A method for quality analysis of Scutellaria baicalensis Georgi based on dual-wavelength equal-baseline differential fusion spectra

Abstract

The present invention discloses a method for quality analysis of Scutellaria baicalensis Georgi in traditional Chinese medicine based on double-wavelength equal-baseline differential fusion spectra, belonging to the technical field of quality control of traditional Chinese medicine. The present invention uses HPLC technology to establish a double-wavelength equal-baseline differential fusion characteristic spectrum of the reference medicinal material of Scutellaria baicalensis Georgi, and compares it with the spectrum of the test medicinal material to identify the authenticity of the medicinal material; an internal standard substance is used to relatively quantify the chemical components of the characteristic peaks to distinguish the quality of the medicinal material. While meeting the "integrity" and "clarity" of the chemical components of traditional Chinese medicine, it reduces the quality control cost of the complex system of traditional Chinese medicine, and can effectively solve the problems of simultaneous rapid determination of the active ingredients of Scutellaria baicalensis Georgi and the high quality control cost of the complex system of traditional Chinese medicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine quality control, and specifically relates to a method for comprehensively controlling the quality of Scutellaria baicalensis Georgi medicinal materials based on double-wavelength equal-baseline differential fusion spectra, taking Scutellaria baicalensis Georgi reference medicinal materials as reference substances, establishing a set of methods that do not rely on multiple reference substances. This method is stable and reasonable, can make up for the deficiencies in the current quality control of Scutellaria baicalensis Georgi medicinal materials, and provides a reference for improving the quality of Scutellaria baicalensis Georgi medicinal materials. Background Art

[0002] Scutellaria baicalensis Georgi is the dried root of the plant Scutellaria baicalensis Georgi Scutellaria baicalensis Georgi., which is included in the first volume of the Chinese Pharmacopoeia 2020 Edition. It is bitter in taste and cold in nature, and has the effects of clearing heat and drying dampness, purging fire and detoxifying, stopping bleeding, and preventing miscarriage. Scutellaria baicalensis Georgi is mainly distributed in vast areas of Northeast, North, Southwest and parts of Central China in China, covering provinces such as Heilongjiang, Jilin, Liaoning, Hebei, Inner Mongolia, Shanxi, Shandong, Henan, Shaanxi, Gansu, and Ningxia. Modern pharmacological research shows that Scutellaria baicalensis Georgi has pharmacological activities such as anti-tumor, anti-virus, anti-inflammatory, antioxidant, liver protection and neuroprotection. At the same time, it is proved that flavonoids and their glycosides are the components with the highest content in Scutellaria baicalensis Georgi and are also its active ingredients, including baicalin, wogonoside, baicalein, wogonin, etc.

[0003] At present, the methods for traditional Chinese medicine quality control mainly include single-index content determination method, multi-index content determination method, one assay multi-evaluation method, and the combination method of fingerprint spectrum and content determination, etc. The above methods have the advantages of high sensitivity, good repeatability, and clear indicators, and can provide quantitative data for the quality control of Scutellaria baicalensis Georgi medicinal materials. However, there are also deficiencies that a single index cannot reflect the overall characteristics of the medicinal materials, multi-index content determination depends on multiple reference substances and some index components cannot reflect the efficacy, fingerprint spectrum can only vaguely evaluate the similarity of the medicinal materials and cannot clearly judge the authenticity, quality and inferiority of the test samples. Moreover, the technical difficulty and high cost of the quality evaluation and control technology for the complex system of traditional Chinese medicine have brought great pressure to traditional Chinese medicine enterprises. Therefore, the guiding and supporting role in the rational clinical use of traditional Chinese medicine and the improvement of clinical efficacy has been difficult to reflect. Summary of the Invention

[0004] In view of the above problems, the present invention provides a method for analyzing the quality of Scutellaria baicalensis Georgi in traditional Chinese medicine based on double-wavelength equal-baseline differential fusion spectra. Taking Scutellaria baicalensis Georgi reference medicinal materials as the reference substances for qualitative analysis, a set of methods that do not rely on multiple reference substances and can comprehensively and scientifically control the quality of Scutellaria baicalensis Georgi medicinal materials are established by using high performance liquid chromatography combined with Q-TOF-MS technology.

[0005] In order to achieve the above object, the present invention provides the following technical solutions.

[0006] The present invention provides a method for analyzing the quality of Scutellaria baicalensis Georgi in traditional Chinese medicine based on a dual-wavelength equal-baseline differential fusion spectrum, characterized in that the method is a dual-wavelength equal-baseline differential fusion method for fusing spectra at two wavelengths.

[0007] Further, the specific steps for establishing a dual-wavelength equal-baseline differential fusion spectrum by the dual-wavelength equal-baseline differential fusion method are as follows:

[0008] (1) Inject the control medicinal material and the test medicinal material for detection, with two samples and two needles for each sample.

[0009] (2) Export the data in CSV and AIA formats at 280 nm and 300 nm from the workstation respectively, and perform full-time equal-baseline fusion on the CSV data at 280 nm and 300 nm by selecting the maximum response value.

[0010] (3) Replace the data with higher responses from 10 to 14 min with the data with slightly lower responses at 300 nm to finally obtain differential fusion data.

[0011] (4) Convert the AIA format data into TXT data by the "Similarity Evaluation Software for Traditional Chinese Medicine Fingerprint", replace the response data in the TXT format with the differential fusion data, and save the TXT data to obtain the dual-wavelength equal-baseline differential fusion spectrum.

