Kangshou pill six-wavelength fusion HPLC fingerprint detection method and application thereof

Abstract

The invention belongs to the technical field of quality detection of traditional Chinese medicines, and particularly relates to a six-wavelength fused HPLC (High Performance Liquid Chromatography) fingerprint detection method for Kangshou pills and application thereof. According to the method, octadecyl silane bonded silica gel chromatography is adopted, and a mobile phase A is a 0.1-0.3% phosphoric acid aqueous solution containing 0.003-0.01 mol / L of sodium heptanesulfonate; the mobile phase A is methanol, the mobile phase B is methanol, gradient elution is carried out, a six-wavelength fusion HPLC fingerprint spectrum of 30 batches of Kangshou pills is established, 26 common peaks are determined, and the similarity of 30 batches of samples is greater than 0.9. According to the present invention, the established Kangshou pill HPLC fingerprint has characteristics of good chromatographic information, high precision and good repeatability, can comprehensively reflect the information characteristics of the overall component of the Kangshou pill sample, can provide the reference for the quality evaluation, and can provide the scientific and reasonable theoretical support for the quality control.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine quality detection, and particularly relates to a six-wavelength fusion HPLC fingerprint detection method for Kangshou pills and application thereof. BACKGROUND

[0002] Kangshou pills (Guojiazhunzi Z44020922) are composed of radix rehmanniae preparatae, radix rehmanniae, cortex moutan, radix rehmanniae, poria cocos, radix asparagi, radix ophiopogonis and radix ginseng, have the effects of tonifying qi and blood, moistening lung and nourishing kidney, and are used for treating body weakness, lassitude, dizziness and forgetfulness, insomnia and dreaminess, polyhidrosis, dry cough with little sputum, palpitation and rapid pulse, and soreness of waist and knees caused by deficiency of qi and blood. The Kangshou pills are mainly suitable for adult patients of various ages with organic lesions and sub-health people, and have good effects on treating asthenia, insomnia and dreaminess. The prescription of the Kangshou pills is composed of eight medicinal materials, and the product has many and complex effective components. The product quality is easily affected by factors such as quality of medicinal materials, production process and storage condition, and thus is prone to fluctuation. The current quality standard of the Kangshou pills only uses the control medicinal materials to carry out thin layer identification, lacks analysis and control of characteristic components, and thus cannot accurately evaluate the product quality level and has quality control risks.

[0003] Fingerprint is widely used as an important means for quality control of traditional Chinese medicine prescriptions, has the characteristics of large information amount and strong characteristic, and can comprehensively reflect the quality stability and uniformity of medicinal materials and traditional Chinese medicine preparations. Meanwhile, high performance liquid chromatography (HPLC) has the characteristics of high separation efficiency, fast analysis speed, high quantitative precision, multiple types of detectors and good stability, and is not limited by volatility and thermal stability of samples, and most components in samples can be analyzed and detected on the high performance liquid chromatograph, and thus is one of main methods for constructing fingerprint.

[0004] It is of great significance to carry out the research on the fingerprint of the Kangshou pills, select multiple characteristic component indexes in the Kangshou pills to establish a “fingerprint” information base, comprehensively and systematically analyze various components in the product, improve the standardization of quality control of traditional Chinese medicine, ensure the stability of clinical curative effect, and promote the internationalization of traditional Chinese medicine. The fingerprint technology can comprehensively realize the quality control of different batches of products, ensure the consistency and stability of product quality, and reduce the medication risk caused by quality fluctuation.

[0005] Therefore, it is necessary to research and design an HPLC fingerprint determination method for objectively, accurately and comprehensively detecting the quality of the Kangshou pills on the basis of the prior art, so as to more comprehensively and effectively control the quality of the Kangshou pills, improve the quality control level, and ensure the safety and curative effect of clinical medication. SUMMARY

[0006] The application aims to provide a Kangshou pill HPLC fingerprint construction method, and the peak intensity, number and overall separation degree of the common peaks of the established Kangshou pill HPLC fingerprint are good, the information characteristics of the overall components of the sample can be comprehensively reflected, and more scientific and reasonable theoretical support is provided for quality control.

