A method for quality control of Jiangtangning granules by fingerprinting and multi-component quantification

Abstract

This invention discloses a quality control method for fingerprinting and multi-component quantification of Jiangtangning granules, belonging to the field of pharmaceutical analysis technology. This method employs high-performance liquid chromatography (HPLC) to simultaneously achieve fingerprint determination and quantitative analysis of 10 components under the same chromatographic conditions. The steps include the preparation of test and reference solutions, gradient elution with acetonitrile-phosphoric acid aqueous solution, and detection under optimized chromatographic conditions. The fingerprint spectrum is based on the identification of common peaks from multiple batches of samples, with a similarity ≥0.9; simultaneously, 10 components, including verrucoside isoflavone glucoside, are determined, with a resolution ≥1.5 and good linearity. This method solves the problems of limited control indicators and low efficiency in existing technologies, comprehensively characterizes product quality, improves detection efficiency, reduces costs, and is suitable for the quality control of Jiangtangning granules.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and specifically proposes a method for quality control of fingerprint spectroscopy and multi-component quantification of Jiangtangning granules. Background Technology

[0002] Jiangtangning Granules (National Drug Approval Number Z20205004) is a traditional Chinese medicine compound preparation composed of seven herbs: Astragalus membranaceus, Rehmannia glutinosa, Trichosanthes kirilowii, Schisandra chinensis, Codonopsis pilosula, Glycyrrhiza uralensis, and pumpkin powder. It has the effects of invigorating qi and nourishing yin, promoting body fluid production and quenching thirst. Jingpai Chizhengtang Pharmaceutical Co., Ltd. is the exclusive marketing authorization holder for this product. The active ingredients in the Jiangtangning Granules formula are complex, containing astragaloside, verbascoside glucoside, verbascoside, schisandrol A, schisandrol B, glycyrrhizin, apigenin glycyrrhizin, glycyrrhizic acid, glycyrrhizin, styracin, and styracin, among others, making quality control challenging.

[0003] Chinese invention patent application CN1698705A discloses a preparation method for Jiangtangning tablets, describing the tablet preparation process, but lacks a multi-component quality control method. Liu Yuan et al., in "Simultaneous Determination of the Content of Five Active Ingredients in Jiangtangning Capsules by HPLC and Cluster Analysis," described a quantitative analysis method for five components in Jiangtangning capsules, but this method had poor separation of highly polar components, thus failing to construct a fingerprint spectrum for the product, and the number of quantitative components was limited, failing to cover important components such as schisandrol A, schisandrol B, glycyrrhizin, and glycyrrhizic acid.

[0004] The complex composition of the seven medicinal herbs in Jiangtangning preparations makes it difficult to simultaneously characterize the fingerprint spectrum and detect multiple indicators of traditional Chinese medicine compound preparations under a single chromatographic condition. This typically requires the development of multiple detection methods, which leads to a large workload and low efficiency. Furthermore, research on quality evaluation and control methods for Jiangtangning granules is still relatively weak, resulting in a limited number of controlled indicator components, which is detrimental to the comprehensive quality evaluation of the product.

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

[0006] In view of this, the present invention proposes an analytical method that can achieve both fingerprint spectroscopy determination and multi-index quantification under the same chromatographic conditions, thereby improving the quality control level of Jiangtangning granules and solving the problem of 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 measurement to achieve quantitative analysis of 10 index components of Jiangtangning granules, and can also control the overall quality attributes of the chemical components of Jiangtangning granules through fingerprint spectroscopy, which is of great significance for improving the quality control of Jiangtangning granules described in this invention.