[0012] Further, the method uses the characteristic peaks in the characteristic spectrum of Scutellaria baicalensis Georgi as the qualitative standard for Scutellaria baicalensis Georgi medicinal materials, and can clearly identify the authenticity of the medicinal materials.

[0013] Further, the lower limit of the relative content of each characteristic peak determined by the calculation method for the relative content of the established characteristic peaks can distinguish the quality of Scutellaria baicalensis Georgi.

[0014] The present invention also provides a method for analyzing the quality of Scutellaria baicalensis Georgi in traditional Chinese medicine based on a dual-wavelength equal-baseline differential fusion spectrum, characterized in that the method includes the following steps:

[0015] Step 1 Qualitative research on Scutellaria baicalensis Georgi medicinal materials based on the control medicinal materials.

[0016] (1) Preparation of the solution: Take the powder of Scutellaria baicalensis Georgi control medicinal material or the medium powder of Scutellaria baicalensis Georgi medicinal material, sieve through a No. 4 sieve, weigh 0.3 g accurately, add 40 mL of 70% ethanol, heat under reflux for 3 hours, cool, filter, transfer the filtrate to a 100 mL volumetric flask, wash the container and residue with a small amount of 70% ethanol several times, filter the washing liquid into the same volumetric flask, add 70% ethanol to the scale, and shake well; accurately measure 1 mL, place it in a 10 mL volumetric flask, add methanol to the scale, shake well, and filter through a 0.22 μm microporous filter membrane to obtain the solution.

[0017] (2) Chromatographic conditions: Chromatographic column: Agilent Poroshell 120 SB-C18 (4.6 mm × 100 mm, 2.7 μm); Mobile phase: 0.1% formic acid in water (A) - acetonitrile (B), gradient elution; Elution program: 0 - 10 min, 20% → 20% B; 10 - 12 min, 20% → 23% B; 12 - 28 min, 23% → 23% B; 28 - 30 min, 23% → 40% B; 30 - 40 min, 40% → 40% B; 40 - 45 min, 40% → 100% B; 45 - 48 min, 100% → 100% B; Flow rate: 0.8 mL / min; Column temperature: 30 °C; Wavelengths of DAD detector: 280 nm, 300 nm; Injection volume: 10 μL;

[0018] (3) Establishment of dual-wavelength equal-baseline differential fusion chromatogram: Take the reference medicinal material of Scutellaria baicalensis and 10 batches of test medicinal materials, inject samples for detection according to the methods described in (1) and (2), with double samples and double injections for each sample; Export the data in CSV and AIA formats at 280 nm and 300 nm from the workstation respectively. Select the maximum response value of the CSV data at 280 nm and 300 nm for full-time equal-baseline fusion. In order to make the chromatogram more comprehensively and clearly reflect the quality of the medicinal material, replace the data with higher responses from 10 - 14 min with the data with slightly lower responses at 300 nm, and finally obtain the differential fusion data; Convert the AIA format data into TXT data by the "Similarity Evaluation Software for Traditional Chinese Medicine Fingerprint Chromatogram", replace the response data in the TXT format with the differential fusion data, and save the TXT data to obtain the dual-wavelength equal-baseline differential fusion chromatogram;

[0019] (4) Establishment of characteristic chromatogram: Import the dual-wavelength equal-baseline differential fusion chromatogram obtained in (3) into the "Similarity Evaluation Software for Traditional Chinese Medicine Fingerprint Chromatogram" to obtain the HPLC fingerprint chromatograms of 10 batches of test medicinal materials; Take the chromatogram of the reference medicinal material of Scutellaria baicalensis as the reference chromatogram, use the median for automatic matching, perform multi-point calibration, mark the characteristic peaks, generate the common pattern R, and compare it with the characteristic chromatogram of the reference medicinal material;

[0020] (5) Similarity evaluation: Determine the authenticity of Scutellaria baicalensis medicinal materials based on the similarity of characteristic peaks; The similarity between the test medicinal materials and the reference medicinal material is greater than 0.995;

[0021] (6) Chemical composition analysis of characteristic peaks: Adopt Q-TOF-MS technology, analyze the retention time, primary ion mass-to-charge ratio, and secondary ion fragment information of the compounds, and match them with the information reported in relevant literatures. Peak 2 is baicalin, peak 7 is wogonoside, peak 8 is baicalein, and peak 9 is wogonin;

[0022] Step 2 Relative quantitative study on the chemical components of characteristic peaks based on internal standard substances;

[0023] Peak 2, "Baicalin", is the index specified in the "Content Determination" section of Scutellaria baicalensis Georgi in Part I of the Chinese Pharmacopoeia (2020 Edition). This peak has good separation effect, stable retention time in the middle, and low price of the reference substance. Therefore, it is used as the internal standard substance to carry out the relative quantitative study of the chemical components of characteristic peaks based on the internal standard substance; taking "average - standard deviation" as the lower limit of the relative content of the chemical components of the characteristic peaks relative to the chemical components of the internal standard substance, it is considered excellent if the relative content of the characteristic peaks measured from the test medicinal materials according to this method is not lower than this content lower limit;

[0024] Step 3 Methodology investigation:

[0025] (1) Specificity test: Respectively take the mixed reference substance solution and the test solution, inject samples for analysis according to the above chromatographic conditions. The results show that the separation degree between each main peak and impurity peak is good, and the specificity is strong;