[0007] In a first aspect, the application provides a Kangshou pill six-wavelength fusion HPLC fingerprint detection method, which comprises the following steps:

[0008] Step 1: Preparation of test sample solution: Kangshou pill powder is added into a methanol aqueous solution, ultrasonic extraction is performed, filtration is performed, and the filtrate is taken to obtain the test sample solution;

[0009] Step 2: Preparation of control sample solution: 2,3,5,4'-tetrahydroxystilbene glucoside control sample (THSG) and emodin are dissolved in a methanol aqueous solution to prepare a mixed control sample solution;

[0010] Step 3: Determination: the test sample solution and the control sample solution are determined by high performance liquid chromatography with the same chromatographic conditions to obtain the fingerprint of the test sample solution and the fingerprint of the control sample solution, the fingerprint of the test sample solution is compared with the fingerprint of the control sample solution, the index components in the fingerprint of the test sample solution are attributed and positioned, and thus the fingerprint of the Kangshou pill is obtained,

[0011] The determination conditions of the high performance liquid chromatography are as follows:

[0012] Chromatographic column: octadecylsilane bonded silica gel chromatographic column;

[0013] Column temperature: 30-40 DEG C;

[0014] Injection volume: 5-10 mu L;

[0015] Mobile phase: mobile phase A is 0.1-0.3% phosphoric acid aqueous solution containing 0.003-0.01 mol / L sodium heptanesulfonate; mobile phase B is methanol;

[0016] Gradient elution is adopted, the proportion of the mobile phase in the elution process is volume percent, and the elution program is as follows:

[0017] Time (min) Mobile phase A (%) Mobile phase B (%) 0 - 5 min 95％-85％ 5％-15％ 5 - 25 min 85％-75％ 15％-25％ 25 - 35 min 75％-50％ 25％-50％ 35 - 50 min 50％-20％ 50％-80％ 50 - 60 min 20％-10％ 80％-90％ 60 - 65 min 10％-95％ 90％-5％

[0018] Detector: diode array detector;

[0019] Detection wavelength: 220 nm, 237 nm, 246 nm, 265 nm, 278 nm and 300 nm, wherein 265 nm is used as the quantitative detection wavelength, and the contents of THSG and emodin are determined at 265 nm.

[0020] As an optional way, in the above detection method, in step 1, about 1.0 g of Kangshu pill powder is precisely weighed, placed in a 10 mL volumetric flask, 80% methanol solution is added to the calibration mark, ultrasonic treatment is performed for 30 min, after cooling to room temperature, shaking is performed, 0.45 μm microporous filter membrane is used for filtration, and the filtrate is taken as the test sample solution.

[0021] As an optional way, in the above detection method, in step 2, THSG and emodin reference substances are precisely weighed, dissolved in 80% methanol solution to prepare a reference solution containing THSG 0.12 mg / mL and emodin 0.04 mg / mL.

[0022] As an optional way, in the above detection method, in step 3, the determination conditions of the high performance liquid chromatography are as follows:

[0023] The chromatographic column is Packed Column 5C18-MS-II, the inner diameter is 4.6 mm x 250 mm, the particle size is 5 μm;

[0024] The column temperature is 35°C;

[0025] The injection volume is 10 μL;

[0026] The mobile phase is that mobile phase A is 0.2% phosphoric acid aqueous solution containing 0.005 mol / L sodium heptanesulfonate; and mobile phase B is methanol.

[0027] As an optional way, in the above detection method, the detection method further comprises collecting a three-dimensional spectrum chromatogram of 190-400 nm by using a diode array detector.

[0028] In a second aspect, the application provides a construction method of a standard fingerprint spectrum of Kangshu pill, characterized in that: a plurality of batches of Kangshu pill samples are detected by using the six-wavelength fusion HPLC fingerprint spectrum detection method of Kangshu pill in the first aspect, a common mode control spectrum is generated by obtaining the fingerprint spectra of the plurality of batches of Kangshu pill samples, the common characteristic peaks existing in the spectrum are taken as the common characteristic peaks, the relative retention time of the common characteristic peaks, the ratio of the area of each common characteristic peak to the total peak area are determined, the index components in the Kangshu pill fingerprint spectrum are attributed and positioned according to the relative retention time, and the standard fingerprint spectrum of Kangshu pill is established.

