Vigour-preserving decoction fingerprint spectrum, multi-component quantitative analysis method and application of vigour-preserving decoction fingerprint spectrum

Abstract

The invention discloses a vitality-preserving decoction fingerprint spectrum and multi-component quantitative analysis method and application thereof, a DAD and evaporative light-scattering detector (ELSD) combined scheme is adopted, according to different properties of components, variable wavelengths are adopted for determination in different time periods, the detection process is simplified, single sample introduction is realized through series connection of detectors, and the detection accuracy is improved. Meanwhile, a plurality of medicinal components are represented, and a multi-component fingerprint spectrum can be directly generated, so that the quality control is more comprehensive and efficient, the detection efficiency is improved, and time and resources are saved.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug and food detection, and specifically relates to the establishment and application of a fingerprint spectrum of Baoyuan Decoction product and a multi-component quantitative analysis method. Background Art

[0002] Baoyuan Decoction, a traditional classic prescription, possesses significant medicinal value and extensive clinical application. From the Ming Dynasty to the present day, its indications range from childhood acne to relieving vital energy deficiency. It has since evolved through medical practice into a classic TCM formula for tonifying Qi and nourishing the vital energy. Its formula, which includes five herbs: ginseng, astragalus, licorice, cinnamon, and ginger, has a wide range of clinical indications, including those for wasting conditions such as coronary heart disease, heart failure, aplastic anemia, chronic hepatitis, and chronic nephritis. It is also used for postpartum weakness and post-heatstroke conditioning. Its mechanism of action not only replenishes Qi and nourishes the vital energy, but also, through its antioxidant properties, slows the aging process and enhances overall body function. With the continuous development of the health industry, the five herbs used in Baoyuan Decoction are also being incorporated into health supplements and foods, offering promising prospects for development in both pharmaceutical and food products.

[0003] Baoyuan Decoction, a classic traditional Chinese medicine formula, requires quality control to consider the multiple active ingredients of various herbs, including ginseng, astragalus, and licorice. Due to its complex composition, current testing and analysis methods suffer from difficulties in balancing testing conditions for different components, lengthy testing times, incomplete quality control indicators, and a cumbersome testing process.

[0004] Chinese invention patent application CN 112903882A discloses a method for constructing an HPLC characteristic spectrum of a Baoyuan Decoction preparation. However, the detection time is as long as 110 minutes. The detection wavelength is only measured at 203 nm, and the absorption of the chromatographic peaks of some components is weak. At the same time, quantitative analysis of multiple components is not carried out simultaneously.

[0005] Chinese invention patent CN 114184719B discloses a method for establishing a dual-wavelength fingerprint of Baoyuan Decoction. This method requires complex pre-processing, is time-consuming and labor-intensive, and takes up to 120 minutes to detect. The method only identifies six characteristic peaks at dual wavelengths of 203nm and 254nm, resulting in fewer components that can characterize product quality. Furthermore, the chromatographic peaks of some components exhibit weak absorption at the aforementioned wavelengths. Simultaneously, quantitative analysis of multiple components is not performed simultaneously, making it impossible to conduct comprehensive quality control of Baoyuan Decoction.

[0006] Chinese invention patent CN 111443142B discloses a method for the simultaneous detection of multiple index components in Baoyuan Decoction preparations. This method uses a DAD detector to simultaneously determine the content of different index components at 203 nm, 260 nm, 290 nm, and 360 nm. This method requires different sample pretreatment methods and repeated injections to achieve simultaneous quantification of multiple index components, making the operation relatively cumbersome. Furthermore, the chromatographic peak shape of high-polarity and low-polarity components at 203 nm is poor, making it difficult to construct a fingerprint that characterizes product quality attributes. Chromatographic conditions that simultaneously achieve fingerprint analysis and multi-component quantitative analysis remain to be developed.

[0007] Therefore, the development of a convenient, efficient and stable analytical method for the simultaneous determination of Baoyuan Decoction fingerprint and multiple key indicator components is of great significance for the quality control of Baoyuan Decoction material standards, preparations and related foods. Summary of the Invention

[0008] The present invention addresses the problems of the prior art and provides a fingerprint and multi-component quantitative analysis method for Baoyuan Decoction, as well as its application. Using a dual detector system (DAD variable wavelength-ELSD) coupled with a detector, rapid characterization of multiple active ingredients in ginseng, astragalus, licorice, and ginger is achieved. Quantitative analysis of 11 components, including calycosin glucoside, apigenin, liquiritin, formononetin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, formononetin, ammonium glycyrrhizate, 6-gingerol, and astragaloside IV, is achieved. A fingerprint of Baoyuan Decoction is established, and this analytical method can serve as an efficient tool for comprehensive quality control of Baoyuan Decoction as a pharmaceutical or food product.

[0009] One of the purposes of the present invention is to provide a convenient, efficient and stable analytical method for the simultaneous determination of the fingerprint of Baoyuan Decoction and multiple key indicator components. A DAD and an evaporative light scattering detector (ELSD) are used in combination, and variable wavelengths are used for determination at different time periods according to the different properties of the components, which simplifies the detection process. A single injection is achieved by connecting the detectors in series, and multiple medicinal components are characterized at the same time. A multi-component fingerprint can be directly generated, making quality control more comprehensive and efficient, improving detection efficiency, and saving time and resources.