[0026] (2) Linearity and range: Take the baicalin reference substance solution, dilute it step - by - step by 2 times to obtain 6 baicalin mass concentration solutions. Respectively inject samples for analysis according to the above chromatographic conditions. Taking mass (X) as the abscissa and peak area (Y) as the ordinate, draw the standard curve and conduct linear regression to obtain the linear regression equation (Y = 579.96X - 27.552), and the correlation coefficient r = 0.9999. The results show that baicalin has a good linear relationship with the peak area in the range of 0.0784 - 2.51 μg;

[0027] (3) Precision test: Take the same test solution, inject samples continuously 6 times under the above chromatographic conditions, measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD value; the results show that the RSD values of the relative retention time and relative peak area of each chromatographic peak are all less than 0.92%, indicating good instrument precision;

[0028] (4) Stability test: Take the same test solution, inject samples at 0, 2, 4, 8, 12, and 24 h after preparation respectively under the above chromatographic conditions, measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD value; the results show that the RSD values of the relative retention time and relative peak area of each chromatographic peak are all less than 1.6%, indicating that the test solution is stable within 24 h;

[0029] (5) Repeatability test: Take the same batch of samples, prepare 6 test solutions in parallel, inject samples for detection under the above chromatographic conditions, measure the content of baicalin and the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD value; the results show that the average content of baicalin is 12.34%, and the RSD is 1.3%; the RSD values of the relative retention time and relative peak area of each chromatographic peak are all less than 2.0%, indicating good method repeatability;

[0030] (6) Sample recovery rate: Six portions of Scutellaria baicalensis powder were accurately weighed, each with a weight of 0.15 g. A baicalin reference substance equivalent to 0.15 g of the powder was accurately added to each portion. The test solution was prepared according to the above method and tested under the above chromatographic conditions. The average sample recovery rate of baicalin was calculated to be 99.5% (n=6), with an RSD of 1.1%.

[0031] Furthermore, the characteristic peaks of step 2 are all characteristic active chemical components that enable Scutellaria baicalensis to exert its efficacy and pharmacological effects.

[0032] Furthermore, the method uses the characteristic peaks in the characteristic spectrum of the Scutellaria baicalensis medicinal material as the qualitative standard of the Scutellaria baicalensis medicinal material, and can clearly identify the authenticity of the medicinal material.

[0033] Furthermore, the method can distinguish the quality of Scutellaria baicalensis by determining the lower limit of the relative content of each characteristic peak through the calculation method of the relative content of characteristic peaks established.

[0034] Compared with the prior art, the present invention has the following beneficial effects.

[0035] (1) The present invention closely follows the quality connotation and characteristics of multi-component, multi-functional and integrated effects of traditional Chinese medicine, and takes the chemical components that can reflect its efficacy as indicators; a self-reference is used to relatively quantify the chemical components of characteristic peaks, without the need to purchase a large number of reference substances, and the content of effective chemical components in the test medicinal materials can be controlled as much as possible. At the same time, in view of the low response values ​​of some components of a single wavelength and the excessively high response values ​​of the main components of different wavelengths, which lead to incomplete information on the medicinal material components reflected in the spectrum, the dual-wavelength equal baseline differential fusion method is used to fuse the spectra at two wavelengths. While meeting the "integrity" and "clarity" of the chemical components of traditional Chinese medicine, the cost of quality evaluation and control of the complex system of traditional Chinese medicine is reduced. The present invention constructs a characteristic spectrum with reference medicinal materials as the reference material, and uses a limited number of representative components that can represent the efficacy of Scutellaria baicalensis as characteristic peaks to evaluate the similarity of the characteristic peaks of the reference medicinal materials and the test medicinal materials, which can be used to judge the authenticity of Scutellaria baicalensis; selects an internal standard substance with a stable retention time and low price and easy to obtain as a quantitative evaluation index, conducts a relative quantitative study of the chemical components of the characteristic peaks based on the internal standard substance, and calculates the relative content of the chemical components of the characteristic peaks of the test medicinal materials through accurate quantification of the chemical components of the internal standard substance. This method can effectively solve the problems of rapid simultaneous determination of the effective components of Scutellaria baicalensis and high cost of quality evaluation and control of complex systems of traditional Chinese medicine.

[0036] (2) The present invention discloses for the first time a quality analysis method for the traditional Chinese medicine Scutellaria baicalensis based on dual-wavelength equal baseline differential fusion spectrum, which can make up for the shortcomings of the current quality control methods for Scutellaria baicalensis medicinal materials, such as a single indicator cannot reflect the overall characteristics of the medicinal material, multi-indicator content determination depends on multiple reference substances and some indicator components cannot reflect the efficacy, and the fingerprint spectrum can only vaguely evaluate the similarity of medicinal materials and cannot clearly judge the authenticity and quality of the test samples.

[0037] (3) In this study, chromatographic peaks with good resolution and signal-to-noise ratio greater than 10 were selected as characteristic peaks, and a total of 11 characteristic peaks were determined. The chemical components of the characteristic peaks of Scutellaria baicalensis identified by mass spectrometry technology are flavonoids. Some studies have shown that the flavonoid components of Scutellaria baicalensis can significantly inhibit α-glucosidase. In addition, baicalin can induce apoptosis of tumor cells, block the tumor cell cycle and inhibit its proliferation; wogonoside has various pharmacological activities such as anti-tumor, anti-inflammatory, antibacterial, antioxidant, cardiovascular protection, and neuroprotection; baicalein has strong antibacterial efficacy and a broad antibacterial spectrum; wogonin also shows good anti-tumor effects. The above chemical components are considered to be the main active components in Scutellaria baicalensis and can comprehensively and scientifically reflect the internal quality of traditional Chinese medicine Scutellaria baicalensis. Description of the Drawings

[0038] Figure 1 It is the superimposed chromatogram of 10 batches of Scutellaria baicalensis test medicinal materials.