[0029] As an optional mode, in the above method for constructing the standard fingerprint spectrum, the number of common fingerprint peaks of the Kangshou pill sample at the detection wavelengths of 220 nm, 237 nm, 246 nm, 265 nm, 278 nm and 300 nm is 14, 8, 9, 13, 14 and 11 respectively, and the macro qualitative similarity of the fingerprint spectrum of the Kangshou pill sample should be no less than 0.90 and the macro quantitative similarity should be between 80% and 120% according to the peak area and retention time of the common fingerprint peaks.

[0030] As an optional mode, in the above method for constructing the standard fingerprint spectrum, the peak of 2,3,5,4'-tetrahydroxystilbene-2-O-β-glucopyranoside is used for identification of the common fingerprint peak and is also a reference quantitative measurement correction peak.

[0031] The peak No. 5 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-glucopyranoside when detected at the wavelength of 220 nm;

[0032] The peak No. 3 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-glucopyranoside when detected at the wavelength of 237 nm;

[0033] The peak No. 4 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-glucopyranoside when detected at the wavelength of 246 nm;

[0034] The peak No. 9 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-glucopyranoside when detected at the wavelengths of 265 nm and 278 nm; and

[0035] The peak No. 9 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-glucopyranoside when detected at the wavelength of 300 nm.

[0036] In a third aspect, the present application provides a quality detection method for the fingerprint spectrum of the Kangshou pill, which comprises obtaining the fingerprint spectrum of the Kangshou pill sample by using the six-wavelength fusion HPLC fingerprint spectrum detection method for the Kangshou pill according to the first aspect, comparing the similarity with the standard fingerprint spectrum of the Kangshou pill obtained by using the method for constructing the standard fingerprint spectrum of the Kangshou pill according to the second aspect, and comparing the content of THSG and emodin in the Kangshou pill sample with the standard preparation of the Kangshou pill.

[0037] As an optional mode, in the above quality detection method, the system quantitative fingerprint method (SQFM) is performed, the sample fingerprint spectrum (SFP) is compared with the reference fingerprint spectrum (RFP), the similarity parameter (S m ) is calculated, and the full-spectrum component quantitative evaluation is further realized through the fingerprint projection content (P m ).

[0038] As an optional mode, in the above quality detection method, the calculation formula of the SQFM is:

[0039]

[0040] The variables x and y respectively represent the fingerprint peak area of a sample fingerprint spectrum (SFP) and a reference fingerprint spectrum (RFP), the variables S and S' respectively represent the similarity before and after fingerprint peak correction, the variables C and P respectively represent the projection content similarity and the quantitative similarity, and the fingerprint signal homogenization coefficient γ is a reliable index for measuring the homogenization degree of the chemical fingerprint signal distribution. i i F F y As a comparison standard, the relative deviation α of the sample γ x is defined as the coefficient of variation of fingerprint homogeneity, and by using S m , P m and α, the SQFM divides the samples into multiple quality grades, and samples with the same quality grade have similar chemical fingerprint characteristics, and a significant change in the quality grade indicates a significant difference in quality.

[0041] As an optional mode, in the above quality detection method, the similarity is ≥ 0.90.

[0042] Compared with the prior art, the present application has the following advantages and positive effects:

[0043] The HPLC fingerprint spectrum of the Kangshou pill established by the present application has good chromatographic information, high precision and good repeatability, comprehensively reflects the information characteristics of the overall components of the Kangshou pill sample, and can provide a reference for the quality evaluation and more scientific and reasonable theoretical support for the quality control. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The HPLC characteristic fingerprint spectrum of the control was obtained by detection at 220 nm;

[0045] Figure 2 The HPLC characteristic fingerprint spectrum of the standard preparation of the Kangshou pill was obtained by detection at 220 nm;

[0046] Figure 3 The HPLC characteristic fingerprint spectrum of 30 batches of Kangshou pill preparations was obtained by detection at 220 nm;

[0047] Figure 4 The HPLC characteristic fingerprint spectrum of the control was obtained by detection at 237 nm;

[0048] Figure 5 ​​​​HPLC characteristic fingerprint of the reference of Kangshou pills obtained with detection at 237 nm;

[0049] Figure 6 HPLC characteristic fingerprint of 30 batches of Kangshou pills obtained with detection at 237 nm;

[0050] Figure 7 HPLC characteristic fingerprint of the reference obtained with detection at 246 nm;

[0051] Figure 8 HPLC characteristic fingerprint of the reference of Kangshou pills obtained with detection at 246 nm;

[0052] Figure 9 HPLC characteristic fingerprint of 30 batches of Kangshou pills obtained with detection at 246 nm;