[0010] A fingerprint spectrum and multi-component quantitative analysis method of Baoyuan Decoction comprises the following steps:

[0011] (1) Preparation of test solution: Weigh the Baoyuan Decoction substance standard, preparation or food sample to prepare the test solution.

[0012] The present invention determines the best test sample preparation method by comparing the extraction rate, peak appearance and separation degree of multiple index components in the test sample solution obtained by different test sample pretreatment methods.

[0013] The present invention does not require the design of independent sample pretreatment methods for different index components, and the content determination of multiple index components can be achieved by sharing the same sample pretreatment method.

[0014] Preferably, the preparation method of the test solution is as follows: accurately weigh about 0.5 g of the Baoyuan Decoction substance standard, preparation or food sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 80% methanol containing 4% concentrated ammonia test solution, weigh it, ultrasonically treat it for 30 minutes, cool it, make up for the weight loss, shake it well, filter it, accurately measure 25 mL of the filtrate, evaporate it to dryness, dissolve the residue with 80% methanol and transfer it to a 10 ml volumetric flask, add 80% methanol to the volume, shake it well, and filter it with a 0.22 um filter membrane to obtain the test solution.

[0015] (2) Preparation of reference solution: Accurately weigh appropriate amounts of calycosin glucoside, apiosyl liquiritin, liquiritin, formononetin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, formononetin, ammonium glycyrrhizate, 6-gingerol, and astragaloside IV reference substances, and add solvent to prepare mixed reference solutions of different concentrations.

[0016] Preferably, the solvent for preparing the reference substance is methanol.

[0017] Preferably, the concentrations of the index components in the mixed reference substance are: 1.828-18.2758 mg / L of calycoside isoflavone glucoside, 10.1774-101.7739 mg / L of apiosyl liquiritin, 22.2680-222.6800 mg / L of liquiritin, 2.1364-21.3640 mg / L of formononetin, 11.7312-117.312 mg / L of ginsenoside Rg1, 9.8304-98.3040 mg / L of ginsenoside Rb1, 21.3785-213.7848mg / L, formononetin 2.4883-24.8832mg / L, ammonium glycyrrhizate 30.2863-302.8628mg / L, 6-gingerol 2.1029-21.0290mg / L, astragaloside IV 60.6060-606.06mg / L.

[0018] (3) Chromatographic analysis conditions: octadecylsilane bonded silica gel as the filler of the chromatographic column, acetonitrile as the mobile phase A and water as the mobile phase B for gradient elution, dynamic switching wavelength as the detection wavelength, injection volume of 1 μl, and detector using a DAD-ELSD combination.

[0019] The present invention investigates the mobile phase system, and the results show that when acetonitrile-water is used as the mobile phase system, the separation effect of the target components in the chromatogram is better. When acetic acid and formic acid are added, the chromatogram baseline drifts seriously, and when phosphoric acid is added, the requirements of the ELSD detector cannot be met. Therefore, acetonitrile is preferably used as mobile phase A and water is used as mobile phase B for gradient elution.

[0020] Preferably, the gradient elution program is as follows:

[0021]

[0022] In the present invention, the full wavelength scanning of the DAD detector is performed and the detection wavelength is dynamically switched during the analysis process according to the absorption characteristics of the target component, so as to improve the response sensitivity of different components and reduce the interference of non-target components.

[0023] According to the gradient elution procedure, the present invention automatically switches to the corresponding wavelength range 1 minute before the target chromatographic peak appears, avoiding baseline drift and reducing data acquisition delay, while achieving continuous and accurate quantification of multiple target components.

[0024] Preferably, the specific method of dynamically switching the wavelength is: 0-23 min, 237 nm; 23-26 min, 203 nm; 26-28 min, 237 nm; 28-35 min, 203 nm; this method can also be applied to VWD detectors.

[0025] The present invention adopts a scheme of combining DAD and evaporative light scattering detector (ELSD). The two different detectors can simultaneously detect compounds of different properties. The two detectors can also work simultaneously on the same instrument to analyze and process the same sample, eliminating the need for multiple injections, improving detection efficiency and saving time and resources.

[0026] Preferably, the detector is a combination of DAD and ELSD. The test sample first passes through the DAD detector for multiple component analysis. The measurement process can be used to construct and synchronously obtain the Baoyuan Decoction fingerprint. After DAD variable wavelength detection, it flows through the ELSD detector for astragaloside analysis.

[0027] The present invention also investigated and screened different models and brands of chromatographic columns. Taking into account the characteristics of each target component and instrument, Waters ACQUITY UPLC HSS T3, Agilent AQ-C18, Endeavorsil C18, Excsep TM Separation of target components using UHPLC SCB-C18 and other columns.

[0028] Preferably, the chromatographic column model is Excsep TMUHPLC SCB-C18.

[0029] Preferably, the chromatographic column has a specification of 2.1*150 mm and a filler diameter of 1.8 μm.

[0030] The present invention also investigates the effects of different column temperatures (20°C, 25°C, 30°C) and different flow rates (0.2 mL / min, 0.3 mL / min, 0.4 mL / min) on the separation degree of each target component.