[0039] Figure 2 It is the characteristic chromatogram of the control medicinal material of Scutellaria baicalensis and the common pattern of the test medicinal materials.

[0040] Figure 3 It is the total ion current chromatogram of Scutellaria baicalensis medicinal material under the positive ion mode.

[0041] Figure 4 It is the 283 nm chromatogram of Scutellaria baicalensis in batch S1.

[0042] Figure 5 It is the 300 nm chromatogram of Scutellaria baicalensis in batch S1.

[0043] Figure 6 It is the differential fusion chromatogram of Scutellaria baicalensis in batch S1.

[0044] Figure 7 It is the full-time equal-baseline fusion chromatogram of Scutellaria baicalensis in batch S1. Specific Embodiments

[0045] The following examples will help to understand the present invention, but these examples are only for illustrating the present invention, and the present invention is not limited to these contents.

[0046] Example 1.

[0047] Agilent-1260Ⅱ high performance liquid chromatograph (Agilent Technologies, USA); Agilent 6550 Q-TOF-MS mass spectrometer (Agilent Technologies, USA); HS6150 ultrasonic cleaner (Tianjin Hengao Technology Development Co., Ltd.); ME55 one in a hundred thousandth electronic analytical balance (Shanghai Mettler-Toledo Instruments Co., Ltd.).

[0048] The producing areas of the ten batches of Scutellaria baicalensis Georgi. test medicinal materials were Inner Mongolia, Shanxi, Gansu, northern Shaanxi, Heilongjiang, Liaoning, Jilin, Shaanxi, Inner Mongolia, and Hebei, numbered S1 - S10. Identified by Professor Zhang Hui of Liaoning University of Traditional Chinese Medicine, they were all the dried roots of the plant Scutellaria baicalensis Georgi. of the Labiatae family; Scutellaria baicalensis Georgi. reference medicinal material (batch number: 120955 - 201810, National Institutes for Food and Drug Control); reference substances of baicalin (batch number: 110715 - 201821), baicalein (batch number: 111595 - 201607), and wogonin (batch number: 111514 - 201706) were all provided by the National Institutes for Food and Drug Control, with a mass fraction ≥ 98%; the reference substance of wogonoside (batch number: O0903AS) was provided by Dalian Meilun Biotechnology Co., Ltd., with a mass fraction ≥ 98%; methanol, acetonitrile (mass spectrometry grade, Merck, Germany); formic acid (chromatographic pure, Tianjin Kemiou Chemical Reagent Co., Ltd.); water (purified water, Hangzhou Wahaha Group Co., Ltd.).

[0049] 1 Qualitative study of Scutellaria baicalensis Georgi. medicinal materials based on reference medicinal materials.

[0050] 1.1 Preparation of reference substance solutions.

[0051] Appropriately weigh the reference substances of baicalin, wogonoside, baicalein, and wogonin and transfer them to 10 mL volumetric flasks. Precisely weigh them and make up the volume with methanol to prepare single reference substance solutions of baicalin at 0.251 mg / mL, wogonoside at 0.239 mg / mL, baicalein at 0.219 mg / mL, and wogonin at 0.298 mg / mL respectively. Then, take appropriate amounts of each to prepare a mixed reference substance solution of baicalin at 0.179 mg / mL, wogonoside at 0.0341 mg / mL, baicalein at 0.0156 mg / mL, and wogonin at 0.0213 mg / mL.

[0052] 1.2 Preparation of reference medicinal material solution.

[0053] Take about 0.3 g of the powder of Scutellaria baicalensis Georgi. reference medicinal material, precisely weigh it, add 40 mL of 70% ethanol, heat under reflux for 3 hours, cool, filter, transfer the filtrate to a 100 mL volumetric flask, wash the container and residue with a small amount of 70% ethanol in several portions, filter the washing liquid into the same volumetric flask, add 70% ethanol to the scale, and shake well. Precisely measure 1 mL and transfer it to a 10 mL volumetric flask, add methanol to the scale, shake well, and filter through a 0.22 μm microporous filter membrane to obtain the solution.

[0054] 1.3 Preparation of test sample solutions.

[0055] Take about 0.3 g of the medium powder of Scutellaria baicalensis Georgi. medicinal material (sieved through No. 4 sieve), and prepare the test sample solution according to the method under item "1.1" to obtain the solution.

[0056] 1.4 Chromatographic conditions.

[0057] Chromatographic column: Agilent Poroshell 120 SB-C18 (4.6 mm × 100 mm, 2.7 μm); Mobile phase: 0.1% formic acid in water (A) - acetonitrile (B), gradient elution; Elution program: 0 - 10 min, 20% → 20% B; 10 - 12 min, 20% → 23% B; 12 - 28 min, 23% → 23% B; 28 - 30 min, 23% → 40% B; 30 - 40 min, 40% → 40% B; 40 - 45 min, 40% → 100% B; 45 - 48 min, 100% → 100% B; Flow rate: 0.8 mL / min; Column temperature: 30 °C; Wavelength of DAD detector: 280 nm, 300 nm; Injection volume: 10 μL.

[0058] 1.5 Establishment of characteristic fingerprint.

