A method for analyzing the quantitative fingerprint of a large yin tonic pill

Abstract

The application discloses a method for analyzing a quantitative fingerprint of a Dabuyin pill, and belongs to the field of traditional Chinese medicine quality control research. The method comprises the following steps: preparing a control sample solution and a test sample solution; injecting liquid chromatography analysis and determination, selecting a Silversil C18 chromatographic column, gradient elution, column temperature is 29.5-30.5 DEG C, and the detection wavelength is 290 nm; recording chromatograms of the test sample solution and the control sample solution of multiple batches of Dabuyin pills, determining common peaks and quantitative detection indexes, and evaluating the quality of the Dabuyin pill through the fingerprint similarity and the content of each component. The application can be used for determining 5-hydroxymethyl furfural, 3-O-feruloyl quinic acid, neomangiferoline and berberine hydrochloride in the Dabuyin pill, and has a wide application prospect in the field of Dabuyin pill quality control.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality control research, and relates to a quantitative fingerprint analysis method for the components of Dabuyin Pill. Background Technology

[0002] Da Bu Yin Wan is a traditional Chinese medicine formula originating from *Danxi Xinfa*. It nourishes Yin and reduces internal heat, primarily treating Yin deficiency with excessive internal heat. Symptoms include bone steaming fever, night sweats, seminal emission, cough with hemoptysis, irritability, and pain and heat in the feet and knees. Clinically, it is often used for hyperthyroidism, pulmonary tuberculosis, and bone tuberculosis.

[0003] Currently, most existing research reports on Dabuyin Pill focus on its clinical efficacy, lacking quantitative fingerprint studies on its characteristic components. Compared to detecting individual components, multi-index quantitative fingerprinting can more comprehensively present the overall condition of traditional Chinese medicine. Therefore, it is necessary to develop a quantitative fingerprinting method for Dabuyin Pill.

[0004] The concept of "Quality by Design" (QbD) was first proposed by the U.S. Food and Drug Administration (FDA) and is now widely used in the field of analytical methods research, known as "Analytical Quality by Design" (AQbD). The implementation steps of AQbD typically include defining the objectives of the analytical method, identifying key quality attributes, identifying key method parameters, establishing a mathematical model of the relationship between key quality attributes and key method parameters, constructing the MODR (Meaning of Objectives and Decisions), method validation, and implementing control strategies. Summary of the Invention

[0005] The purpose of this invention is to address the gaps in existing technologies by developing a quantitative fingerprinting method for Dabuyin Pills (especially concentrated pill formulations) based on the AQbD concept, in order to provide a reference for the quality control of Dabuyin Pills.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention establishes a quantitative fingerprint analysis method for four components in Dabuyin Pill using high-performance liquid chromatography-ultraviolet detection. First, potential critical method parameters (CMPs) are identified. Then, critical quality attributes (CQAs) are identified through deterministic screening design (DSD). Next, a quantitative relationship model between CMPs and CQAs is established using stepwise regression. The p-values ​​of the relationship models are all <0.05, explaining most of the variance. The operable design region (MODR) of the method is established using the Monte Carlo method and successfully validated. The robustness of the analytical method is evaluated using Plackett-Burman design.

[0007] Based on the above research, the quantitative fingerprint analysis method for the components of Dabuyin Pill determined by this invention includes the following steps: Using 5-hydroxymethylfurfural, 3-O-feruloylquinic acid, neomangiferin and berberine hydrochloride as reference standards, reference solutions were prepared separately and then mixed to prepare a mixed reference solution; Dabuyin pills were ground into powder, fully dispersed in water, centrifuged, and the supernatant was collected to obtain the test solution; The reference solution and the test solution were injected into the liquid chromatography system for analysis. The chromatographic conditions were as follows: Silversil C18 column, 250 mm in length × 4.6 mm in inner diameter, 5 μm particle size; mobile phase A was 0.03 mol / L potassium dihydrogen phosphate solution, and mobile phase B was acetonitrile; gradient elution was used, with the following elution conditions: 0–15 min, the volume fraction of mobile phase B changed by 4.5%–5.5%; 15–35 min, the volume fraction of mobile phase B changed by 5.5%–11%; 35–70 min, the volume fraction of mobile phase B changed by 11%–40%, and a run time of 10 min; mobile phase flow rate was 0.78 mL / min–0.82 mL / min; column temperature was 29.5℃–30.5℃; detection method was DAD; detection wavelength was 290 nm; and injection volume was 10 μL. By comparing the chromatograms of multiple batches of Dabuyin Pill test solutions and reference solutions, common peaks and quantitative detection indicators were determined. The quality of Dabuyin Pill was evaluated by fingerprint chromatogram similarity and the content of each component.