[0031] Preferably, the flow rate is 0.3 mL / min and the column temperature is 25°C.

[0032] Preferably, the carrier gas flow rate of the ELSD detector in step (3) is 2.8 L / min, and the drift tube temperature is 105°C.

[0033] The above-mentioned preferred gradient elution program and various chromatographic conditions can improve the separation, symmetry and sensitivity of the chromatographic peaks of each target component, reduce the interference of non-target components, and thus increase the accuracy of the quantitative determination of each target component.

[0034] The second purpose of the present invention relates to the application of the above-mentioned analytical method in the quality control of Baoyuan Decoction material standards, Baoyuan Decoction preparations, and Baoyuan Decoction foods.

[0035] The beneficial effects produced by the present invention are:

[0036] 1. More comprehensive quality characterization: The present invention adopts a combination of DAD variable wavelength and evaporative light scattering detector (ELSD). The analysis method is fast and efficient, with good chromatographic separation effect. It can realize the determination of multiple active ingredients such as ginseng, astragalus, licorice, and ginger, and simultaneously generate the Baoyuan Decoction fingerprint, which is more conducive to the comprehensive evaluation of Baoyuan Decoction product quality.

[0037] 2. The detection process is more convenient and efficient: It can simultaneously take into account the chemical components with different absorption wavelengths and the index components without UV absorption in Baoyuan Decoction. The components that need to be measured multiple times in the early stage can be measured once, avoiding multiple handling of the test samples, effectively reducing the number of injections, and significantly shortening the analysis time. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The chromatograms of the blank solution, mixed reference solution, and test solution under the specificity item of Example 2 under the DAD detector are as follows: for the blank solution (a), the mixed reference solution (b), and the test solution (c), peak 1 is calycosin glucoside, peak 2 is apigenin, peak 3 is liquiritin, peak 4 is formononetin, peak 5 is ginsenoside Rg1, peak 6 is ginsenoside Re, peak 7 is ginsenoside Rb1, peak 8 is formononetin, peak 9 is glycyrrhizic acid, and peak 10 is 6-gingerol;

[0039] Figure 2 The chromatograms of the blank solution, astragaloside IV reference solution, and test solution under the specificity item of Example 2 under the ELSD detector are shown, wherein the blank solution (a-1), astragaloside IV reference solution (b-1), and test solution (c-1), peak 11 is astragaloside IV;

[0040] Figure 3 The chromatograms of the blank solution, mixed reference solution, test solution, negative test solution (d) without ginseng, and single ginseng medicinal material test solution (e) under the specificity item of Example 2 under the DAD detector conditions are shown;

[0041] Figure 4 The chromatograms of the blank solution, mixed reference solution, test solution, negative test solution (f) without astragalus, and single astragalus test solution (g) under the specificity item of Example 2 under DAD detector conditions are shown;

[0042] Figure 5 The chromatograms of the blank solution, mixed reference solution, test solution, negative test solution (h) without licorice, and single licorice medicinal material test solution (i) under the specificity item of Example 2 under DAD detector conditions are shown;

[0043] Figure 6 The chromatograms of the blank solution, mixed reference solution, test solution, negative test solution without ginger (j) and ginger single medicinal material test solution (k) under the specificity item of Example 2 under the DAD detector conditions are shown;

[0044] Figure 7 This is the superposition of the standard fingerprints of 18 batches of Baoyuan Decoction substances in Example 4;

[0045] Figure 8 This is an overlay of fingerprints of 9 batches of samples of different Baoyuan decoctions in Example 5;

[0046] Figure 9 This is the chromatogram of the test solution of Comparative Example 1 under DAD detector conditions;

[0047] Figure 10 This is the chromatogram of the test solution of Comparative Example 2 under DAD detector conditions;

[0048] Figure 11 This is the chromatogram of the test solution of Comparative Example 3 under DAD detector conditions;

[0049] Figure 12 This is the chromatogram of the test solution of Comparative Example 4 under DAD detector conditions;

[0050] Figure 13This is the chromatogram of the test solution of Comparative Example 5 under DAD detector conditions;

[0051] Figure 14 This is the chromatogram of the test solution of Comparative Example 6 under DAD detector conditions;

[0052] Figure 15 This is the chromatogram of the test solution of Comparative Example 7 under DAD detector conditions;

[0053] Figure 16 This is the chromatogram of the test solution of Comparative Example 8 under DAD detector conditions;

[0054] Figure 17 This is the chromatogram of the test solution of Comparative Example 9 under DAD detector conditions;

[0055] Figure 18 This is a comparison chart of the astragaloside reference solution of Comparative Example 10 and Comparative Example 11 under the conditions of DAD and ELSD detectors;

[0056] Figure 19 The chromatogram of the mixed reference solution (b-2) and the test solution (c-1) of Comparative Example 11 under ELSD detector conditions;

[0057] Figure 20 This is the chromatogram of the astragaloside reference solution (b-1) and the test solution (c-2) of Comparative Example 12 under ELSD detector conditions. DETAILED DESCRIPTION

[0058] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0059] Example 1

[0060] 1.1 Preparation of test solution

[0061] Add 11.19g of ginseng, 22.38g of astragalus, 5.61g of licorice, 2.25g of cinnamon, and 9g of ginger to 1200mL of pure water in a decoction pot and soak for 30 minutes. Heat to boiling and then simmer on low heat until the volume reaches 500mL. Filter the extract while it is hot. Add 900mL of pure water to the residue and decoct according to the above steps until the extract volume reaches 400mL. Filter and combine the extracts while they are hot and freeze-dry them to obtain freeze-dried powder, which is the material reference sample of Baoyuan Decoction.