[0059] Take the reference medicinal material of Scutellaria baicalensis and 10 batches of test medicinal materials, and prepare the reference medicinal material solution and test sample solution respectively according to the methods under "1.2" and "1.3". Inject and detect according to the chromatographic conditions under "1.4", with two samples and two injections for each sample. Export the data in CSV and AIA formats at 280 nm and 300 nm from the liquid chromatography workstation respectively. Select the maximum response value of the CSV data at 280 nm and 300 nm for full-time equal baseline fusion. In order to make the fingerprint more comprehensively and clearly reflect the quality of the medicinal material, replace the data with higher response values from 10 - 14 min with the data with slightly lower response values at 300 nm, and finally obtain the differential fusion data. The AIA format data is converted into TXT data by the "Similarity Evaluation Software for Traditional Chinese Medicine Fingerprints", replace the response data in the TXT format with the differential fusion data, save the TXT data, and import it into the "Similarity Evaluation Software for Traditional Chinese Medicine Fingerprints" again to obtain the HPLC fingerprints of 10 batches of test medicinal materials. Take the fingerprint of the reference medicinal material of Scutellaria baicalensis as the reference fingerprint, use the median for automatic matching, and perform multi-point calibration to mark the characteristic peaks. The HPLC fingerprints of 10 batches of test medicinal materials are shown in Figure 1 , a total of 11 characteristic peaks are calibrated, and the total peak area accounts for more than 87% of the fingerprint. Among them, the stability, repeatability, and resolution of peak 2 are good, so it is used as the reference peak S; Compare the common mode R with the fingerprint of the reference medicinal material, as shown in Figure 2 . In the chromatogram of the test sample, 11 common peaks should be presented and should correspond to the retention times of the 11 common peaks in the chromatogram of the reference medicinal material, as shown in Table 1.

[0060] Table 1 Retention times and relative retention times of characteristic peaks ( )

[0061] 。

[0062] 1.6 Similarity evaluation.

[0063] Data processing was carried out using the "Similarity Evaluation Software for Traditional Chinese Medicine Fingerprint" (version 2012.130723) of the Pharmacopoeia Commission of the People's Republic of China. This software uses the cosine of the included angle method to calculate the similarity of each chromatogram compared with the reference medicinal material chromatogram. The authenticity of Scutellaria baicalensis Georgi medicinal materials was determined by the similarity of the characteristic peaks of the reference Scutellaria baicalensis Georgi and the test medicinal materials. According to the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, the similarity between the test sample and the reference medicinal material fingerprint should not be lower than 0.900. The similarity between the test Scutellaria baicalensis Georgi medicinal materials and the reference medicinal materials was greater than 0.990, indicating that the quality of the collected test Scutellaria baicalensis Georgi medicinal materials was relatively stable. The similarity calculation results are shown in Table 2.

[0064] Table 2 Results of similarity evaluation of test medicinal materials

[0065] 。

[0066] 1.7 Chemical composition analysis of characteristic peaks.

[0067] 1.7.1 Chromatographic analysis conditions.

[0068] Chromatographic column: Agilent Poroshell 120 SB-C 18 Chromatographic column (2.1 mm × 100 mm, 2.7 μm); Mobile phase: 0.1% formic acid water (A) - acetonitrile (B), gradient elution; Elution program: 0 - 10 min, 20% → 20% B; 10 - 12 min, 20% → 23% B; 12 - 28 min, 23% → 23% B; 28 - 30 min, 23% → 40% B; 30 - 40 min, 40% → 40% B; 40 - 45 min, 40% → 100% B; 45 - 48 min, 100% → 100% B; Flow rate: 0.3 mL / min; Column temperature: 30 °C; Injection volume: 2 μL.

[0069] 1.7.2 Mass spectrometry analysis conditions.

[0070] Electrospray ionization source (ESI), positive ion mode, capillary voltage 3500 V, drying gas flow rate 11 L / min, drying gas temperature 150 °C, nebulizer pressure 25 psi, sheath gas temperature 350 °C, sheath gas flow rate 10 L / min, mass scanning range 100 - 1000 m / z. The auto MS / MS mode was used, and methods such as comparison with reference substances and database query were used to analyze the chemical composition of characteristic peaks.

[0071] 1.7.3 Composition analysis.

[0072] On the basis of initially using an Agilent-1260Ⅱ high performance liquid chromatograph to compare with the reference substance by relative retention time, an Agilent 6550 Q-TOF-MS mass spectrometer was used for mass spectrometry analysis. The Agilent MassHunter Qualitative Analysis software was used to analyze the chemical components in the positive ion mode by comparing with the reference substance, and further clarify the accurate chemical components of the common peaks. Methods such as comparing with the reference substance and querying the database were used to analyze the chemical components of the characteristic peaks. Four chemical components in 11 common peaks were identified in the positive ion mode, and the total ion current chromatogram is shown in Figure 3 . Among them, peak 2 is baicalin, peak 7 is wogonoside, peak 8 is baicalein, and peak 9 is wogonin. The identification results are shown in Table 3.

[0073] Table 3 Analysis results of chemical components of characteristic peaks in positive ion mode

[0074] .

[0075] A method for analyzing the quality of Scutellaria baicalensis Georgi in traditional Chinese medicine based on a dual-wavelength equal-baseline differential fusion chromatogram can be used to judge the authenticity of Scutellaria baicalensis Georgi medicinal materials.

[0076] 2 Relative quantitative study of chemical components of characteristic peaks based on internal standard substances.

