solution contains 2-20 μg of formononetin; and / or, Each 1 ml of the reference solution contains 2-20 μg of glycyrrhizic acid; and / or, Each 1ml of the reference solution contains 10-50μg of glycyrrhizin; and / or, Each 1ml of the reference solution contains 10-50μg of schisandrin A; and / or, Each 1 ml of the reference solution contains 50-200 μg of schisandra chinensis ethyl.

[0013] In some embodiments, the mobile phase system was investigated, and the results showed that when acetonitrile-phosphoric acid aqueous solution was used as the mobile phase system, the separation effect of the target components in the chromatogram was better, the number of chromatographic peaks of methanol-phosphoric acid aqueous solution was small and the separation effect was poor, and the baseline drift of the acetonitrile-formic acid aqueous solution system was serious. Therefore, acetonitrile is preferably used as mobile phase A and phosphoric acid aqueous solution is used as mobile phase B for gradient elution.

[0014] In some embodiments, the chromatographic conditions in step (3) include at least one of the following conditions: The phosphoric acid concentration in mobile phase B is 0.05%-0.2%, preferably, the phosphoric acid concentration is 0.1%; The mobile phase flow rate is 0.8-1.2 ml / min, preferably, the mobile phase flow rate is 1.0 ml / min; The column oven temperature is 25-35 ° C, preferably, the column oven temperature is 30 ° C; The injection volume is 5-20 μL, preferably, the injection volume is 10 μL; The detection wavelength is 220-260 nm, preferably, the detection wavelength is 237 nm; The column length of the chromatographic column is 100-300 mm, the inner diameter is 3.0-5.0 mm, and the particle size is 3.5-10 μm. Preferably, the chromatographic column model is SB-Aq, with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0015] In some embodiments, in step (3), the gradient elution conditions are: from 0 to 18 min, the volume ratio of mobile phase B is reduced from 99% to 81%; from 18 to 40 min, the volume ratio of mobile phase B is reduced from 81% to 60%; from 40 to 55 min, the volume ratio of mobile phase B is reduced from 60% to 46%; from 55 to 60 min, the volume ratio of mobile phase B is reduced from 46% to 5%.

[0016] In some embodiments, in step (4), the fingerprint is measured based on 15 batches of test samples, and the control fingerprint is generated by fitting the fingerprints of the 15 batches of test samples by the mean method. In the fingerprint, the similarity between the common peak used to evaluate the similarity and the control fingerprint is not less than 0.9, and the proportion of peaks with a separation degree ≥1.5 in the common peak is not less than 70%.

[0017] In some embodiments, under the above-mentioned chromatographic conditions, the separation effect of the 10 components in step (5), namely, peak 11 (calycosin isoflavone glucoside), peak 13 (glycyrrhizin), peak 14 (glycyrrhizin), peak 16 (formononetin), peak 21 (calycosin isoflavone), peak 22 (glycyrrhizin), peak 25 (glycyrrhizic acid), peak 26 (formononetin), peak 27 (schisandrin A), and peak 28 (schisandrin B), is good, with good linear relationship and quantitative limit, and can be used for content determination.

[0018] The present invention has the following beneficial effects compared to the prior art: (1) Provide a more comprehensive and reliable quality control method for Jiangtangning Granules: By combining fingerprint spectrum with multi-index component content determination method, the quality of the drug can be described and evaluated as a whole from the two aspects of fingerprint spectrum and multi-component quantification, which can achieve more comprehensive and reliable quality control of Jiangtangning Granules and improve the quality control level.

[0019] (2) High detection efficiency and low cost: One test can simultaneously achieve quantitative analysis of 10 components, effectively avoiding the need to use different methods to detect components in batches. On the premise of improving the quality control level, it can significantly shorten the detection time, greatly improve the detection efficiency, and reduce the cost of labor, reagents, etc.

[0020] (3) Simple and fast operation: Through the optimization of chromatographic conditions, quantitative analysis and fingerprint analysis are determined at one time using the same method, which is simple, fast and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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 or the description of the prior art. 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.