[0008] Furthermore, the Da Bu Yin Wan is a concentrated pill.

[0009] Furthermore, the fingerprint spectrum contains 9 fingerprint peaks, with peak 9 as the reference peak, and the relative retention times are as follows: peak 1: 0.142; peak 2: 0.194; peak 3: 0.249; peak 4: 0.416; peak 5: 0.501; peak 6: 0.547; peak 7: 0.601; peak 8: 0.680; peak 9: 1.000; wherein peaks 2, 4, 6, and 9 correspond to 5-hydroxymethylfurfural, 3-O-feruloylquinic acid, neomangiferin, and berberine hydrochloride, respectively.

[0010] The present invention has the following advantages over the prior art: This invention establishes for the first time a quantitative fingerprint spectral detection method for four components in Dabuyin Pills. This method can be used to determine 5-hydroxymethylfurfural, 3-O-feruloylquinic acid, neomangiferin, and berberine hydrochloride in Dabuyin Pills (especially the concentrated pill form), or for fingerprint spectral similarity assessment. The method has undergone methodological validation, with relative standard deviations (RSDs) all less than 5.0%, demonstrating good precision and repeatability. Furthermore, the elution solvent is simple, indicating broad application prospects in the quality control of Dabuyin Pills. Attached Figure Description

[0011] Figure 1 This is a contour plot of the interactions between various factors, where Figure 1 (A) is a contour plot showing the interaction between column temperature and the proportion of phase B at the end of the first gradient on the separation of 5-hydroxymethylfurfural and the first peak. Figure 1 (B) in the figure is a contour plot showing the interaction between column temperature and flow rate on 3-O-feruloylquinic acid and the resolution of the first peak; Figure 1 (C) in the figure is a contour plot showing the interaction between column temperature and the proportion of phase B at the end of the first gradient on 3-O-feruloylquinic acid and the resolution of the first peak. Figure 1 (D) in the figure is a contour plot of the interaction between column temperature and the proportion of phase B at the end of the second gradient on 3-O-feruloylquinic acid and the resolution of the first peak. Figure 1 (E) is a contour plot of the interaction between flow rate and the proportion of phase B at the end of the first gradient on 3-O-feruloylquinic acid and the resolution of the first peak. Figure 1 (F) in the figure is a contour plot showing the interaction between the flow rate and the proportion of phase B at the end of the first gradient on the retention time of berberine hydrochloride. Figure 2 The MODR is calculated for each factor under constraints of three key quality attributes and an acceptable risk of 0.1. Figure 2 The factor in (A) is the column temperature, flow rate, and the proportion of phase B at the end of the first gradient. Figure 2 The factor (B) in the equation is the ratio of column temperature, flow rate, and B at the end of the second gradient. Figure 2 The factor (C) in the equation represents the column temperature, the proportion of phase B at the end of the first gradient, and the proportion of phase B at the end of the third gradient. Figure 2 The factors in (D) are column temperature and flow rate; Figure 3 It is a chromatogram of the solution; in which Figure 3 (A) in the figure is the chromatogram of the test sample solution. Figure 3 (B) in the figure is the chromatogram of the mixed reference solution; Figure 4 These are fingerprint chromatograms of multiple batches of Dabuyin Pills (concentrated pills). Detailed Implementation

[0012] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0013] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0014] The instruments used are as follows: High Performance Liquid Chromatograph (Agilent 1260 Infinity II, Agilent Technologies, USA); Centrifuge (Minispin, Eppendorf, Germany); Ultrapure Water Treatment System (Synergy UVMilli-Q, Merck Millipore, USA); Analytical Balance (MCA125P-2CCN-U, Sartorius, Germany); Digital Display Thermostatic Water Bath (WB-2, Changde Bickman Biotechnology Co., Ltd.)