[0053] Figure 10 HPLC characteristic fingerprint of the reference obtained with detection at 265 nm;

[0054] Figure 11 HPLC characteristic fingerprint of the reference of Kangshou pills obtained with detection at 265 nm;

[0055] Figure 12 HPLC characteristic fingerprint of 30 batches of Kangshou pills obtained with detection at 265 nm;

[0056] Figure 13 HPLC characteristic fingerprint of the reference obtained with detection at 278 nm;

[0057] Figure 14 HPLC characteristic fingerprint of the reference of Kangshou pills obtained with detection at 278 nm;

[0058] Figure 15 HPLC characteristic fingerprint of 30 batches of Kangshou pills obtained with detection at 278 nm;

[0059] Figure 16 HPLC characteristic fingerprint of the reference obtained with detection at 300 nm;

[0060] Figure 17 HPLC characteristic fingerprint of the reference of Kangshou pills obtained with detection at 300 nm;

[0061] Figure 18 HPLC characteristic fingerprint of 30 batches of Kangshou pills obtained with detection at 300 nm;

[0062] Figure 19 HPLC characteristic fingerprint of the reference of Kangshou pills obtained with detection at 6 wavelengths;

[0063] Figure 20 To obtain the HPLC characteristic fingerprints of 30 batches of Kangshou Pill preparations using six-wavelength fusion;

[0064] Figure 21 The TQ values ​​of Kangshou Pill under different conditions;

[0065] Figure 22 The UV absorption spectra of THSG and emodin are shown.

[0066] Figure 23 A three-dimensional spectral chromatogram;

[0067] Figure 24 The chromatograms of S1 before and after six-wavelength fusion are shown.

[0068] Figure 25 SWFFP and reference fingerprint (RFP) of 30 batches of Kangshou Pill samples;

[0069] Figure 26 Hierarchical cluster analysis (HCA) of 30 batches of samples;

[0070] Figure 27 For different wavelengths and SWIFFP S m P m and the change of α;

[0071] Figure 28 For S m P m PCA load diagrams for α and α. Detailed Implementation

[0072] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0073] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0074] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0075] Example:

[0076] 1. Preparation of sample and reference solutions

[0077] Reference solution: THSG (purity ≥98%, from Chengdu Desite Biomedical) and emodin (purity ≥98%, from China Institute for Food and Drug Control) reference substances were accurately weighed, dissolved in 80% methanol aqueous solution to prepare a reference solution containing THSG 0.12 mg / mL and emodin 0.04 mg / mL. About 1.0 g of Kangshou pills (Kangshou pill samples (30 batches) and standard preparations were both from Guangzhou Baiyunshan Jingmengtang Pharmaceutical Co., Ltd.) powder (passed through a 50-mesh sieve) was accurately weighed into a 10-mL volumetric flask, 80% methanol was added to the mark, and ultrasonic treatment (power 240 W, frequency 40 kHz) was performed for 30 min. After cooling to room temperature, it was shaken uniformly, and then filtered through a 0.45-μm microporous filter. The filtrate was taken as the test solution.

[0078] 2. Instruments and HPLC chromatographic conditions

[0079] Agilent 1100 high-performance liquid chromatography system (Agilent Technologies, USA) was used, equipped with an online degassing machine, a low-pressure four-element gradient pump, an automatic sampler, and a diode array detector (DAD). The chromatographic column was Packed Column 5C18-MS-II (inner diameter 4.6 mm x 250 mm, particle size 5 μm), and the column temperature was 35°C. The mobile phase: A phase was 0.2% phosphoric acid aqueous solution (v / v) containing 5 mM 1-heptanesulfonic acid sodium, and B phase was methanol; the flow rate was 1.0 mL / min, and the gradient elution program was: 0-5 min (5%-15% B), 5-25 min (15%-25% B), 25-35 min (25%-50% B), 35-50 min (50%-80% B), 50-60 min (80%-90% B), and 60-65 min (90%-5% B). The injection volume was 10 μL. The DAD detection wavelength was set to 220, 237, 246, 265, 278, 300 nm, of which 265 nm was used as the quantitative detection wavelength.