[0022] Figure 1 The fingerprints of 15 batches of Jiangtangning granules in Example 3 of the present invention are as follows; Figure 2 This is the reference fingerprint of Jiangtangning Granules obtained in Example 3 of the present invention; Figure 3The chromatogram of the mixed control sample for content determination of the embodiment 4 of the present application, wherein peak 1 is calycosin-7-glucoside, peak 2 is licuraside, peak 3 is glycyrrhizin, peak 4 is ononin, peak 5 is calycosin, peak 6 is glycyrrhizinic acid, peak 7 is glycyurin, peak 8 is formononetin, peak 9 is schisandrin A, and peak 10 is schisandrin B; Figure 4 The chromatogram of the test sample for content determination of the embodiment 5 of the present application, wherein peak 1 is calycosin-7-glucoside, peak 2 is licuraside, peak 3 is glycyrrhizin, peak 4 is ononin, peak 5 is calycosin, peak 6 is glycyrrhizinic acid, peak 7 is glycyurin, peak 8 is formononetin, peak 9 is schisandrin A, and peak 10 is schisandrin B; Figure 5 The 3D graph of the full wavelength scanning in the embodiment 1 of the present application; Figure 6 The chromatogram of the process of the mobile phase system research in the embodiment 1 of the present application; Figure 7 The chromatogram of the process of the research on the preparation method of the test sample solution in the embodiment 1 of the present application; Figure 8 The chromatogram of the process of the research on the flow rate in the embodiment 1 of the present application; Figure 9 The chromatogram of the process of the research on the column temperature in the embodiment 1 of the present application; Figure 10 The chromatogram of the process of the research on the injection volume in the embodiment 1 of the present application; Figure 11 The chromatogram of the Zangtangning in the comparative example 1 of the present application; Figure 12 The chromatogram of the Zangtangning in the comparative example 2 of the present application; Figure 13 The chromatogram of the Zangtangning in the comparative example 3 of the present application; Figure 14 The chromatogram of the Zangtangning in the comparative example 4 of the present application. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, all the other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0025] Unless otherwise specified, the methods used in the following examples are conventional methods. The materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art, unless otherwise specified, and can be obtained commercially by those skilled in the art.

[0026] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value with any lower range limit or preferred value, regardless of whether the range is disclosed alone. For example, when a range "1 to 5" is disclosed, the described range should be interpreted as including the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within the range. In the present specification and claims, range definitions may be combined and / or interchanged, and if not otherwise stated, such ranges include all subranges contained therein.

[0027] Example 1 Fingerprint Detection Method for Jiangtangning Granules Take about 4 g of the powder of the Jiangtangning granules to be tested, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of methanol, stopper it, weigh it, heat and reflux it for 60 minutes, take it out, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0028] Prepare 0.05-0.5 mg / mL mixed reference solution of calycosin glucoside, apigenin, liquiritin, formononetin, calycosin, liquiritigenin, ammonium glycyrrhizate, formononetin, schisandrin A and schisandrin B for qualitative analysis.

[0029] Octadecylsilane bonded silica gel was used as the filler; gradient elution was performed using acetonitrile (A)-0.1% phosphoric acid solution (B) as specified in the table below; the detection wavelength was 237 nm, the column was an SB-AqC18 (250 mm × 4.6 mm, 5 μm), the column temperature was 30°C, and the flow rate was 1.0 ml / min. The theoretical plate number calculated based on calycosin glucoside should be no less than 3000.

[0030] Table 1 HPLC gradient elution conditions of JTNKL01 granules

[0031] Respectively, 10 μl of the control solution and the test solution were precisely taken and injected into the liquid chromatograph for determination, and the chromatogram was recorded.

[0032] The chromatographic conditions were determined by multi-parameter optimization, wherein the detection wavelength was screened by full-wavelength scanning 3D graph (attached Figure 5 ), the response values of each component were balanced at 237 nm, and the interference was less; the mobile phase system was determined by acetonitrile-carbonic acid aqueous solution, acetonitrile-water comparative test (attached Figure 6 ), acetonitrile-0.1% phosphoric acid aqueous solution was the optimal system; the test sample preparation method was verified by chromatogram comparison of methanol heating reflux extraction, ethanol reflux extraction and the like (attached Figure 7 ), and the effectiveness of methanol heating reflux extraction was verified; the flow rate, column temperature and injection volume were respectively investigated at 0.8-1.2 ml / min (attached Figure 8 ), 25-35℃ (attached Figure 9 ), 5-20 μL (attached Figure 10 ), and 1.0 ml / min, 30℃, 10 μL were determined as the optimal parameters.

[0033] Example 2 Methodology investigation of JTNKL01 granule fingerprint Precision: about 4 g of JTNKL01 granule powder of the same batch was precisely weighed, and the preparation of the test sample solution and the chromatographic condition method experiment were carried out according to the preparation of the test sample solution and the chromatographic condition method experiment in Example 1, and the sample was continuously injected for 6 times.