[0015] The following materials were used: acetonitrile (chromatographic grade, ASTOON, USA); methanol (chromatographic grade, ASTOON, USA); ultrapure water was prepared using a Milli-Q ultrapure water system; 5-hydroxymethylfurfural (batch number: C14007429, purity 97.0%, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.); neomangiferin (batch number: M17IB209878, purity ≥98%, purchased from Shanghai Yuanye Biotechnology Co., Ltd.); 3-O-feruloylquinic acid (batch number: FR241201, purity >98%, purchased from Shanghai Fuyu Biotechnology Co., Ltd.); berberine hydrochloride (batch number: 2024121821, purity ≥98.8%, purchased from Northern Weiye Metrology Group Co., Ltd.).

[0016] It should be noted that the Da Bu Yin Wan used in the following embodiments is Da Bu Yin Wan (concentrated pill), which was provided by Zhejiang Hangzhou Hu Qing Yu Tang Pharmaceutical Co., Ltd.

[0017] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with techniques or conditions described in common knowledge in the art or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0018] Example 1: Study on the quantitative fingerprint detection method of four components in Dabuyin Pill (concentrated pill).

[0019] This embodiment studies a quantitative fingerprint spectral detection method for four components in Dabuyin Pill (concentrated pill), including the following steps: 1. Reagent preparation.

[0020] 1.1 Preparation of test solution: Grind Dabuyin Pill (concentrated pill) into powder, accurately weigh 0.5 g, add 30 ml of ultrapure water, sonicate for 30 min, centrifuge at 12000 r / min for 10 min, and take the supernatant to obtain the test solution.

[0021] 1.2 Preparation of reference solutions: Accurately weigh 12.08 mg of 5-hydroxymethylfurfural, 2.48 mg of 3-O-feruloylquinic acid, 1.14 mg of neomangiferin, and 1.42 mg of berberine hydrochloride, respectively. Dissolve 5-hydroxymethylfurfural in ultrapure water, and dissolve 3-O-feruloylquinic acid, neomangiferin, and berberine hydrochloride in methanol, respectively, to prepare reference stock solutions. Dilute the stock solutions with the corresponding solvents to obtain a series of reference solutions of different concentrations. Accurately weigh 0.46 mg of 5-hydroxymethylfurfural, 2.39 mg of 3-O-feruloylquinic acid, 1.79 mg of neomangiferin, and 0.93 mg of berberine hydrochloride, respectively, and place them in a 10 mL volumetric flask. Dissolve in pure methanol to prepare a mixed reference solution.

[0022] 2. HPLC-DAD analysis.

[0023] 2.1 Key method parameter selection.

[0024] First, potential critical method parameters were identified. Common potential critical method parameters mainly include column length, particle size, column temperature, and packing material; the quantity, type, and ratio of organic phases; and gradient run times. Based on previous experimental results, the detection wavelength for HPLC was set at 290 nm, mobile phase A was an aqueous solution of potassium dihydrogen phosphate, and mobile phase B was pure acetonitrile. Six potential critical method parameters were screened, specifically including: the amount of potassium dihydrogen phosphate added in phase A (X1), column temperature (X2), flow rate (X3), the proportion of phase B at the end of the first gradient (X4), the proportion of phase B at the end of the second gradient (X5), and the proportion of phase B at the end of the third gradient (X6).

[0025] This invention selects a deterministic screening experimental design (DSD) method that can examine more parameters with fewer experiments. Since the resolution of most components, such as 5-hydroxymethylfurfural, did not reach 1.5 in the first DSD optimization results, a second DSD optimization was conducted based on the first DSD experimental study. The parameters of the two DSD optimization analyses and their corresponding levels are shown in Table 1. The mobile phase gradient is shown in Table 2.