[0080] 3. Data processing

[0081] HPLC fingerprint data was analyzed by "Traditional Chinese Medicine Spectrum Quantization Consistency Digital Evaluation System 4.0" (TCM-SQPC-DES4.0, registration number 7037415, Beijing Yada Consistency Technology). SPSS 22.0 statistical software (IBM Corporation, USA) and Origin 2021 were used for data visualization.

[0082] 4. System quantitative fingerprint method (SQFM) theory

[0083] SQFM is a quality control technology based on the evaluation of sample quality consistency of the whole fingerprint chemical composition. This method compares the sample fingerprint (SFP) with the reference fingerprint (RFP), calculates the similarity parameter (S m ), and further calculates the fingerprint projection content (P).m The mathematical expression for achieving quantitative assessment of full-spectrum components is as follows:

[0084]

[0085] In equations (1) and (2), the variable x i and y i These represent the fingerprint peak areas of the sample fingerprint (SFP) and the reference fingerprint (RFP), respectively. Variable S F and S′ F , representing the similarity before and after fingerprint peak correction, respectively. Variables C and P represent the projection content similarity and quantitative similarity, respectively. The fingerprint signal homogenization coefficient γ is a reliable indicator for measuring the degree of homogenization of the chemical fingerprint signal distribution. When γ approaches 1, the size of each fingerprint signal becomes more uniform. As shown in equation (3), the γ of the sample x Compared with standard fingerprint spectrum γ y The closer the samples are, the higher their similarity to the standard fingerprint. Based on γ... y As a comparison standard, the sample's γ x The relative deviation α is defined as the coefficient of variation of fingerprint homogeneity. Using S... m P m SQFM classifies samples into eight quality grades, as shown in Table 1. Grades 1 to 8 represent spectra from best to worst quality. Samples of the same quality grade have similar chemical fingerprint characteristics, while significant changes in quality grade indicate significant differences in quality.

[0086] Table 1. Quality Grades Assessed by SQFM and Corresponding Acceptance Standards

[0087]

[0088] 5. Experimental Results

[0089] (1) HPLC chromatogram

[0090] The HPLC characteristic fingerprints of the reference standard, Kangshouwan standard preparation, and 30 batches of Kangshouwan preparations obtained by detection at 220 nm are as follows: Figures 1 to 3 As shown, the HPLC characteristic fingerprints of the reference standard, Kangshou Pill standard preparation, and 30 batches of Kangshou Pill preparations obtained by detection at 237 nm are as follows: Figures 4 to 6 As shown; the HPLC characteristic fingerprints of the reference standard, Kangshou Pill standard preparation, and 30 batches of Kangshou Pill preparations obtained by detection at 246 nm are as follows. Figures 7 to 9 As shown, the HPLC characteristic fingerprints of the reference standard, Kangshou Pill standard preparation, and 30 batches of Kangshou Pill preparations obtained by detection at 265nm are as follows: Figures 10 to 12As shown, the HPLC characteristic fingerprints of the reference standard, Kangshou Pill standard preparation, and 30 batches of Kangshou Pill preparations obtained by detection at 278 nm are as follows: Figures 13 to 15 As shown, the HPLC characteristic fingerprints of the reference standard, Kangshou Pill standard preparation, and 30 batches of Kangshou Pill preparations obtained by detection at 300 nm are as follows: Figures 16 to 18 As shown, the HPLC characteristic fingerprints of the Kangshou Pill standard preparation and 30 batches of Kangshou Pill preparations were obtained by six-wavelength fusion. Figure 19 and Figure 20 As shown.

[0091] (2) Condition optimization and methodology verification

[0092] The effects of three mobile phases and four extraction methods on fingerprint peak resolution and response values ​​were investigated using high-performance liquid chromatography (HPLC). Total mass (TQ) values ​​under different conditions were calculated using software. Three dilution methods and four extraction methods were evaluated to optimize chromatographic information acquisition and peak separation while shortening analysis time. Higher TQ values ​​indicated better peak resolution and response values. Results are as follows: Figure 21 A to Figure 21 As shown in B, mobile phase 2 and extraction method 4 had the highest TQ values, so these two methods were ultimately chosen for analysis.

[0093] By comparing the retention times and UV absorption spectra of the sample and standard at 265 nm, two biomarkers were identified: peak 9 was THSG, and peak 12 was emodin. Figure 22 A to Figure 22 B). The THSG peak exhibited excellent resolution and a large response value at all wavelengths, making it suitable as a reference peak. The applicability of the established HPLC method was evaluated using S10, examining precision, stability, and reproducibility. The RSD values ​​for relative peak areas were all below 3.7%, and the RSD values ​​for retention times were all below 0.66%, indicating good instrument precision, good method reproducibility, and good sample stability. These results demonstrate that the developed HPLC method is feasible and efficient for evaluating the quality of Kangshou Pills.