[0062] Accurately weigh about 0.5 g of Baoyuan Decoction material reference sample, place it in a stoppered conical flask, accurately add 50 mL of 80% methanol containing 4% concentrated ammonia test solution, weigh, ultrasonicate for 30 minutes, cool, make up to the weight loss, shake well, filter, accurately measure 25 mL of the filtrate, evaporate to dryness, dissolve the residue with 80% methanol and transfer it to a 10 ml volumetric flask, add 80% methanol to the volume, shake well, and filter with a 0.22 μm filter membrane to obtain the test solution.

[0063] 1.2 Preparation of reference solution

[0064] Accurately weigh 4.72 mg of reference substances, including calycosin glucoside, 4.11 mg of apiosyl liquiritin, 11.72 mg of liquiritin, 2.18 mg of formononetin, 6.24 mg of ginsenoside Rg1, 5.12 mg of ginsenoside Re, 11.24 mg of ginsenoside Rb1, 2.56 mg of formononetin, 16.62 mg of glycyrrhizic acid, and 4.21 mg of 6-gingerol, and place them in different 5 mL volumetric flasks. Accurately weigh 6.30 mg of astragaloside IV and place it in a 10 mL volumetric flask. Methanol was added to prepare the reference substance stock solutions of each index component.

[0065] Precisely measure 0.4 mL, 2.5 mL, 1 mL, 2 mL, 2 mL, 2 mL, 1 mL, 2 mL, 2 mL, 1 mL, 2 mL, and 0.5 mL of the reference substance stock solutions of calycosin glucoside, apigenin, liquiritin, formononetin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, formononetin, glycyrrhizic acid, and 6-gingerol, respectively, and place them in a single 20 mL volumetric flask. Dilute to the mark with methanol and shake well to obtain the above 10 mixed reference substance stock solutions. Precisely measure 6 mL, 5 mL, 4 mL, 2 mL, and 1 mL of the mixed reference substance stock solutions, respectively, and place them in separate 10 mL volumetric flasks. Add methanol to prepare mixed reference substance solutions of varying concentrations. Separately, dilute the astragaloside IV reference substance stock solution to obtain reference substance solutions of varying concentrations.

[0066] 1.3 Preparation of negative control solution

[0067] According to the preparation method of Baoyuan Decoction substance reference sample, negative freeze-dried samples without ginseng, astragalus, licorice and ginger slices were prepared respectively, and then the test solution of each missing medicinal flavor was prepared according to the preparation method of the test solution.

[0068] 1.4 Preparation of single medicinal material solution

[0069] According to the preparation method of Baoyuan Decoction material reference sample, freeze-dried samples of ginseng, astragalus, licorice and ginger slices were prepared respectively, and then the test solution of each single medicinal flavor was prepared according to the preparation method of the test solution.

[0070] 1.5 Chromatographic conditions:

[0071] Chromatographic column: Excsep TM UHPLC SCB-C18 (2.1 mm*150 mm, 1.8 μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in Table 1; flow rate, 0.3 mL / min; column temperature, 25°C; injection volume, 1 μL; dynamic wavelength switching: 0-23 min, 237 nm; 23-26 min, 203 nm; 26-28 min, 237 nm; 28-35 min, 203 nm; detector: DAD-ELSD; carrier gas flow rate of the ELSD detector, 2.8 L / min, drift tube temperature: 105°C.

[0072] Table 1 Gradient elution program

[0073]

[0074]

[0075] Example 2

[0076] Verification of the methodological approach for determining the content of multiple index components:

[0077] 2.1 Exclusivity

[0078] According to the chromatographic conditions under item 1.5, the blank solution, test solution, reference solution, negative control solution, and single medicinal sample solution were sampled and analyzed respectively. The results showed that ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 were from ginseng; liquiritin, glycyrrhizic acid, apigenin, and formononetin were from licorice; calycosin glucoside, formononetin, formononetin, and astragaloside IV were from astragalus; 6-gingerol was from ginger; the blank and negative control solutions had no interference at the retention time of each target peak, indicating that the method has good specificity.

[0079] 2.2 Linearity and range

[0080] Accurately pipette the mixed reference solution and astragaloside IV solution of different concentrations under 1.2 respectively, analyze them under the chromatographic conditions under 1.5, record the peak area of ​​each chromatographic peak, draw a standard curve with the logarithm of the reference concentration of astragaloside IV as the abscissa and the logarithm of the corresponding peak area as the ordinate. Draw standard curves for the remaining index components with the reference concentration as the abscissa and the corresponding peak area as the ordinate. Obtain the regression equations for each reference substance. The results are shown in Table 2.

[0081] Table 2 Linear regression equations of each indicator component

[0082]

[0083]

[0084] The results showed that the coefficient of determination of each indicator component within the investigation range was greater than 0.9990, and the linear relationship was good.