[0034] Repeatability: about 4 g of JTNKL01 granule powder of the same batch was precisely weighed, and 6 test sample solutions were prepared in parallel according to the preparation method of the test sample solution in Example 1, and the same chromatographic condition was determined.

[0035] Stability: about 4 g of JTNKL01 granule powder of the same batch was precisely weighed, and the preparation of the test sample solution and the chromatographic condition method experiment were carried out according to the preparation of the test sample solution and the chromatographic condition method experiment in Example 1, and the sample was injected for determination after being placed for 0, 2, 4, 8, 16 and 24 hours, respectively.

[0036] The precision, repeatability and stability methodology experiments were investigated with the control fingerprint as a reference, and the similarity evaluation methodology experiment was feasible. The experimental results showed that the similarity of the precision test was above 0.999, the similarity of the repeatability test was above 0.998, and the similarity of the stability test was above 0.998. The above results showed that the method was stable and feasible, and could be used for fingerprint analysis of the technical scheme. The detailed experimental results are shown in Table 2.

[0037] Table 2 Precision, repeatability, and stability test results (n=6)

[0038] Example 3 Fingerprint determination and common peak calibration of 15 batches of Jiangtangning granules Each herbal decoction piece in the formula was randomly combined into 15 batches according to the batch number. Equal amounts of each herbal decoction piece from each batch were weighed and extracted, concentrated, and granulated to prepare finished granules. Fifteen batches of Jiangtangning Granules were sampled and tested on an HPLC chromatograph according to the test sample preparation method and chromatographic conditions described in Example 1. Fingerprints of Jiangtangning Granules from different batches were obtained by integrating the total peak area with a minimum peak area ≥ 0.5‰. The 15 batches of chromatograms were imported into the 2012 version of the similarity software "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine". The mean method and multi-point correction were used for peak matching, and a total of 32 common peaks were obtained. Among them, 11 common peaks were identified by reference substances and mass spectrometry, namely peak 11 (calycosin isoflavone glucoside), peak 13 (apigenin), peak 14 (liquiritin), peak 16 (formononetin), peak 19 (isoliquiritin), peak 21 (calycosin isoflavone), peak 22 (liquiritigenin), peak 25 (glycyrrhizic acid), peak 26 (formononetin), peak 27 (schisandrin A), and peak 28 (schisandrin B). Finally, the reference fingerprint R was generated, as shown in Figure 2. Figure 1 、 Figure 2 The similarity matching results between the 15 batches of samples and the control fingerprints are shown in Table 3 below.

[0039] Table 3 Similarity matching results of fingerprints of 15 batches of samples

[0040] Example 4 Investigation of the methodological approach for determining the content of 10 characteristic components Linear range: A series of 10 reference sample solutions were prepared as follows: calycosin glucoside (1.96-98.20µg / mL), formononetin (1.92-96.11µg / mL), calycosin (1.74-86.95µg / mL), formononetin (1.72-86.21µg / mL), apiosylliquiritin (5.00-249.78µg / mL), liquiritin (4.95-247.52µg / mL), glycyrrhizic acid (2.86-143.15µg / mL), liquiritigenin (1.80-90.08µg / mL), schisandrin A (3.01-150.35µg / mL), and schisandrin B (0.64-31.83µg / mL). 10 μl of the above 10 reference sample mixed solutions were injected into the HPLC liquid chromatograph, and the test solution and chromatographic conditions were tested according to Example 1. The standard curve was drawn with the injection concentration as the horizontal axis X and the peak area as the vertical axis Y. The results are shown in Figure 1. Figure 3 As shown, all 10 components have good linear relationships within the investigated range, with correlation coefficients greater than 0.999. Detailed experimental results are shown in Table 4 below.

[0041] Precision: Take about 4 g of Jiangtangning Granules (JTNKL01) powder from the same batch, accurately weigh it, and experiment according to the preparation of the test solution and chromatographic conditions and methods under Example 1, and inject the sample continuously for 6 times.

[0042] Repeatability: Take about 4 g of Jiangtangning Granules (JTNKL01) powder from the same batch, weigh accurately, and prepare 6 test solutions in parallel according to the preparation method of the test solution in Example 1, and measure under the same chromatographic conditions as above.

[0043] Stability: Take about 4 g of Jiangtangning Granules (JTNKL01) powder from the same batch, weigh it accurately, and test it according to the preparation and chromatographic conditions of the test solution in Example 1. Samples are injected and measured after 0, 4, 8, 16, 24, and 48 hours after preparation.