[0094] (3) Establishment and evaluation of SWFFP

[0095] Fingerprints were acquired at six detection wavelengths (220 nm, 237 nm, 246 nm, 265 nm, 278 nm, and 300 nm) and analyzed using the software listed in the "Data Processing" section. The "cdf" files of 30 batches of samples at each single wavelength were loaded into the software, and peaks were aligned to generate reference fingerprints (RFPs). SQFM was used to evaluate the quantitative similarity index P. m An evaluation was conducted. Significant changes in sample grade were observed at different wavelengths. A three-dimensional spectral chromatogram was generated. Figure 23To provide more comprehensive peak information, fingerprint spectra from 30 batches of samples at six wavelengths were fused to overcome the limitations and biases of single-wavelength evaluation. The fusion process was performed using software, employing a maximum peak mode lasting 0.2 minutes. SWFFP exhibited UV absorption peaks at all wavelengths. The fingerprint spectra of the test subjects before and after fusion are shown below. Figure 24 As shown, the combined fingerprint profiles of 30 batches of samples are as follows: Figure 25 As shown. Twenty-six peaks of SWFFP were designated as representative peaks. The peak areas of these 26 representative peaks were used as variables, and hierarchical cluster analysis (HCA) was performed on 30 batch samples using software. Figure 26 The results were divided into three distinct groups: Group 1 (S1, S2, S6, S17, S18, and S19), Group 2 (S3, S4, and S5), and Group 3 (the remaining samples). Samples within each group showed roughly similar compositional levels.

[0096] SWFFP was evaluated using SQFM (Table 2). SWFFP provides more extensive peak information, enabling the fusion evaluation to more accurately reflect changes in sample quality levels compared to a single wavelength. Figure 27 ). All 30 batches of Kangshou Pills - HPLC S m The values ​​all exceeded 0.9, while P m The value is between 80% and 120%. In Origin 2021, using S... m P m Principal component analysis (PCA) was performed with α as variables. The cumulative variance contribution of PC1 (53.0%) and PC2 (28.8%) was 81.8%, capturing most of the sample information. Figure 28 In the PCA analysis, S21 was identified as an outlier, while the remaining groups were consistent with the HCA results, providing evidence for the accuracy of the fingerprint results in the SQFM assessment.

[0097] Table 2. SWFFP Assessment

[0098]

[0099]

[0100] (4) Reliability assessment of the HPLC fingerprint of Kangshou Pill

[0101] The SWFFP data was evaluated by examining the uncertainty and reliability of the reference fingerprint (RFP) and the fingerprints of each batch of samples being evaluated. The error propagation principle was used to define a standard fingerprint and accurately and quantitatively assess the reliability of the identification results for each sample batch. SWFFP data was obtained using 30 sample batches. m and P mThe uncertainty and reliability of the Kangshouwan-HPLC-reference fingerprint (RFP) were calculated. Qualitative reliability exceeded 0.9962 (Table 3), indicating high reliability of the reference fingerprint in terms of the number and proportion of chemical fingerprints. Quantitative reliability exceeded 0.9697, confirming the high reliability of the full content provided by the reference fingerprint when using this reference fingerprint (RFP) for quantitative assessment. Furthermore, the macroscopic qualitative and macroscopic quantitative reliability of the Kangshouwan-HPLC-reference fingerprint (RFP) based on the average fingerprint of 30 sample batches exceeded 0.9820, indicating that the reference fingerprint is accurate and reliable as a standard for evaluating individual sample batches.

[0102] Table 3. Qualitative and quantitative uncertainties and reliability of Kangshou Pill-HPLC-reference fingerprint (RFP)

[0103]

[0104] Based on the uncertainties of 30 batches of Kangshou Pill-HPLC fingerprint chromatograms and their corresponding control fingerprint chromatograms, the uncertainty and reliability of each batch's fingerprint chromatogram were evaluated. Qualitative uncertainty results (Table 4) were significantly higher (>0.1) for S1 (level 4), S15 (level 3), S16 (level 3), S17 (level 4), S18 (level 4), S19 (level 3), and S23 (level 3). In contrast, quantitative reliability values ​​remained consistently high (>0.9), and the overall evaluation results for all 30 sample batches showed high reliability (S... Ri >0.85, P Ri >0.9). Reliability is independent of quality grade, indicating that it is an independent indicator in the quality assessment of traditional Chinese medicine. Therefore, reliability analysis is an important component of quality assessment, providing a more comprehensive and accurate evaluation for the quality control of traditional Chinese medicine.