[0077] Take Scutellaria baicalensis Georgi medicinal materials, prepare the test solution according to the method under item "1.3", and inject the sample for analysis according to the chromatographic conditions under item "1.4". Peak 2 "baicalin" is the index specified in the "Content Determination" item of Scutellaria baicalensis Georgi in Part I of the Chinese Pharmacopoeia 2020 Edition. This peak has good separation effect, stable retention time in the middle, and low price of the reference substance. Therefore, it is used as the internal standard substance for relative quantification of characteristic peaks. The calculation method for the relative content of chemical components of characteristic peaks in each test sample is: relative content of chemical components of characteristic peaks % = peak area of characteristic peak × (content % of internal standard substance / peak area of internal standard substance). The accurate quantification results of baicalin are shown in Table 4. The relative content of chemical components of characteristic peaks in each test sample is shown in Table 5. Take "average - standard deviation" as the lower limit of the relative content of chemical components of characteristic peaks relative to the chemical components of the internal standard substance. It is preferable that the relative content of characteristic peaks measured for the test medicinal materials according to this method is not lower than this content lower limit.

[0078] Table 4 Accurate quantification results of internal standard substance baicalin

[0079] .

[0080] Table 5 Relative quantitative results of characteristic peaks based on internal standard substances

[0081] 。

[0082] A method for analyzing the quality of Scutellaria baicalensis Georgi based on a dual-wavelength equal-baseline differential fusion spectrum according to the present invention. The relative quantitative results of the characteristic peaks of Scutellaria baicalensis Georgi can be used to judge the quality of Scutellaria baicalensis Georgi medicinal materials.

[0083] 3 Methodology investigation.

[0084] 3.1 Specificity test.

[0085] Take the mixed reference substance solution and the test solution respectively, and inject samples for analysis according to the chromatographic conditions in item "1.4". The results show that the resolution between each main peak and the impurity peak is good, and the specificity is strong.

[0086] 3.2 Linearity and range.

[0087] Take the reference substance solution of baicalin, dilute it step by step by 2 times to obtain 6 mass concentration solutions of baicalin. Inject samples for analysis respectively according to the above chromatographic conditions. Take the mass (X) as the abscissa and the peak area (Y) as the ordinate, draw the standard curve and perform linear regression to obtain the linear regression equation (Y = 579.96X - 27.552), and the correlation coefficient r = 0.9999. The results show that baicalin has a good linear relationship with the peak area in the range of 0.0784 - 2.51 μg.

[0088] 3.3 Precision test.

[0089] Take the same test solution, inject samples continuously 6 times under the chromatographic conditions in item "1.4", measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD value. The results show that the RSD values of the relative retention time and relative peak area of each chromatographic peak are all less than 0.92%, indicating that the instrument precision is good.

[0090] 3.4 Stability test.

[0091] Take the same test solution, inject samples at 0, 2, 4, 8, 12, and 24 h after preparation respectively under the chromatographic conditions in item "1.4", measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD value. The results show that the RSD values of the relative retention time and relative peak area of each chromatographic peak are all less than 1.6%, indicating that the test solution is stable within 24 h.

[0092] 3.5 Repeatability test.

[0093] Take the same batch of samples, prepare 6 test solutions in parallel, inject samples for detection under the chromatographic conditions in item "1.4", measure the content of baicalin and the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD value. The results show that the average content of baicalin is 12.34%, and the RSD is 1.3%. The RSD values of the relative retention time and relative peak area of each chromatographic peak are all less than 2.0%, indicating that the method has good repeatability.

[0094] 3.6 Sample addition recovery rate.

[0095] Accurately weigh 6 portions of Scutellaria baicalensis Georgi medicinal material powder, each portion being 0.15 g. Accurately add baicalin reference substance equivalent to the content of 0.15 g of the medicinal material. Prepare the test solution according to the above method. Inject the sample for detection under the chromatographic conditions in item "1.4". The average sample addition recovery rate of baicalin is calculated to be 99.5% (n = 6), and the RSD is 1.1%.

[0096] Example 2 Differential fusion and non-fusion comparison test.

[0097] Take the Scutellaria baicalensis Georgi medicinal material chromatograms of batch S1 in Example 1 and process them as follows: (1) Export the AIA data at 280 nm and perform visualization processing with the similarity evaluation software for traditional Chinese medicine fingerprints; (2) Export the AIA data at 300 nm and perform visualization processing with the similarity evaluation software for traditional Chinese medicine fingerprints; (3) Export the AIA data at 280 nm and 300 nm respectively, and obtain the differential fusion chromatogram according to the differential fusion method in Example 1; (4) Export the AIA data at 280 nm and 300 nm respectively, and perform full-time equal baseline fusion to obtain the full-time equal baseline fusion chromatogram. The results are shown in the appendix Figures 4 - 7 . The results show that the chromatograms obtained by this method show more information than the single-wavelength chromatograms and the conventional full-time equal baseline fusion chromatograms, and can more comprehensively evaluate the quality of Scutellaria baicalensis Georgi medicinal materials.

[0098] The method for controlling the quality of Scutellaria baicalensis Georgi medicinal materials proposed by the present invention can effectively judge the authenticity, quality and inferiority of the medicinal materials, and form a scientific and controllable traditional Chinese medicine quality control system, aiming to provide an exploratory solution for traditional Chinese medicine quality control and the sustainable development of the traditional Chinese medicine industry.