[0044] The RSD values ​​of the peak areas of each compound were used to evaluate the feasibility of the precision, repeatability, and stability method. The experimental results showed that the method was stable and feasible and could be used to determine the contents of the 10 characteristic components of the technical solution of the present invention. Detailed experimental results are shown in Table 4 below.

[0045] Accuracy: 9 samples with known index component contents were taken and accurately weighed. The recovery rate of unequal additions was determined using the high, medium, and low (1:1.5, 1:1, and 1:0.5) concentration method. Three replicates were prepared for each concentration and measured according to the chromatographic conditions of Example 1. The average recovery and RSD of each component were calculated. The results are shown in Table 4.

[0046] Table 4 Methodological study results of 10 characteristic components

[0047] Example 5 Determination of 10 characteristic components in 15 batches of samples According to the established method, the preparation of the test solution and the chromatographic conditions under Example 1 were used to determine the contents of 10 characteristic components in 15 batches of samples. The chromatograms of the content determination of 15 batches of samples are shown in the attached Figure 4 , showing that the 10 characteristic components had good separation under the optimized chromatographic conditions (Ri≥1.5), which was consistent with the chromatographic parameters determined in Example 1 and the attached Figure 4 The separation effect was consistent with that of the control group, which verified the reliability of the quantitative analysis method. The results are shown in Table 5.

[0048] Table 5 Determination results of 10 characteristic components in 15 batches of samples

[0049] The above determination results show that the method for determining the content of 10 characteristic components is stable and feasible, with high selectivity, fast analysis speed and high sensitivity, which is of great significance for improving the quality control of the Jiangtangning granules described in the present invention.

[0050] Comparative Example 1 High-performance liquid chromatography (HPLC) conditions: Column: XSelect HSST3 (250 mm × 4.6 mm, 5 μm); Mobile phase: Acetonitrile (A) 0.1% phosphoric acid in water; Flow rate: 1.0 mL / min; Gradient elution conditions are shown in Table 1; Column temperature: 30°C; Injection volume: 10 μL; Detection wavelength: 260 nm. The same test solution and mixed reference working solution as in Example 1 were injected into the HPLC for determination, and the chromatograms were recorded. See Appendix. Figure 11 .

[0051] It can be proved that when the chromatographic column in the detection method provided by the present invention is replaced with other chromatographic columns, the multiple components of Jiangtangning granules cannot be effectively separated, thereby affecting the quantitative analysis of the components.

[0052] Comparative Example 2 Preparation method of test solution: take about 4g of the powder of the Jiangtangning granules to be tested, accurately weigh it, put it into a stoppered conical flask, accurately add 25ml of ethanol, seal it, weigh it, heat and reflux for 60 minutes, take it out, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate. In Example 1, the same chromatographic conditions were used for HPLC analysis, and the chromatogram was recorded. The peak height of each component was obviously low, indicating that the test sample component was not fully extracted. See the attached figure. Figure 12 .

[0053] It can be proved that replacing the extraction solvent methanol in the detection method provided by the present invention with other solvents cannot achieve sufficient extraction of multiple components in Jiangtangning granules and accurate control of their content.

[0054] Comparative Example 3 High performance liquid chromatography (HPLC) conditions: mobile phase: methanol (A) 0.1% phosphoric acid aqueous solution; flow rate: 1.0 mL / min; gradient elution conditions see Table 1; column temperature: 30°C; injection volume: 10 μL; detection wavelength: 260 nm (other conditions are the same as in Example 1). The same test solution as in Example 1 was injected into the HPLC for determination, and the chromatogram was recorded, see Appendix. Figure 13 .

[0055] It can be proved that replacing the mobile phase acetonitrile in the detection method provided by the present invention with methanol cannot achieve the elution and separation of multiple components in Jiangtangning granules.

[0056] Comparative Example 4 High-performance liquid chromatography (HPLC) conditions: Detection wavelength: 203 nm. Chromatographic column: XSelect HSST3 (250 mm × 4.6 mm, 5 μm); Mobile phase: Acetonitrile (A) 0.1% phosphoric acid aqueous solution; Flow rate: 1.0 mL min1; Gradient elution conditions are shown in Table 1; Column temperature: 30°C; Injection volume: 10 μL (other conditions are the same as in Example 1). The same test solution as in Example 1 was injected into the HPLC for determination, and the chromatogram was recorded. See Appendix. Figure 14 .