[0105] Table 4. Qualitative and quantitative uncertainties and reliability data of 30 batches of Kangshou Pills-HPLC-SWFFP.

[0106]

[0107] (5) Determination of the content of two quality markers

[0108] A standard curve was established at 265 nm (Table 5), and the contents of two quality markers (Q-markers), THSG and emodin, were determined simultaneously to assess sample quality. The content values ​​(μg / mL) are listed in Table 6. THSG contents were relatively high, ranging from 163.20 to 102.19 μg / mL, with S1 and S18 exceeding 160.00 μg / mL. THSG contents of S16, S23, and S28 were all below 105.00 μg / mL. Emodin contents fluctuated less, ranging from 31.62 to 49.96 μg / mL. The content of the two major components (m...) of each sample was calculated. i ) and the average content of the two components in all samples (m i ,P i The ratio of equation (4) is used to calculate P using equation (5). 2C (%), the results are listed in Table 6. Evaluation parameters (P) m and P 2C Correlation analysis between the components yielded a Pearson correlation coefficient of 0.752, which is significantly correlated at the 0.01 level (two-tailed). Therefore, the quantitative components have a significant impact on the quality control of Kangshou Pills.

[0109]

[0110] Table 5. Linear equations, correlation (r), linear range, and recovery rate results of the two-index components.

[0111]

[0112] Table 6. Content of 2 quality markers (μg / mL)

[0113]

[0114]

[0115] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for detecting Kangshou Pill using a six-wavelength fusion HPLC fingerprint, characterized in that: The detection method includes the following steps: Step 1: Preparation of test solution: Take Kangshou pill powder, add methanol aqueous solution, and extract by ultrasonic treatment. Filter, take the filtrate, and obtain the test solution. Step 2: Preparation of reference solution: Take 2,3,5,4'-tetrahydroxystilbene glycoside reference standard (THSG) and emodin, and dissolve them in methanol aqueous solution to prepare a mixed reference solution; Step 3: Determination: The test solution and the reference solution were determined separately using high-performance liquid chromatography (HPLC) under the same chromatographic conditions to obtain the fingerprint chromatograms of the test solution and the reference solution. The fingerprint chromatograms of the test solution and the reference solution were compared, and the index components in the fingerprint chromatogram of the test solution were assigned and located to obtain the fingerprint chromatogram of Kangshou Pill. The determination conditions for the high-performance liquid chromatography method are as follows: Chromatographic column: Octadecylsilane-bonded silica gel column; Column temperature: 30-40℃; Injection volume: 5-10 μL; Mobile phase: Mobile phase A is a 0.1-0.3% aqueous solution of phosphoric acid containing 0.003-0.01 mol / L sodium heptanesulfonate; Mobile phase B is methanol; Gradient elution was used, with all mobile phase proportions being volume percentages. The elution procedure is as follows: Detector: Diode array detector; Detection wavelengths: 220nm, 237nm, 246nm, 265nm, 278nm and 300nm, of which 265nm is used as the quantitative detection wavelength to determine the content of THSG and emodin.

2. The detection method according to claim 1, characterized in that: In step 1, take about 1.0g of Kangshou Pill powder, accurately weigh it, put it in a 10mL volumetric flask, add 80% methanol aqueous solution to the mark, sonicate for 30min, cool to room temperature and shake well, filter through a 0.45μm microporous membrane, and take the filtrate as the test solution.

3. The detection method according to claim 1, characterized in that: In step 2, THSG and emodin reference standards are accurately weighed and dissolved in 80% methanol aqueous solution to prepare a reference solution containing 0.12 mg / mL THSG and 0.04 mg / mL emodin.