[0007] The technical solution of this invention is implemented as follows: This invention provides a quality control method for fingerprinting and multi-component quantification of Jiangtangning granules, comprising the following steps: (1) Preparation of test solution: Take the test granules of Jiangtangning, add the extraction solvent to extract them, filter them, take the filtrate to obtain the test solution; (2) Preparation of reference solution: Take verbascoside, styracin, apigenin, glycyrrhizin, glycyrrhizin, glycyrrhizic acid, verbascoside, styracin, glycyrrhizin, schisandrol A, and schisandrol B to prepare a mixed reference solution. (3) Chromatographic analysis conditions: The chromatographic column was packed with octadecylsilane-bonded silica gel, and gradient elution was performed with acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B. The sample was detected by ultraviolet detector. (4) Fingerprint chromatogram preparation: The test solution and the reference solution are respectively injected into the HPLC chromatograph, and the total peak area is integrated with the minimum peak area ≥ 0.5‰. The fingerprint chromatogram is obtained based on the common peaks in the chromatograms of multiple batches of test samples. The number of multiple batches of test samples is ≥ 10 batches and covers the typical variations in raw material source and production process. (5) Quantitative analysis of multiple components: Ten components with a resolution (Ri) ≥ 1.5, namely verbascoside, styracin, apigenin, glycyrrhizin, glycyrrhizin, glycyrrhizic acid, verbascoside, styracin, glycyrrhizin, schisandrol A, and schisandrol B, were selected for content determination. Product quality control was carried out by 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 mass ratio of the test sample to the volume of the extraction solvent used is (1-4):(12.5-50), and the unit of the mass ratio is g / ml.

[0011] In some embodiments, the extraction step employs reflux extraction at a temperature of 75-85°C for 30-60 minutes.

[0012] In some implementations, in step (2): Each 1 ml of the reference solution contains 150-250 μg of verbascoside glucoside; and / or, Each 1 ml of the reference solution contains 100-200 μg of verbascoside isoflavone; and / or, Each 1 ml of the reference solution contains 500-1500 μg of apigenin; and / or, Each 1 ml of the reference solution contains 50-150 μg of glycyrrhizin; and / or, Each 1 ml of the reference solution contains 50-150 μg of gentianin; and / or Each 1 ml of the reference solution contains 2-20 μg of gentianin; and / or, Each 1 ml of the reference solution contains 2-20 μg of glycyrrhizic acid; and / or, Each 1 ml of the reference solution contains 10-50 μg of glycyrrhizin; and / or, Each 1 ml of the reference solution contains 10-50 μg of schisandrol A; and / or, Each 1 ml of the reference solution contains 50-200 μg of schisandrin ethyl.

[0013] In some embodiments, the mobile phase system was investigated, and the results showed that the separation effect of each target component in the chromatogram was better when acetonitrile-phosphoric acid aqueous solution was used as the mobile phase system. Methanol-phosphoric acid aqueous solution had fewer chromatographic peaks and poor separation effect. The baseline drift of acetonitrile-formic acid aqueous solution system was serious. Therefore, acetonitrile was preferred as mobile phase A and phosphoric acid aqueous solution was preferred 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 concentration of phosphoric acid in mobile phase B is 0.05%-0.2%, preferably 0.1%; The mobile phase flow rate is 0.8-1.2 ml / min, preferably 1.0 ml / min; The column oven temperature is 25-35℃, preferably 30℃; The injection volume is 5-20 μL, preferably 10 μL; The detection wavelength is 220-260nm, preferably 237nm; The column length is 100-300 mm, the inner diameter is 3.0-5.0 mm, and the particle size is 3.5-10 μm. Preferably, the column model is SB-Aq, the column length is 250 mm, the inner diameter is 4.6 mm, and the particle size is 5 μm.

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

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

[0017] In some embodiments, under the above chromatographic conditions, the separation effect of the 10 marker components in step (5) – peak 11 (verrucium isoflavone glucoside), peak 13 (apigenin glycyrrhizin), peak 14 (glycyrrhizin), peak 16 (mangosteenin), peak 21 (verrucium isoflavone), peak 22 (glycyrrhizin), peak 25 (glycyrrhizic acid), peak 26 (mangosteenin), peak 27 (schisandrol A), and peak 28 (schisandrol B) – is good, with good linearity and limit of quantitation, and can be used for content determination.

[0018] The present invention has the following advantages over 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 in an overall manner from both fingerprint spectrum and multi-component quantification, which can realize more comprehensive and reliable quality precision control of Jiangtangning granules and improve the quality control level.