[0057] It can be proved that when the detection wavelength of 260 nm in the detection method provided by the present invention is replaced with other detection wavelengths, the separation of the chromatographic peaks of high-polarity components is poor, and the characterization and quantitative detection of multiple components in Jiangtangning granules cannot be achieved.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for fingerprint and multi-component quantitative quality control of Jiangtangning granules, characterized in that: Fingerprint construction and quantification of 10 index components were achieved simultaneously through a single HPLC injection, including the following steps: (1) Preparation of test solution: Take the Jiangtangning granules to be tested, add the extraction solvent to extract, filter, and take the filtrate to obtain the test solution; (2) Preparation of reference solution: Take calycosin glucoside, formononetin, apiosyl liquiritin, liquiritin, glycyrrhizic acid, calycosin, formononetin, liquiritin, schisandrin A, and schisandrin B to prepare a mixed reference solution; (3) Chromatographic analysis conditions: octadecylsilane bonded silica gel as the filler of the chromatographic column, acetonitrile as the mobile phase A and phosphoric acid aqueous solution as the mobile phase B for gradient elution, and ultraviolet detector for detection; (4) Preparation of fingerprint: aspirate the test sample solution and the reference sample solution separately, inject them into the HPLC chromatograph, integrate the minimum peak area ≥ 0.5‰ of the total peak area, and obtain the fingerprint based on the common peaks in the spectra measured by multiple batches of test samples, the number of which is ≥ 10 batches; (5) Multi-component quantitative analysis: 10 components with a separation degree (Ri) ≥ 1.5, namely, calycosin glucoside, formononetin, apiosyl liquiritin, liquiritin, glycyrrhizic acid, calycosin, formononetin, liquiritigenin, schisandrin A, and schisandrin B, were selected for content determination, and product quality control was performed based on the content of each component.

2. The method according to claim 1, wherein In step (1), the extraction solvent is at least one of water, methanol and ethanol, the material-liquid ratio of the mass of the test sample to the volume of the extraction solvent used is (1-4): (12.5-50) g / ml, the extraction method is heating reflux extraction, the reflux temperature is 75-85°C, and the extraction time is 30-60 min.

3. The method according to claim 1, wherein In step (2), each 1 ml of the reference solution contains 150-250 μg of calycosin glucoside, 100-200 μg of calycosin, 500-1500 μg of liquiritin, 50-150 μg of formononetin, 2-20 μg of formononetin, 2-20 μg of glycyrrhizic acid, 10-50 μg of glycyrrhizin, 10-50 μg of schisandrae alcohol A, and 50-200 μg of schisandrae alcohol B.

4. The method according to claim 1, wherein In step (3), the chromatographic analysis conditions include: The phosphoric acid concentration in mobile phase B is 0.05%-0.2%; The mobile phase flow rate is 0.8-1.2 ml / min; The column oven temperature is 25-35°C; The injection volume is 5-20 μL; The detection wavelength is 220-260nm; The column length of the chromatographic column is 100-300 mm, the inner diameter is 3.0-5.0 mm, and the particle size is 3.5-10 μm.

5. The method according to claim 4, wherein In step (3), the chromatographic analysis conditions include: The phosphoric acid concentration in mobile phase B was 0.1%; The mobile phase flow rate was 1.0 ml / min; The column oven temperature was 30°C; The injection volume was 10 μL; The detection wavelength is 237 nm; The chromatographic column model is SB-Aq, with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

6. The method according to claim 1, wherein In step (3), the gradient elution conditions are as follows: from 0 to 18 min, the volume ratio of mobile phase B is reduced from 99% to 81%; from 18 to 40 min, the volume ratio of mobile phase B is reduced from 81% to 60%; from 40 to 55 min, the volume ratio of mobile phase B is reduced from 60% to 46%; from 55 to 60 min, the volume ratio of mobile phase B is reduced from 46% to 5%.

7. The method according to claim 1, wherein In step (4), the fingerprint is measured based on 15 batches of test samples, and the control fingerprint is generated by fitting the fingerprints of the 15 batches of test samples by the mean method. In the fingerprint, the similarity between the common peak used to evaluate the similarity and the control fingerprint is not less than 0.9, and the proportion of peaks with a separation degree of ≥1.5 in the common peak is not less than 70%.

8. The method according to claim 1, wherein The Jiangtangning granules are composed of 40 parts of astragalus, 20 parts of rehmannia, 10 parts of liquorice, 10 parts of schisandra, 8 parts of pseudoginseng, 20 parts of trichosanthes, and 5 parts of pumpkin powder.

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