[0026] Table 1. DSD Optimization Factors and Corresponding Levels Table 2 Analytical Conditions Gradient Elution Procedure The above parameters were optimized using DSD, and experimental conditions were generated using Design-Expert 13.0 software. The two DSD optimization conditions are shown in Table 3. Since the resolution of the new mangiferin and the surrounding peaks were both greater than 1.5, they were not considered as critical quality attributes. This invention uses the resolution between 5-hydroxymethylfurfural and the preceding peak, and the resolution between 3-O-feruloylquinic acid and the preceding peak, as method evaluation indicators Y1 and Y2. Furthermore, to shorten the analytical time, the retention time of the last eluting peak, berberine hydrochloride, was used as method evaluation indicator Y3.

[0027] Table 3. Experimental conditions and data for DSD optimization The experimental design results were analyzed using Design Expert 13.0 software. Formula (1) was used to model and screen key method parameters and key quality attributes.

[0028] (1); Where a0 is a constant term, a i X is the partial regression coefficient. i Here, m represents the number of parameters to be studied, and Y represents the method evaluation index. A stepwise regression method was used to simplify the model, and the significance level for adding and deleting terms in the model was set to 0.05. The remaining parameters in the model were considered key method parameters.

[0029] A second-order mathematical model is used to describe the relationship between key method parameters and key quality attributes, and a quantitative model is established using formula (2).

[0030] (2); Where b0 is a constant term, b i b ii b ij These are the regression coefficients for the first-order term, the second-order term, and the interaction terms of each factor, respectively, where n is the number of key method parameters, and X is the regression coefficient. c，i X j，i The significance level for each key method parameter, as well as for adding and deleting terms in the model, was set to 0.05.

[0031] The MODR was calculated using the Monte Carlo method, with a minimum risk of 0.1 and 1000 simulations.

[0032] The experimental results are shown in Table 3. Multiple linear regression equations were used to model the data in Table 3, and analysis of variance (ANOVA) was used to determine the influence of parameters on the response variable. Table 4 lists the regression coefficients and p-values ​​of the regression model. According to the p-values ​​of the linear terms in the ANOVA results, the amount of potassium dihydrogen phosphate added in phase A had no significant effect on Y1, Y2, and Y3; column temperature had a significant effect on Y2 and Y3; mobile phase flow rate had a significant effect on Y3; the proportion of phase B at the end of the first gradient had a significant effect on Y1; and the proportion of phase B at the end of the second and third gradients both had a significant effect on Y3.

[0033] Table 4. Results of ANOVA for the Multiple Linear Regression Model In the table, "-" indicates that the item is not significant, "*" indicates that the p-value of the item is less than 0.05, and "**" indicates that the p-value of the model item is less than 0.01.

[0034] 2.2 Establishment of a quantitative model between key method parameters and key quality attributes Formula (2) was used to model the key method parameters and key quality attributes. Table 5 lists the regression coefficients and p-value ranges of the regression models. The p-values ​​of all three models are less than 0.05, indicating that each model fits well and can explain most of the variance.

[0035] Table 5 Regression coefficients and variance analysis for each model The interactions between factors can be represented using contour plots, such as... Figure 1 As shown. Figure 1 (A) shows that as the column temperature decreases and the proportion of phase B decreases at the end of the first gradient, the separation between 5-hydroxymethylfurfural and the front peak gradually increases. Figure 1 Figures (B) to (D) show that as the column temperature increases, the flow rate of the mobile phase decreases, and the proportion of phase B increases at the end of the first and second gradients, the separation of 3-O-feruloylquinic acid and the front peak gradually increases. Figure 1 (E) shows that as the mobile phase flow rate increases, the proportion of phase B decreases at the end of the first gradient, and the separation between 3-O-feruloylquinic acid and the front peak gradually increases. Figure 1 (F) shows that the retention time of berberine hydrochloride gradually decreases as the flow rate of the mobile phase increases.