[0019] (2) High detection efficiency and low cost: A single test can simultaneously perform quantitative analysis of 10 components, effectively avoiding the need to use different methods to detect components multiple times. Under the premise of improving quality control, it can significantly shorten the detection time, greatly improve detection efficiency, and reduce labor, reagent and other costs.

[0020] (3) Simple and quick operation: Through optimization of chromatographic conditions, quantitative analysis and fingerprint chromatogram are determined in one go using the same method, which is simple, quick and efficient. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The fingerprint chromatograms of 15 batches of Jiangtangning granules in Example 3 of this invention; Figure 2 This is a reference fingerprint of Jiangtangning granules obtained in Example 3 of the present invention; Figure 3The chromatogram of the mixed reference standard for content determination in Example 4 of the present invention is shown below. Peak 1 is verbascoside, peak 2 is apigenin glycyrrhizin, peak 3 is glycyrrhizin, peak 4 is schisandrin, peak 5 is verbascoside, peak 6 is glycyrrhizin, peak 7 is glycyrrhizic acid, peak 8 is schisandrin, peak 9 is schisandrol A, and peak 10 is schisandrol B. Figure 4 The chromatogram of the test sample for content determination in Example 5 of the present invention is shown below. Peak 1 is verbascoside, peak 2 is apigenin glycyrrhizin, peak 3 is glycyrrhizin, peak 4 is schisandrin, peak 5 is verbascoside, peak 6 is glycyrrhizin, peak 7 is glycyrrhizic acid, peak 8 is schisandrin, peak 9 is schisandrol A, and peak 10 is schisandrol B. Figure 5 This is a full-wavelength scan 3D image from Embodiment 1 of the present invention; Figure 6 The colorimetric spectrum of the mobile phase system in Example 1 of this invention is shown. Figure 7 This is a chromatogram of the test sample solution preparation method in Example 1 of the present invention. Figure 8 This is the chromatogram for flow rate determination in Example 1 of the present invention; Figure 9 This is the chromatogram for column temperature determination in Example 1 of the present invention; Figure 10 This is the chromatogram for investigating the injection volume in Example 1 of the present invention; Figure 11 The chromatogram of cinnamon in Comparative Example 1 of this invention; Figure 12 The chromatogram of cinnamon in Comparative Example 2 of this invention; Figure 13 The chromatogram of cinnamon in Comparative Example 3 of this invention; Figure 14 The chromatogram of cinnamon in Comparative Example 4 of this invention is shown. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[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 the embodiments of this invention pertain. If any definition stated in this section is contrary to or otherwise inconsistent with a definition stated in a patent, patent application, published patent application, or other publication incorporated herein by reference, the definitions listed here shall prevail over those incorporated herein by reference.

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

[0026] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1 to 5” is disclosed, the described range should be interpreted as including ranges “1 to 4”, “1 to 3”, “1 to 2”, “1 to 2 and 4 to 5”, “1 to 3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range. In this specification and claims, range definitions may be combined and / or interchanged, unless otherwise stated, these ranges include all subranges contained therein.

[0027] Example 1 Fingerprint spectroscopy detection method for Jiangtangning granules Take approximately 4g of the powdered Jiangtangning granules to be tested, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of methanol, seal tightly, weigh, heat under reflux for 60 minutes, remove, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0028] Prepare a 0.05-0.5 mg / mL mixed reference solution by disoprostol glucoside, apigenin glycyrrhizin, glycyrrhizin, schisandrol A, and schisandrol B for qualitative analysis.

[0029] The chromatographic column was octadecylsilane-bonded silica gel; gradient elution was performed using acetonitrile (A) and 0.1% phosphoric acid solution (B) as specified in the table below; the detection wavelength was 237 nm; the column was 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 verbascoside glucoside, should be no less than 3000.

[0030] Table 1. HPLC gradient elution conditions for Jiangtangning granules

[0031] Accurately pipette 10 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, measure and record the chromatograms to obtain the solution.