[0085] 2.3 Precision test

[0086] Take the Baoyuan Decoction substance reference sample, prepare the test solution according to the method under 1.1, and continuously inject and analyze 6 times according to the chromatographic conditions under 1.5. Record the peak area of ​​each chromatographic peak of the target component and calculate their respective RSD values. The results show that the RSD values ​​of each target component are all less than 2.0%, indicating that the precision of the instrument is good.

[0087] 2.4 Repeatability test

[0088] According to the method under 1.1, 6 test solutions were prepared from the Baoyuan Decoction substance reference sample, and the solutions were determined and analyzed according to the chromatographic conditions under 1.5. The average content and RSD value of each target component were calculated. The results showed that the RSD value of the average content of each target component was 0.21%-1.06%, indicating that the method had good repeatability.

[0089] 2.5 Stability test

[0090] According to the chromatographic conditions under item 1.5, the test sample solution under item 2.3 was sampled and analyzed at 0, 6, 8, 12, 14, 18, and 24 hours, respectively. The peak area of ​​the chromatographic peak of each target component was recorded, and the RSD value of each was calculated to be 0.12%-0.97%. The results showed that the test sample had good stability within 24 hours.

[0091] 2.6 Accuracy test

[0092] Accurately weigh about 0.25 g of Baoyuan Decoction substance reference samples with known content of each target component, a total of 6 portions, and accurately add 100% of the known content of each reference substance stock solution. All of them are prepared into test sample solutions according to the method under 1.1. At the same time, the determination and analysis are carried out under the chromatographic conditions under 1.5. The average recovery rate and RSD value of each index component are calculated. The results are shown in Table 3.

[0093] Table 3 Experimental results of spiked recovery of target components

[0094]

[0095]

[0096]

[0097] The above results show that the recoveries of the target components are between 92.41% and 102.45%, and the RSD values ​​are between 0.49% and 2.83%, indicating that the method has good accuracy.

[0098] Example 3

[0099] Determination of the content of multiple batches of Baoyuan Decoction substance reference samples

[0100] Eighteen batches of Baoyuan Decoction substance reference samples were prepared according to the test sample preparation method under 1.1. At the same time, the mixed reference solution under 1.2 was taken and analyzed under the chromatographic conditions under 1.5. The content of each target component in the 18 batches of test samples was calculated according to the external standard method. The content determination results are shown in Table 4.

[0101] Table 4 Content results of multiple target components in 18 batches of Baoyuan Decoction reference samples

[0102]

[0103]

[0104] From the results in the above table, it can be concluded that there are certain differences in the contents of calycosin glucoside, apigenin, liquiritin, formononetin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, formononetin, glycyrrhizic acid, 6-gingerol and astragaloside IV among different batches of Baoyuan Decoction material benchmarks. In order to carry out comprehensive quality control of Baoyuan Decoction material benchmarks, it is very necessary to accurately quantify the above-mentioned multiple indicator components.

[0105] Example 4

[0106] Fingerprint determination of multiple batches of Baoyuan Decoction substance reference samples

[0107] The Chinese medicine fingerprint similarity evaluation software (version 2012.130723) was used to comprehensively analyze the 18 batches of Baoyuan Decoction substance reference fingerprints measured under the DAD detector in Example 3, and an overlay of the fingerprints between the batches was generated. The similarity results are shown in Table 5.

[0108] Table 5 Similarity results of the reference fingerprints of 18 batches of Baoyuan Decoction substances

[0109]

[0110] From the results in the above table, it can be seen that the similarities of the 18 batches of Baoyuan Decoction substance benchmarks are all above 0.9, indicating that the quality stability among the 18 batches of Baoyuan Decoction substance benchmark samples is good, and also indicating that the fingerprint spectrum detection and analysis method can provide a guarantee for the quality control of the Baoyuan Decoction substance benchmark.

[0111] Example 5

[0112] Comparison of similarity of fingerprints of different Baoyuan Decoction samples

[0113] Three batches of Baoyuan Decoction material reference, Baoyuan Decoction preparation, and Baoyuan Decoction food samples were prepared according to the test sample preparation method under 1.1. At the same time, the mixed reference solution under 1.2 was taken and measured under the chromatographic conditions under 1.5. The DAD detector spectra were then processed and analyzed using the traditional Chinese medicine fingerprint similarity evaluation software to generate an overlay of the fingerprint spectra of different samples. The similarity results are shown in Table 6.

[0114] Table 6 Similarity results of fingerprints of different Baoyuan Decoction samples

[0115]

[0116]

[0117] From the results in the above table, it can be seen that the similarities between different Baoyuan Decoction samples are all greater than 0.9, indicating that this fingerprint analysis method can be widely used in the quality control of Baoyuan Decoction samples.

[0118] Comparative Example 1

[0119] Chromatographic conditions: Column: Excsep TM UHPLC SCB-C18 (2.1 mm*150 mm, 1.8 μm); acetonitrile as mobile phase A, 0.1% acetic acid solution as mobile phase B, gradient elution as specified in Table 1; flow rate, 0.3 mL / min; column temperature, 25°C; injection volume, 1 μL; dynamic wavelength switching: 0-23 min, 237 nm; 23-26 min, 203 nm; 26-28 min, 237 nm; 28-35 min, 203 nm; detector: DAD-ELSD; carrier gas flow rate of the ELSD detector, 2.8 L / min, drift tube temperature: 105°C.