[0036] 2.3 Establishment and Verification of MODR When calculating MODR, the target range and acceptable risk were set after optimization of key quality attributes (Table 6). The proportion of potassium dihydrogen phosphate in the stationary mobile phase was 0.03 mol·L⁻¹. -1 The calculated MODR is as follows Figure 2As shown in (A) to (D) in the figure. For ease of operation, the recommended operating parameters are: column temperature 29.5℃ ~ 30.5℃, flow rate 0.78 mL / min ~ 0.82 mL / min, B phase ratio at the end of the first gradient 5.3% ~ 5.7%, B phase ratio at the end of the second gradient 10.8% ~ 11.2%, and B phase ratio at the end of the third gradient 39.5% ~ 40.5%.

[0037] Table 6. Scope and Risks of Each Key Quality Attribute To verify the reliability of MODR, three points were randomly selected within MODR for verification. The verification conditions and results are as follows: Figure 2 (D) and Table 7. Based on the validation results, the relative error between the predicted and experimental values ​​is within 3%, therefore the established MODR is considered relatively reliable.

[0038] Table 7. Experimental conditions and results at the verification points 2.4 Durability Test The durability test primarily assesses the method's resistance to interference. A two-level Plackett-Burman test design was selected to determine the experimental plan and examine the durability of MODR (Table 8).

[0039] Table 8. Results of the Plackett-Burman experiment The results showed that within the investigated range, the resolution of 5-hydroxymethylfurfural and its peak, and 3-O-feruloylquinic acid and its peak were all greater than 1.5, and the retention times of berberine hydrochloride were all within 67 min. These results indicate that the method exhibits good robustness within this range. Based on the obtained MODR, the chromatographic conditions were determined as follows: 5.5% B-phase ratio at the end of the first gradient, 11% B-phase ratio at the end of the second gradient, 40% B-phase ratio at the end of the third gradient, 0.03 mol / L potassium dihydrogen phosphate in the aqueous phase, 0.80 mL / min mobile phase flow rate, and 30℃ column temperature.

[0040] 3. Validation of fingerprinting methodology.

[0041] 3.1 Chromatogram and the position of common peaks.

[0042] Chromatograms of Dabuyin Pill (concentrated pill) and mixed reference standards are shown below. Figure 3As shown. 5-Hydroxymethylfurfural, 3-O-feruloylquinic acid, neomangiferin, and berberine hydrochloride were confirmed using reference standards. The remaining common peaks were determined by liquid chromatography-mass spectrometry (LC-MS) using an AB Sciex X500B instrument under the following conditions: IDA mode was used for data acquisition in both positive and negative ion modes. Source gas parameters: Nebulized gas (GS1): 379 kPa; Nebulized gas (GS2): 379 kPa; Curtain gas (CUR): 241 kPa; Ion source temperature (TEM): 600℃.

[0043] Positive ions: TOF MS: Scan range: m / z 100~1500; Ion source voltage (IS): 5500 V; Declustering voltage (DP): 80 V; Collision energy (CE): 10 V; Collision energy variation (CES): 0 V. TOF MS / MS: Scan range: m / z 50~1500; Ion source voltage (IS): 5500 V; Declustering voltage (DP): 80 V; Collision energy (CE): 35 V; Collision energy variation range: 15 V.

[0044] Negative ions: TOF MS: Scan range: m / z 100~1500; Ion source voltage (IS): -4500 V; Declustering voltage (DP): -100 V; Collision energy (CE): -10 V; Collision energy variation (CES): 0 V. TOF MSMS: Scan range: m / z 50~1500; Ion source voltage (IS): -4500 V; Declustering voltage (DP): -80 V; Collision energy (CE): -35 V; Collision energy variation range: 15 V.

[0045] Preliminary identification results were obtained by comparing the LC-MS results with the standard spectral library LibraryView (SCIEX, USA). Further confirmation was achieved by comparing the mass-to-charge ratios of the parent ion and characteristic daughter ions reported in current literature. Figure 3 The composition of the compounds with peaks in the middle is inferred as shown in Table 9.