[0032] The chromatographic conditions were determined through multi-parameter optimization, wherein the detection wavelength was determined by full-wavelength scan 3D plot (attached). Figure 5 Screening showed that the response values ​​of each component were balanced and had little interference at 237 nm; the mobile phase system was compared with acetonitrile-formic acid aqueous solution and acetonitrile-water systems (see attached). Figure 6 The optimal system was determined to be acetonitrile-0.1% phosphoric acid aqueous solution; chromatograms of extraction methods for the test samples using methanol reflux and ethanol reflux were compared (see attached). Figure 7 The effectiveness of methanol heating and reflux extraction was verified; the flow rate, column temperature, and injection volume were set at 0.8-1.2 ml / min (see attached diagram). Figure 8 ), 25-35℃ (with) Figure 9 ), 5-20μL (with) Figure 10 After investigation, 1.0 ml / min, 30℃, and 10 μL were determined to be the optimal parameters.

[0033] Example 2 Methodological Investigation of Fingerprint Spectroscopy for Jiangtangning Granules Precision: Take about 4g of the same batch of Jiangtangning granules (JTNKL01) powder, weigh it accurately, and conduct the experiment according to the preparation of the test solution and chromatographic conditions and methods in Example 1, and inject the sample 6 times consecutively.

[0034] Repeatability: Take about 4g of the same batch of Jiangtangning granules (JTNKL01) powder, accurately weigh it, and prepare 6 test solutions in parallel according to the preparation method of the test solution in Example 1. Determine the test solution under the same chromatographic conditions as described above.

[0035] Stability: Take about 4g of the same batch of Jiangtangning granules (JTNKL01) powder, accurately weigh it, and conduct experiments according to the preparation of the test solution and chromatographic conditions and methods in Example 1. After preparation, the sample is injected and measured at 0, 2, 4, 8, 16 and 24 hours.

[0036] The methodological feasibility was evaluated using precision, repeatability, and stability experiments, with reference fingerprint spectra. The similarity scores were used to assess the methodological feasibility. Experimental results showed that the similarity scores for precision, repeatability, and stability were all above 0.999, 0.998, and 0.998, respectively. These results demonstrate that the method is stable and feasible and can be used for fingerprint analysis of the technical solution of this invention. Detailed experimental results are shown in Table 2 below.

[0037] Table 2 Results of precision, repeatability, and stability tests (n=6)

[0038] Example 3 Fingerprint analysis and common peak calibration of 15 batches of Jiangtangning granules Each herb in the formula was randomly combined into 15 batches according to different batches. Equal amounts of each herb from each batch were weighed, extracted, concentrated, and granulated to prepare finished granules. The 15 batches of Jiangtangning granules were then analyzed by HPLC according to the preparation method and chromatographic conditions described in Example 1. The fingerprint chromatograms of different batches of Jiangtangning granules were obtained by integrating the minimum peak area ≥ 0.5‰ of the total peak area. Fifteen batches of chromatograms were imported into the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" software. Peak matching was performed using the mean method and multi-point correction, yielding 32 common peaks. Eleven of these common peaks were identified using reference standards and mass spectrometry: peak 11 (verrucoside glucoside), peak 13 (apigenin glycyrrhizin), peak 14 (glycyrrhizin), peak 16 (mangosteenin), peak 19 (isoglycyrrhizin), peak 21 (verrucoside), peak 22 (glycyrrhizin), peak 25 (glycyrrhizic acid), peak 26 (mangosteenin), peak 27 (schisandrol A), and peak 28 (schisandrol B). Finally, a reference fingerprint R was generated, as shown below. Figure 1 , Figure 2 The similarity matching results of the fingerprint spectra among the 15 batches of samples and with the control are shown in Table 3 below.