[0120] The same test solution and mixed reference solution as in Example 1 were injected into an ultra-high performance liquid chromatograph for analysis and the chromatograms were recorded simultaneously.

[0121] The test results show that when the water in the mobile phase B in the chromatographic conditions of the present invention is replaced with 0.1% acetic acid and eluted according to the gradient in Table 1, the overall baseline drift in the chromatogram is severe, making it impossible to perform quantitative analysis of the target components.

[0122] Comparative Example 2

[0123] Chromatographic conditions: Column: Excsep TMUHPLC SCB-C18 (2.1 mm*150 mm, 1.8 μm); acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution as specified in Table 1; flow rate 0.3 mL / min; column temperature 25°C; injection volume 1 μL; dynamic switching wavelength: 0-23 min, 237 nm; 23-26 min, 203 nm; 26-28 min, 237 nm; 28-35 min, 203 nm; detector: DAD-ELSD; carrier gas flow rate of the ELSD detector was 2.8 L / min, and the drift tube temperature was 105°C.

[0124] The same test solution and mixed reference solution as in Example 1 were injected into an ultra-high performance liquid chromatograph for analysis and the chromatograms were recorded simultaneously.

[0125] The test results show that when the water in the mobile phase B in the chromatographic conditions of the present invention is replaced with 0.1% formic acid and eluted according to the gradient in Table 1, the overall baseline in the chromatogram drifts severely, making it impossible to perform quantitative analysis of the target components.

[0126] Comparative Example 3

[0127] Chromatographic conditions: Column: Excsep TM UHPLC SCB-C18 (2.1 mm*150 mm, 1.8 μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in Table 1; flow rate, 0.4 mL / min; column temperature, 25°C; injection volume, 1 μL; dynamic wavelength switching: 0-23 min, 237 nm; 23-26 min, 203 nm; 26-28 min, 237 nm; 28-35 min, 203 nm; detector: DAD-ELSD; carrier gas flow rate of the ELSD detector, 2.8 L / min, drift tube temperature: 105°C.

[0128] The same test solution and mixed reference solution as in Example 1 were injected into an ultra-high performance liquid chromatograph for analysis and the chromatograms were recorded simultaneously.

[0129] The experimental results show that when the flow rate in the chromatographic conditions of the present invention is adjusted to 0.4 mL / min, the components such as formononetin, ginsenoside Rg1, and ginsenoside Re in the DAD chromatogram of the test sample cannot be effectively separated.

[0130] Comparative Example 4

[0131] Chromatographic conditions: Column: Excsep TMUHPLC SCB-C18 (2.1 mm*150 mm, 1.8 μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in Table 1; flow rate, 0.2 mL / min; column temperature, 25°C; injection volume, 1 μL; dynamic wavelength switching: 0-23 min, 237 nm; 23-26 min, 203 nm; 26-28 min, 237 nm; 28-35 min, 203 nm; detector: DAD-ELSD; carrier gas flow rate of the ELSD detector, 2.8 L / min, drift tube temperature: 105°C.

[0132] The same test solution and mixed reference solution as in Example 1 were injected into an ultra-high performance liquid chromatograph for analysis and the chromatograms were recorded simultaneously.

[0133] The experimental results show that when the flow rate in the chromatographic conditions of the present invention is adjusted to 0.2 mL / min, the retention times of multiple target peaks in the DAD chromatogram of the test sample shift backward, among which the separation of formononetin and glycyrrhizic acid is poor, and accurate quantification cannot be performed.

[0134] Comparative Example 5

[0135] Chromatographic conditions: Column: Excsep TM UHPLC SCB-C18 (2.1 mm*150 mm, 1.8 μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in Table 1; flow rate, 0.3 mL / min; column temperature, 30°C; injection volume, 1 μL; dynamic wavelength switching: 0-23 min, 237 nm; 23-26 min, 203 nm; 26-28 min, 237 nm; 28-35 min, 203 nm; detector: DAD-ELSD; carrier gas flow rate of the ELSD detector, 2.8 L / min, drift tube temperature: 105°C.

[0136] The same test solution and mixed reference solution as in Example 1 were injected into an ultra-high performance liquid chromatograph for analysis and the chromatograms were recorded simultaneously.

[0137] The test results show that when the column temperature is set to 30°C in the chromatographic conditions of the present invention, formononetin and glycyrrhizic acid synthesize a peak in the DAD chromatogram of the test sample, and cannot be quantified.

[0138] Comparative Example 6

[0139] Chromatographic conditions: Column: Excsep TMUHPLC SCB-C18 (2.1 mm*150 mm, 1.8 μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in Table 1; flow rate, 0.3 mL / min; column temperature, 20°C; injection volume, 1 μL; dynamic wavelength switching: 0-23 min, 237 nm; 23-26 min, 203 nm; 26-28 min, 237 nm; 28-35 min, 203 nm; detector: DAD-ELSD; carrier gas flow rate of the ELSD detector, 2.8 L / min, drift tube temperature: 105°C.