[0046] Table 9. Common peaks corresponding to compounds in Dabuyin Pill (concentrated pill) preparation. 3.2 Investigation of fingerprinting methodology Using peak 9 (berberine hydrochloride) as the reference peak, the relative retention time (RRT) and relative peak area (RPA) of each common peak were calculated, and the experimental results were evaluated by RSD (Table 10). The fingerprinting methodology was investigated under the following chromatographic conditions: Column: Silversil C18 column (250 mm × 4.6 mm, 5 μm); Mobile phase: Phase A was 0.03 mol / L potassium dihydrogen phosphate solution, and Phase B was acetonitrile. Gradient elution program: 0–15 min, 4.5%–5.5% B; 15–35 min, 5.5%–11% B; 35–70 min, 11%–40% B, followed by a 10 min run. Mobile phase flow rate: 0.80 mL / min; Column temperature: 30℃; Detection mode: DAD; Detection wavelength: 290 nm; Injection volume: 10 μL.

[0047] Precision: The RSD of the RRT for each common peak is less than 1.0%, and the RSD of the RPA is less than 3.0%, indicating good injection precision. Repeatability: The RSD of the RRT for each common peak is less than 1.0%, and the RSD of the RPA is less than 2.0%, indicating good method repeatability. Sample Stability: The RSD of the RRT for each common peak is less than 1.0%, and the RSD of the RPA is less than 3.0%, indicating good sample stability.

[0048] Table 10. Relative retention time and relative peak area of ​​common peaks in Dabuyin Pills (concentrated pills) 3.3 Examination of fingerprint similarity The original chromatograms of 11 batches of Dabuyin Pills (concentrated pills) were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Software (2012 version)". The first batch (S1) was used as the reference chromatogram. The method was median, and the time window width was set to 0.5 min to generate a control chromatogram. The fingerprint chromatograms of each batch of Dabuyin Pills (concentrated pills) are shown below. Figure 4 The similarity of fingerprints between 11 batches of Dabuyin Pills (concentrated pills) and the control was calculated (Table 11). The similarity between different batches of Dabuyin Pills (concentrated pills) and between each batch and the control fingerprint was greater than 0.95, indicating that the consistency of each batch of Dabuyin Pills (concentrated pills) was good.

[0049] Table 11. Similarity of fingerprint chromatograms between different batches of Dabuyin Pills (concentrated pills) and between each batch and the control. 3.4 Methodological Investigation of Content Determination Precision: The RSD of the peak areas of all four components was less than 3.0%, indicating good injection precision. Repeatability: The RSD of the contents of all four components was less than 2.0%, indicating good repeatability of the method. Sample stability: The RSD of the contents of all four components was less than 2.0%, indicating good sample stability. The linearity and recovery results of the method are shown in Tables 12 and 13, respectively. Overall, this method has good accuracy and can be used for the quantitative detection of Dabuyin Pill (concentrated pill).

[0050] Table 12 Linear relationships, limits of detection, and limits of quantitation for four components of Dabuyin Pill (concentrated pill). Table 13 Spike recovery results of each component in Dabuyin Pill (concentrated pill) (n=6) In summary, the final analytical conditions determined in this embodiment are as follows: Column: Silversil C18 column (250 mm × 4.6 mm, 5 μm); Mobile phase: Phase A is 0.03 mol / L potassium dihydrogen phosphate solution, and Phase B is acetonitrile. Gradient elution program: 0-15 min, 4.5%-5.5% B; 15-35 min, 5.5%-11% B; 35-70 min, 11%-40% B, followed by a 10 min run time. Mobile phase flow rate: 0.80 mL / min; Column temperature: 30℃; Detection method: DAD; Detection wavelength: 290 nm; Injection volume: 10 μL. This method has passed methodological validation and can be used for the determination of four components in Dabuyin Pill (concentrated pill), or for fingerprint chromatographic similarity assessment.

[0051] Example 2: Application example of quantitative fingerprint detection method.