[0039] Table 3. Similarity matching results of fingerprint spectra of 15 batches of samples

[0040] Example 4 Methodological investigation of the determination of 10 marker components Linear range: A series of mixed solutions of 10 reference standards were prepared as follows: verbenafil glucoside (1.96-98.20 µg / mL), gentianin (1.92-96.11 µg / mL), verbenafil (1.74-86.95 µg / mL), gentianin (1.72-86.21 µg / mL), apigenin glycyrrhizin (5.00-249.78 µg / mL), glycyrrhizin (4.95-247.52 µg / mL), glycyrrhizic acid (2.86-143.15 µg / mL), glycyrrhizin (1.80-90.08 µg / mL), schisandrol A (3.01-150.35 µg / mL), and schisandrol B (0.64-31.83 µg / mL). 10 μl of each of the 10 reference standard solutions was injected into an HPLC system, and the test was conducted according to the preparation of the test solution and chromatographic conditions described in Example 1. A standard curve was plotted with the injection concentration as the x-axis and the peak area as the y-axis. The results are as follows: Figure 3 As shown, all 10 components exhibited good linear relationships within the scope of the study, with correlation coefficients all greater than 0.999. Detailed experimental results are shown in Table 4 below.

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

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

[0043] Stability: Take about 4g of the same batch of Jiangtangning granules (JTNKL01) powder, accurately weigh it, and conduct experiments according to the preparation of the test solution and chromatographic conditions and methods in Example 1. After preparation, the sample is injected and measured at 0, 4, 8, 16, 24 and 48 hours.

[0044] The precision, repeatability, and stability of the methodology were evaluated using the RSD values ​​of the peak areas of each compound. Experimental results show that the method is stable and feasible, and can be used for the determination of the content of 10 marker components in the technical solution of this invention. Detailed experimental results are shown in Table 4 below.

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

[0046] Table 4. Results of methodological studies on 10 marker components

[0047] Example 5 Determination of the content of 10 marker components in 15 batches of samples According to the established method, the contents of 10 marker components in 15 batches of samples were determined using the preparation of the test solution and chromatographic conditions described in Example 1. The chromatograms of the content determination in the 15 batches of samples are attached. Figure 4 The results showed that the 10 marker components were well separated under the optimized chromatographic conditions (Ri≥1.5), consistent with the chromatographic parameters determined in Example 1 and the attached data. Figure 4 The separation effect was consistent, verifying the reliability of the quantitative analysis method. The measurement results are shown in Table 5 below.

[0048] Table 5. Results of content determination of 10 marker components in 15 batches of samples

[0049] The above test results show that the method for determining the content of 10 marker 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 Jiangtangning granules described in this 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 aqueous solution; Flow rate: 1.0 mL / min; Gradient elution conditions are shown in Table 1; Column temperature: 30℃; Injection volume: 10 μL; Detection wavelength: 260 nm. The same test solution and mixed reference standard 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 demonstrated that if the chromatographic column in the detection method provided by this invention is replaced with another chromatographic column, multiple components of Jiangtangning granules cannot be effectively separated, thus affecting the quantitative analysis of the components.

[0052] Comparative Example 2 Preparation method of test solution: Accurately weigh approximately 4g of the powdered Jiangtangning granules to be tested, place it in a stoppered conical flask, accurately add 25ml of ethanol, seal tightly, weigh, heat under reflux for 60 minutes, remove, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate. Under the same chromatographic conditions as in Example 1, inject the solution into HPLC for determination and record the chromatograms. The peak heights of each component are significantly lower, indicating that the extraction of the test component is insufficient. (See Appendix) Figure 12 .

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

[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 are shown in Table 1; Column temperature: 30℃; 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 demonstrated that replacing the mobile phase acetonitrile with methanol in the detection method provided by this invention 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. 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℃; 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 demonstrated that when the detection wavelength of 260nm in the detection method provided by this invention is replaced with other detection wavelengths, the separation of the chromatographic peaks of high polarity components is poor, and it is impossible to characterize and quantitatively detect multiple components in Jiangtangning granules.

[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 within the protection scope of the present invention.