[0140] The same test solution and mixed reference solution as in Example 1 were injected into an ultra-high performance liquid chromatograph for analysis and the chromatograms were recorded simultaneously.

[0141] The test results show that when the column temperature is set to 20°C in the chromatographic conditions of the present invention, the separation degree of formononetin and glycyrrhizic acid in the DAD chromatogram of the test sample is very poor, and they cannot be accurately quantified.

[0142] Comparative Example 7

[0143] Chromatographic conditions: Chromatographic column: Endeavorsil C18 (2.1mm*150mm, 1.8μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in Table 1; flow rate, 0.3mL / min; column temperature, 25℃; injection volume, 1μL; dynamic switching wavelength: 0-23min, 237nm; 23-26min, 203nm; 26-28min, 237nm; 28-35min, 203nm; detector: DAD-ELSD combination; carrier gas flow rate of ELSD detector is 2.8L / min, and drift tube temperature is: 105℃.

[0144] The same test solution and mixed reference solution as in Example 1 were injected into an ultra-high performance liquid chromatograph for analysis and the chromatograms were recorded simultaneously.

[0145] The experimental results show that when the chromatographic column of the present invention is replaced with Endeavorsil C18 (2.1mm*150mm, 1.8μm), the target components formononetin, ginsenoside Rg1, and ginsenoside Re in the DAD chromatogram of the test sample cannot be effectively separated, and therefore cannot be quantified.

[0146] Comparative Example 8

[0147] Chromatographic conditions: Chromatographic column: Agilent AQ-C18 (2.1×150 mm, 2.7 μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in Table 1; flow rate, 0.3 mL / min; column temperature, 25°C; injection volume, 1 μL; dynamic switching wavelength: 0-23 min, 237 nm; 23-26 min, 203 nm; 26-28 min, 237 nm; 28-35 min, 203 nm; detector: DAD-ELSD combination; carrier gas flow rate of the ELSD detector was 2.8 L / min, and the drift tube temperature was: 105°C.

[0148] The same test solution and mixed reference solution as in Example 1 were injected into an ultra-high performance liquid chromatograph for analysis and the chromatograms were recorded simultaneously.

[0149] The experimental results show that when the chromatographic column of the present invention is replaced with Agilent AQ-C18 (2.1×150 mm, 2.7 μm), apiosyl liquiritin and liquiritin completely overlap in the DAD chromatogram of the test sample, and multiple target peaks cannot be effectively separated.

[0150] Comparative Example 9

[0151] Chromatographic conditions: Chromatographic column: ACQUITY UPLC-HSS T3 (2.1×150 mm, 1.8 μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in Table 1; flow rate, 0.3 mL / min; column temperature, 25°C; injection volume, 1 μL; dynamic switching wavelength: 0-23 min, 237 nm; 23-26 min, 203 nm; 26-28 min, 237 nm; 28-35 min, 203 nm; detector: DAD-ELSD combination; carrier gas flow rate of the ELSD detector was 2.8 L / min, and the drift tube temperature was: 105°C.

[0152] The same test solution and mixed reference solution as in Example 1 were injected into an ultra-high performance liquid chromatograph for analysis and the chromatograms were recorded simultaneously.

[0153] The experimental results show that when the chromatographic column of the present invention is replaced with ACQUITY UPLC-HSS T3 (2.1×150 mm, 1.8 μm), the separation degree of formononetin and glycyrrhizic acid in the DAD chromatogram of the test sample is very poor, and they cannot be accurately quantified.

[0154] Comparative Example 10

[0155] Chromatographic conditions: Column: Excsep TMUHPLC SCB-C18 (2.1 mm*150 mm, 1.8 μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in Table 1; flow rate, 0.3 mL / min; column temperature, 25°C; injection volume, 1 μL; dynamic switching wavelength: 0-23 min, 237 nm; 23-26 min, 203 nm; 26-28 min, 237 nm; 28-35 min, 203 nm; detector: DAD.

[0156] The same test solution and astragaloside IV reference solution as in Example 1 were injected into an ultra-high performance liquid chromatograph for analysis, and the chromatograms were recorded simultaneously.

[0157] The experimental results show that when the detector of the present invention is changed to a separate DAD detector, the chromatographic peak absorption of the target component astragaloside IV is very weak compared to that when an ELSD detector is used, and it cannot be accurately quantified.

[0158] Comparative Example 11

[0159] Chromatographic conditions: Column: Excsep TM UHPLC SCB-C18 (2.1 mm*150 mm, 1.8 μm); acetonitrile as mobile phase A, water as mobile phase B, gradient elution as specified in Table 1; flow rate, 0.3 mL / min; column temperature, 25°C; injection volume, 1 μL; detector, ELSD, carrier gas flow rate, 2.8 L / min, drift tube temperature, 105°C.

[0160] The same test solution and mixed reference solution as in Example 1 were injected into an ultra-high performance liquid chromatograph for analysis and the chromatograms were recorded simultaneously.

[0161] The test results show that when the detection method of the present invention is changed to an ELSD detector, no chromatographic peaks appear for some target components except astragaloside IV, and the separation of some target components is very poor, making it impossible to accurately quantify them.