[0052] The established method was used to detect the target components in 11 batches of Dabuyin Pills (concentrated pills), and the results are shown in Table 14. The results showed that the content of 5-hydroxymethylfurfural ranged from 0.03388 to 0.06792 mg / g, the content of 3-O-feruloylquinic acid ranged from 0.3120 to 1.4761 mg / g, the content of neomangiferin ranged from 0.2758 to 0.9103 mg / g, and the content of berberine hydrochloride ranged from 0.8127 to 1.8231 mg / g. The content of each target component varied among different batches of Dabuyin Pills (concentrated pills), with berberine hydrochloride having the highest content.

[0053] Table 14. Detection results (mg / g) of various target components in 11 batches of Dabuyin Pills (concentrated pills) The above embodiments are only used to illustrate the design concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, all equivalent changes or modifications made based on the principles and design ideas disclosed in the present invention are within the protection scope of the present invention.

Claims

1. A quantitative fingerprint analysis method for components in Dabuyin Pill, characterized in that, The method is used to determine the content of one or more of 5-hydroxymethylfurfural, 3-O-feruloylquinic acid, neomangiferin, and berberine hydrochloride in Dabuyin Pill, and includes the following steps: Using 5-hydroxymethylfurfural, 3-O-feruloylquinic acid, neomangiferin and berberine hydrochloride as reference standards, reference solutions were prepared separately and then mixed to prepare a mixed reference solution. Grind Da Bu Yin Wan into powder, disperse it fully in water, centrifuge, and take the supernatant to obtain the test solution; The reference solution and the test solution were injected into the liquid chromatography system for analysis. The chromatographic conditions were as follows: Silversil C18 column, 250 mm in length × 4.6 mm in inner diameter, 5 μm particle size; mobile phase A was 0.03 mol / L potassium dihydrogen phosphate solution, and mobile phase B was acetonitrile; gradient elution was used, with the following elution conditions: 0–15 min, the volume fraction of mobile phase B changed by 4.5%–5.5%; 15–35 min, the volume fraction of mobile phase B changed by 5.5%–11%; 35–70 min, the volume fraction of mobile phase B changed by 11%–40%, and a run time of 10 min thereafter; mobile phase flow rate was 0.78 mL / min–0.82 mL / min; column temperature was 29.5℃–30.5℃; detection mode was DAD; detection wavelength was 290 nm; and injection volume was 10 μL. By comparing the chromatograms of multiple batches of Dabuyin Pill test solution and reference solution, common peaks and quantitative detection indicators were determined, and the quality of Dabuyin Pill was evaluated by fingerprint chromatogram similarity and the content of each component. The fingerprint spectrum contains nine fingerprint peaks. Taking peak 9 as the reference peak, the relative retention times are as follows: Peak 1: 0.142; Peak 2: 0.194; Peak 3: 0.249; Peak 4: 0.416; Peak 5: 0.501; Peak 6: 0.547; Peak 7: 0.601; Peak 8: 0.680; Peak 9: 1.

000. Peaks 2, 4, 6, and 9 correspond to 5-hydroxymethylfurfural, 3-O-feruloylquinic acid, neomangiferin, and berberine hydrochloride, respectively.

2. The quantitative fingerprint analysis method for components in Dabuyin Pill according to claim 1, characterized in that, The Da Bu Yin Wan is a concentrated pill.

3. The quantitative fingerprint analysis method for components in Dabuyin Pill according to claim 1, characterized in that, In the reference solution, 5-hydroxymethylfurfural was dissolved in water, and 3-O-feruloylquinic acid, neomangiferin, and berberine hydrochloride were dissolved in methanol.

4. The quantitative fingerprint analysis method for components in Dabuyin Pill according to claim 1, characterized in that, After the Dabuyin Pill is ground into powder, it is dissolved in water at a material-to-liquid ratio of 1:60, sonicated for 30 min, centrifuged at 12000 r / min for 10 min, and the supernatant is collected to obtain the test solution.

5. The quantitative fingerprint analysis method for components in Dabuyin Pill according to claim 1, characterized in that, The mobile phase flow rate is 0.80 mL / min, and the column temperature is 30℃.

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