Claims

1. A method for quality analysis of Scutellaria baicalensis Georgi in traditional Chinese medicine based on dual-wavelength equal-baseline differential fusion spectra, characterized in that, the method is a dual-wavelength equal-baseline differential fusion method for fusing spectra at two wavelengths; the specific steps for establishing a dual-wavelength equal-baseline differential fusion spectrum by the dual-wavelength equal-baseline differential fusion method are as follows: (1) Sampling and detecting the control medicinal material and the test medicinal material, with double samples and double needles for each sample: Take the powder of Scutellaria baicalensis Georgi control medicinal material or the medium powder of Scutellaria baicalensis Georgi medicinal material, add 70% ethanol, heat under reflux, cool, filter, place the filtrate in a volumetric flask, wash the container and residue with a small amount of 70% ethanol in portions, filter the washing liquid into the same volumetric flask, add 70% ethanol to the scale, and shake well; accurately measure 1 mL, place it in a 10 mL volumetric flask, add methanol to the scale, shake well, and filter through a 0.22 μm microporous filter membrane to obtain the sample. Sampling and detecting, with double samples and double needles for each sample. Chromatographic conditions: Chromatographic column: Agilent Poroshell 120 SB-C18 (4.6 mm × 100 mm, 2.7 μm); Mobile phase: 0.1% formic acid water (A) - acetonitrile (B), gradient elution; Elution program: 0 - 10 min, 20% → 20% B; 10 - 12 min, 20% → 23% B; 12 - 28 min, 23% → 23% B; 28 - 30 min, 23% → 40% B; 30 - 40 min, 40% → 40% B; 40 - 45 min, 40% → 100% B; 45 - 48 min, 100% → 100% B; Flow rate: 0.8 mL / min; Column temperature: 30 °C; Wavelength of DAD detector: 280 nm, 300 nm; Injection volume: 10 μL; (2) Export the data in CSV and AIA formats at 280 nm and 300 nm from the workstation respectively, and perform full-time equal-baseline fusion on the CSV data at 280 nm and 300 nm by selecting the maximum response value. (3) Replace the data with higher responses from 10 to 14 min with the data with slightly lower responses at 300 nm to finally obtain differential fusion data. (4) Convert the AIA format data into TXT data by the "Similarity Evaluation Software for Traditional Chinese Medicine Fingerprint", replace the response data in the TXT format with the differential fusion data, and save the TXT data to obtain the dual-wavelength equal-baseline differential fusion spectrum.

2. A method for quality analysis of Scutellaria baicalensis Georgi in traditional Chinese medicine based on dual-wavelength equal-baseline differential fusion spectra according to claim 1, characterized in that, the method uses the characteristic peaks in the characteristic spectrum of Scutellaria baicalensis Georgi medicinal material as the qualitative standard for Scutellaria baicalensis Georgi medicinal material, and can clearly identify the authenticity of the medicinal material.

3. A method for quality analysis of Scutellaria baicalensis Georgi in traditional Chinese medicine based on dual-wavelength equal-baseline differential fusion spectra according to claim 1, characterized in that, the method can distinguish the quality of Scutellaria baicalensis Georgi by the lower limit of the relative content of each characteristic peak determined by the established calculation method of the relative content of characteristic peaks.