[0162] Comparative Example 12

[0163] Preparation of test solution

[0164] Weigh about 0.5 g of the substance reference sample, accurately weigh it, place it in a 20 ml volumetric flask, add an appropriate amount of 80% methanol, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, let it cool, dilute to the scale with 80% methanol, shake well, filter it with a 0.22 μm filter membrane, and take the filtrate to obtain the test solution.

[0165] Chromatographic conditions: same as in Example 1.

[0166] The test solution, the mixed reference solution in Example 1, and the astragaloside IV reference solution were respectively injected into an ultra-high performance liquid chromatograph for determination and analysis, and the chromatograms were recorded simultaneously.

[0167] The test results show that when the preparation method of the test solution of the present invention is changed, the chromatographic peak of astragaloside IV cannot be analyzed under the conditions of the ELSD detector, and it cannot be quantified.

[0168] In summary, the above embodiments are merely examples of preferred embodiments of the present invention and do not include all embodiments of the present invention. Persons skilled in the art may make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope defined in the claims.

Claims

1. A fingerprint spectrum and multi-component quantitative analysis method of Baoyuan Decoction, characterized in that: The steps include: (1) Preparation of test solution: Weigh the Baoyuan Decoction standard substance, preparation, or food sample to prepare the test solution; (2) Preparation of reference solution: Accurately weigh appropriate amounts of calycosin glucoside, apigenin, liquiritin, formononetin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, formononetin, ammonium glycyrrhizate, 6-gingerol, and astragaloside IV reference substances, and add solvent to prepare mixed reference solutions of different concentrations; (3) Chromatographic analysis of reference solution and test solution: The detector is a combination of DAD and ELSD. The sample to be tested first passes through the DAD detector for multiple component analysis. The measurement process is used to construct and simultaneously obtain the fingerprint of Baoyuan Decoction. After DAD variable wavelength detection, it flows through the ELSD detector for astragaloside analysis; the chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile is used as mobile phase A, and water is used as mobile phase B for gradient elution. The detection wavelength is a dynamic switching wavelength, and the injection volume is 1 μl.

2. The method according to claim 1, characterized in that The preparation method of the test solution in step (1) is as follows: accurately weigh about 0.5 g of the Baoyuan Decoction substance standard, preparation or food sample, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 80% methanol containing 4% concentrated ammonia test solution, weigh it, ultrasonically treat it for 30 minutes, let it cool, make up for the weight loss, shake it well, filter it, accurately measure 25 mL of the filtrate, evaporate it to dryness, dissolve the residue with 80% methanol and transfer it to a 10 ml volumetric flask, add 80% methanol to the volume, shake it well, and filter it with a 0.22 μm filter membrane to obtain the test solution.

3. The method according to claim 1, characterized in that The solvent for preparing the reference substance in step (2) is methanol; the concentrations of the index components in the mixed reference substance are: 1.828-18.2758 mg / L of calycoside isoflavone glucoside, 10.1774-101.7739 mg / L of apigenin, 22.2680-222.6800 mg / L of liquiritin, 2.1364-21.3640 mg / L of formononetin, 11.7312-117.312 mg / L of ginsenoside Rg1, and 11.7312-117.312 mg / L of ginsenoside Re. 9.8304-98.3040mg / L, ginsenoside Rb121.3785-213.7848mg / L, formononetin 2.4883-24.8832mg / L, ammonium glycyrrhizate 30.2863-302.8628mg / L, 6-gingerol 2.1029-21.0290mg / L, astragaloside IV 60.6060-606.06mg / L.

4. The method according to claim 1, wherein The gradient elution procedure in step (3) is as follows: 。 5. The method according to claim 1, wherein The specific method of dynamically switching the wavelength in step (3) is: 0-23min, 237nm; 23-26min, 203nm; 26-28min, 237nm; 28-35min, 203nm.

6. The method according to claim 1, characterized in that The models of the chromatographic columns in step (3) are: WatersACQUITY UPLC HSS T3, Agilent AQ-C18, Endeavorsil C18, Excsep TM UHPLC SCB-C18; preferably Excsep TM UHPLC SCB-C18; column specifications are 2.1*150mm, and the packing diameter is 1.8um.

7. The method according to claim 1, characterized in that In step (3), the column temperature is 20°C-30°C, preferably 25°C; the flow rate is 0.2 mL / min-0.4 mL / min, preferably 0.3 mL / min.

8. The method according to claim 1, characterized in that In step (3), the carrier gas flow rate of the ELSD detector is 2.8 L / min, and the drift tube temperature is 105°C.

9. Application of the method according to any one of claims 1 to 8 in the quality control of Baoyuantang material standards, Baoyuantang preparations, and Baoyuantang foods, characterized in that: Multiple batches of Baoyuan Decoction material standards, Baoyuan Decoction preparations and Baoyuan Decoction food samples were taken respectively to prepare test sample solutions, which were then subjected to chromatographic analysis with mixed reference sample solutions. The Chinese medicine fingerprint spectrum similarity evaluation software was then used to process and analyze the spectrum under the DAD detector, generating an overlay map of the fingerprint spectra of different samples to judge their similarity results.

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