Claims

1. A method for detecting fingerprint spectroscopy and quantitative analysis of multiple components in Jiangtangning granules, characterized in that, The Jiangtangning granules are composed of Astragalus membranaceus, Rehmannia glutinosa, Glycyrrhiza uralensis, Schisandra chinensis, Pseudostellaria heterophylla, Trichosanthes kirilowii, and pumpkin powder. A single HPLC injection simultaneously constructs fingerprint spectra and quantifies 10 indicator components, including the following steps: (1) Preparation of test solution: Take the test granules of Jiangtangning, add the extraction solvent to extract them, filter them, take the filtrate to obtain the test solution, wherein the extraction solvent is methanol; (2) Preparation of reference solution: Take verbascoside, styracin, apigenin, glycyrrhizin, glycyrrhizin, glycyrrhizic acid, verbascoside, styracin, glycyrrhizin, schisandrol A, and schisandrol B to prepare a mixed reference solution. (3) Chromatographic analysis conditions: A chromatographic column packed with octadecylsilane-bonded silica gel was used, with acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B for gradient elution. Ultraviolet (UV) detector was used for injection detection at a wavelength of 220-260 nm. The gradient elution conditions were as follows: 0-18 min, the volume ratio of mobile phase B decreased from 99% to 81%; 18-40 min, the volume ratio of mobile phase B decreased from 81% to 60%; 40-55 min, the volume ratio of mobile phase B decreased from 60% to 46%; 55-60 min, the volume ratio of mobile phase B decreased from 46% to 5%. (4) Fingerprint chromatogram preparation: The test solution and the reference solution are respectively injected into the HPLC chromatograph, and the total peak area is integrated with the minimum peak area ≥ 0.5‰. The fingerprint chromatogram is obtained based on the common peaks in the chromatograms of multiple batches of test samples, wherein the number of multiple batches of test samples is ≥ 10 batches. (5) Quantitative analysis of multiple components: Ten components with a resolution (Ri) ≥ 1.5, namely verbascoside, styracin, apigenin, glycyrrhizin, glycyrrhizin, glycyrrhizic acid, verbascoside, styracin, glycyrrhizin, schisandrol A, and schisandrol B, were selected for content determination. Product quality control was carried out by the content of each component.

2. The method as described in claim 1, characterized in that, In step (1), the ratio of the mass of the sample to the volume of the extraction solvent is (1-4):(12.5-50)g / ml. The extraction method is heating and reflux extraction, the reflux temperature is 75-85℃, and the extraction time is 30-60min.

3. The method as described in claim 1, characterized in that, In step (2), each 1 ml of the reference solution contains 150-250 μg of verbascoside glucoside, 100-200 μg of verbascoside, 500-1500 μg of apigenin glycyrrhizin, 50-150 μg of glycyrrhizin, 50-150 μg of schisandrin, 2-20 μg of schisandrin, 2-20 μg of glycyrrhizic acid, 10-50 μg of glycyrrhizin, 10-50 μg of schisandrol A, and 50-200 μg of schisandrol B.

4. The method as described in claim 1, characterized in that, In step (3), the chromatographic analysis conditions include: The concentration of phosphoric acid in mobile phase B is 0.05%-0.2%; The mobile phase flow rate was 0.8-1.2 ml / min; The column oven temperature is 25-35℃; The injection volume is 5-20 μL; The column length 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 as described in claim 4, characterized in that, In step (3), the chromatographic analysis conditions include: The concentration of phosphoric acid in mobile phase B is 0.1%; The mobile phase flow rate was 1.0 ml / min; The column oven temperature is 30℃; The injection volume was 10 μL; The detection wavelength is 237nm; The chromatographic column is model SB-Aq, with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

6. The method as described in claim 1, characterized in that, In step (4), the fingerprint spectrum is obtained based on 15 batches of test samples, and the control fingerprint spectrum is generated by fitting the fingerprint spectrum of 15 batches of test samples using the mean method. In the fingerprint spectrum, the similarity between the common peak used to evaluate similarity and the control fingerprint spectrum is not less than 0.9, and the proportion of peaks with a resolution of ≥1.5 in the common peaks is not less than 70%.

7. The method as described in claim 1, characterized in that, The Jiangtangning granules are composed of 40 parts Astragalus membranaceus, 20 parts Rehmannia glutinosa, 10 parts Glycyrrhiza uralensis, 10 parts Schisandra chinensis, 8 parts Codonopsis pilosula, 20 parts Trichosanthes kirilowii, and 5 parts pumpkin powder.

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