4. A method for quality analysis of Scutellaria baicalensis Georgi in traditional Chinese medicine based on dual-wavelength equal-baseline differential fusion spectra, characterized in that, the method includes the following steps: Step 1 Qualitative study of Scutellaria baicalensis Georgi medicinal material based on the control medicinal material. (1)Preparation of solution: Take the powder of Scutellaria baicalensis Georgi reference medicinal material or the medium powder of Scutellaria baicalensis Georgi medicinal material, pass through No. 4 sieve, weigh 0.3 g accurately, add 40 mL of 70% ethanol, heat under reflux for 3 hours, cool, filter, transfer the filtrate into a 100 mL volumetric flask, wash the container and residue with a small amount of 70% ethanol in several portions, filter the washing solution into the same volumetric flask, add 70% ethanol to the scale, shake well; accurately measure 1 mL, transfer it into a 10 mL volumetric flask, add methanol to the scale, shake well, and filter through a 0.22 μm microporous filter membrane to obtain the solution; (2)Chromatographic conditions: Chromatographic column: Agilent Poroshell 120 SB-C18 (4.6 mm×100 mm, 2.7 μm); Mobile phase: 0.1% formic acid water (A) - acetonitrile (B), gradient elution; Elution program: 0 - 10 min, 20% → 20% B; 10 - 12 min, 20% → 23% B; 12 - 28 min, 23% → 23% B; 28 - 30 min, 23% → 40% B; 30 - 40 min, 40% → 40% B; 40 - 45 min, 40% → 100% B; 45 - 48 min, 100% → 100% B; Flow rate: 0.8 mL / min; Column temperature: 30 °C; Wavelength of DAD detector: 280 nm, 300 nm; Injection volume: 10 μL; (3)Establishment of dual-wavelength equal-baseline differential fusion chromatogram: Take the Scutellaria baicalensis Georgi reference medicinal material and 10 batches of tested medicinal materials and inject samples for detection according to the methods described in (1) and (2), with double samples and double needles for each sample; Export the data in CSV and AIA formats at 280 nm and 300 nm from the workstation respectively, perform full-time equal-baseline fusion on the CSV data at 280 nm and 300 nm by selecting the maximum response value. In order to make the chromatogram more comprehensively and clearly reflect the quality of the medicinal material, replace the data with higher responses from 10 - 14 min with the data with slightly lower responses at 300 nm, and finally obtain the differential fusion data; Convert the AIA format data into TXT data by the "Similarity Evaluation Software for Traditional Chinese Medicine Fingerprint Chromatogram", replace the response data in the TXT format with the differential fusion data, and save the TXT data to obtain the dual-wavelength equal-baseline differential fusion chromatogram; (4)Establishment of characteristic chromatogram: Import the dual-wavelength equal-baseline differential fusion chromatogram obtained in (3) into the "Similarity Evaluation Software for Traditional Chinese Medicine Fingerprint Chromatogram" to obtain the HPLC fingerprint chromatograms of 10 batches of tested medicinal materials; Take the chromatogram of the Scutellaria baicalensis Georgi reference medicinal material as the reference chromatogram, perform automatic matching using the median, perform multi-point calibration, mark the characteristic peaks, generate the common pattern R, and compare it with the characteristic chromatogram of the reference medicinal material; (5)Similarity evaluation: Identify the authenticity of Scutellaria baicalensis Georgi medicinal material based on the similarity of characteristic peaks; The similarity between the tested medicinal materials and the reference medicinal material is greater than 0.995; (6)Characteristic peak chemical composition analysis: Using Q-TOF-MS technology, by analyzing the retention time of compounds, the mass-to-charge ratio of primary ions, and the information of secondary ion fragments, and matching with the information reported in relevant literature, peak 2 is baicalin, peak 7 is wogonoside, peak 8 is baicalein, and peak 9 is wogonin; Step 2 Relative quantitative study of characteristic peak chemical components based on internal standard substances; "Baicalin" at peak 2 is the index specified in the "Content Determination" item of Scutellaria baicalensis Georgi in Part I of the Chinese Pharmacopoeia (2020 Edition). This peak has good separation effect, stable retention time in the middle, and low price of the reference substance. Therefore, it is used as the internal standard substance to carry out the relative quantitative study of characteristic peak chemical components based on internal standard substances; Take "average - standard deviation" as the lower limit of the relative content of characteristic peak chemical components relative to the chemical components of the internal standard substance. It is preferred that the relative content of the characteristic peak measured from the test medicinal materials according to this method is not lower than this content lower limit; Step 3 Methodology investigation: (1)Specificity test: Take the mixed reference substance solution and the test sample solution respectively, and inject samples for analysis according to the above chromatographic conditions. The results show that the separation degree between each main peak and impurity peak is good, and the specificity is strong; (2)Linearity and range: Take the baicalin reference substance solution and dilute it step by step by 2 times to obtain 6 baicalin mass concentration solutions. Inject samples for analysis according to the above chromatographic conditions respectively. Take the mass (X) as the abscissa and the peak area (Y) as the ordinate, draw the standard curve and perform linear regression to obtain the linear regression equation (Y = 579.96X - 27.552), and the correlation coefficient r = 0.9999. The results show that baicalin has a good linear relationship with the peak area in the range of 0.0784 - 2.51 μg; (3)Precision test: Take the same test sample solution and inject samples continuously 6 times under the above chromatographic conditions, measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD value; The results show that the RSD values of the relative retention time and relative peak area of each chromatographic peak are all less than 0.92%, indicating good instrument precision; (4)Stability test: Take the same test sample solution and inject samples at 0, 2, 4, 8, 12, and 24 h after preparation respectively under the above chromatographic conditions, measure the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD value; The results show that the RSD values of the relative retention time and relative peak area of each chromatographic peak are all less than 1.6%, indicating that the test sample solution is stable within 24 h; (5)Repeatability test: Take the same batch of samples, prepare 6 test sample solutions in parallel, inject samples for detection under the above chromatographic conditions, measure the baicalin content and the relative retention time and relative peak area of each chromatographic peak, and calculate the RSD value; The results show that the average baicalin content is 12.34% and the RSD is 1.3%; The RSD values of the relative retention time and relative peak area of each chromatographic peak are all less than 2.0%, indicating good method repeatability; (6) Sample addition recovery rate: Accurately weigh 6 portions of Scutellaria baicalensis Georgi medicinal material powder, 0.15 g for each portion, accurately add baicalin reference substance equivalent to the content of 0.15 g of the medicinal material, prepare the test solution according to the above method, inject and detect under the above chromatographic conditions, and calculate that the average sample addition recovery rate of baicalin is 99.5% (n = 6), and the RSD is 1.1%.

5. A method for quality analysis of Scutellaria baicalensis Georgi in traditional Chinese medicine based on a dual-wavelength equal baseline differential fusion spectrum according to claim 4, characterized in that, the characteristic peaks in step 2 are all characteristic active chemical components for Scutellaria baicalensis Georgi to exert its efficacy and pharmacological effects.

6. A method for quality analysis of Scutellaria baicalensis Georgi in traditional Chinese medicine based on a dual-wavelength equal baseline differential fusion spectrum according to claim 4, characterized in that, the method uses the characteristic peaks in the characteristic spectrum of Scutellaria baicalensis Georgi medicinal material as the qualitative standard for Scutellaria baicalensis Georgi medicinal material, and can clearly identify the authenticity of the medicinal material.

7. A method for quality analysis of Scutellaria baicalensis Georgi in traditional Chinese medicine based on a dual-wavelength equal baseline differential fusion spectrum according to claim 4, characterized in that, the lower limit of the relative content of each characteristic peak determined by the calculation method of the relative content of the characteristic peaks established by the method can distinguish the quality of Scutellaria baicalensis Georgi.

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