4. The detection method according to claim 1, characterized in that: In step 3, the determination conditions for the high-performance liquid chromatography are as follows: Chromatographic column: Packed Column 5C18-MS-Ⅱ, inner diameter 4.6mm×250mm, particle size 5μm; Column temperature: 35℃; Injection volume: 10 μL; Mobile phase: Mobile phase A is a 0.2% aqueous solution of phosphoric acid containing 0.005 mol / L sodium heptanesulfonate; Mobile phase B is methanol.

5. A method for constructing a standard fingerprint spectrum for Kangshou Pill, characterized in that: The method includes: detecting multiple batches of Kangshou Pill samples using the six-wavelength fusion HPLC fingerprint chromatogram detection method of Kangshou Pill as described in any one of claims 1 to 4, obtaining fingerprint chromatograms of multiple batches of Kangshou Pill samples to generate a common pattern control chromatogram, using the chromatographic peaks present in all chromatograms as common characteristic peaks, determining the relative retention time of the common characteristic peaks and the ratio of the area of ​​each common characteristic peak to the total peak area, and assigning and locating the index components in the Kangshou Pill fingerprint chromatogram according to the relative retention time, and establishing a standard fingerprint chromatogram of Kangshou Pill.

6. The method for constructing a standard fingerprint spectrum according to claim 5, characterized in that: The number of common fingerprint peaks in the Kangshou Pill sample at detection wavelengths of 220nm, 237nm, 246nm, 265nm, 278nm, and 300nm were 14, 8, 9, 13, 14, and 11, respectively. Based on the peak area and retention time of the common fingerprint peaks, the macro-qualitative similarity of the fingerprint spectrum of the Kangshou Pill sample should not be lower than 0.90, and the macro-quantitative similarity should be between 80% and 120%.

7. The method for constructing a standard fingerprint spectrum according to claim 5, characterized in that: The 2,3,5,4′-tetrahydroxystilbene-2-O-β-glucopyranoside peak is used for the identification of common fingerprint peaks and is also a reference quantitative measurement correction peak. When detected at a wavelength of 220 nm, peak 5 is 2,3,5,4′-tetrahydroxystilbene-2-O-β-glucopyranoside; When detected at a wavelength of 237 nm, peak 3 is 2,3,5,4′-tetrahydroxystilbene-2-O-β-glucopyranoside; When detected at a wavelength of 246 nm, peak 4 is 2,3,5,4′-tetrahydroxystilbene-2-O-β-glucopyranoside; When detected at wavelengths of 265 nm and 278 nm, peak 9 is 2,3,5,4′-tetrahydroxystilbene-2-O-β-glucopyranoside; and When detected at a wavelength of 300 nm, peak 9 is 2,3,5,4′-tetrahydroxystilbene-2-O-β-glucopyranoside.

8. A method for quality testing of Kangshou Pills, characterized in that: The method includes obtaining a fingerprint spectrum of a Kangshou Pill sample using the six-wavelength fusion HPLC fingerprint spectrum detection method of Kangshou Pill as described in any one of claims 1 to 4, comparing the similarity with the Kangshou Pill standard fingerprint spectrum obtained using the Kangshou Pill standard fingerprint spectrum construction method as described in any one of claims 5 to 7, and comparing the contents of THSG and emodin in the Kangshou Pill sample with those of the Kangshou Pill standard preparation.

9. The quality inspection method according to claim 8, characterized in that: Systematic quantitative fingerprinting (SQFM) was performed, comparing the sample fingerprint (SFP) with the reference fingerprint (RFP), and the similarity parameter (S) was calculated. m Further analysis was conducted using fingerprint projection content (P). m To achieve quantitative evaluation of full-spectrum components.

10. The quality inspection method according to claim 9, characterized in that: The formula for calculating SQFM is as follows: variable x i and y i S represents the fingerprint peak areas of the sample fingerprint (SFP) and the reference fingerprint (RFP), respectively. F and S′ F Let represent the similarity before and after fingerprint peak correction, respectively. Variables C and P represent the similarity of projected content and quantitative similarity, respectively. The fingerprint signal homogenization coefficient γ is a reliable indicator of the homogenization degree of chemical fingerprint signal distribution. When γ approaches 1, the magnitude of each fingerprint signal becomes more uniform. y As a comparison standard, the sample's γ x The relative deviation α is defined as the coefficient of variation of fingerprint homogeneity, using S m P m SQFM classifies samples into multiple quality levels, with samples of the same quality level having similar chemical fingerprint characteristics, while significant changes in quality level indicate significant differences in quality.

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