Characteristic chromatogram for detecting Yiyouqing particulate matter basis as well as construction method and application of characteristic chromatogram

Abstract

The invention discloses a characteristic chromatogram for detecting a Yiyouqing particulate matter basis as well as a construction method and application thereof, and belongs to the field of quality control of traditional Chinese medicine compound preparations. According to the method, hydroxysafflor yellow A, rutin, kaempferol-3-O-rutinoside, astragalus smicus glycoside, flatstem milkvetch glycoside and kaempferol are taken as reference substances, multiple batches of samples of the Yiyouqing granules are analyzed by utilizing a high performance liquid chromatography-mass spectrometry technology, and a chemical component characteristic spectrum is established. Compared with a traditional method, the method has good precision and reproducibility, data are accurate and reliable, the six components can be recognized at the same time under the same chromatographic condition, analysis efficiency is greatly improved, synchronous monitoring of multiple components is achieved, and consistency and stability of preparation quality are guaranteed. The method provides support for improving the quality standard of the Yiyouqing granules, and has important significance on the clinical curative effect and medication safety of the Yiyouqing granules.

Description

Technical Field

[0001] This invention belongs to the field of quality control technology for traditional Chinese medicine compound preparations, specifically relating to a characteristic spectrum for detecting the material basis of Yishiqing granules, its construction method, and its application. Background Technology

[0002] Against the backdrop of the inheritance, innovation, and development of traditional Chinese medicine (TCM), TCM compound formulas, with their unique advantages of "multiple components, multiple targets, and synergistic effects," play an important role in the prevention and treatment of chronic diseases and the management of sub-health conditions. However, the material basis of TCM compound formulas is complex and diverse, and their quality stability and controllability are directly related to clinical efficacy and medication safety. Establishing a scientific and comprehensive quality control system has become one of the core demands for the modernization of TCM.

[0003] Eye strain is one of the most common sub-health conditions in today's society. With the widespread use of electronic devices, factors such as prolonged eye strain and irregular work and rest schedules have led to a year-on-year increase in its incidence, and the affected group is gradually becoming younger. Patients often experience symptoms such as dry eyes, soreness, blurred vision, photophobia, and tearing, which seriously affect their quality of life and work efficiency. Traditional Chinese medicine believes that the core pathogenesis of eye strain is mostly related to liver and kidney deficiency and qi and blood deficiency. The liver opens into the eyes, and the kidneys store essence. When qi and blood are deficient, the eyes do not receive proper nourishment; when liver and kidneys are deficient, the spirit cannot focus. Therefore, tonifying the kidneys, invigorating blood, and replenishing qi to improve vision are the key treatment principles of traditional Chinese medicine for this type of condition.

[0004] Yishiqing Granules (National Drug Approval Number B20050061) is an exclusive product of Xi'an Afanggong Pharmaceutical Co., Ltd. It is a compound preparation composed of two Chinese herbal medicines, safflower and astragalus seed. Through scientific formulation, it exerts synergistic effects such as tonifying the kidney, promoting blood circulation, and improving eyesight. Clinically, it is mainly used to improve eye fatigue, relieve dry eyes and blurred vision. It is suitable for eye discomfort caused by liver and kidney deficiency and deficiency of both qi and blood.

[0005] Although Yishiqing granules have clear clinical value, their current quality control system is still imperfect, making it difficult to fully guarantee the product's quality uniformity and stability. Current research primarily focuses on the quality studies of single medicinal herbs, clinical efficacy observations, and simple safety evaluations, with insufficient in-depth exploration of their intrinsic material basis. The efficacy of traditional Chinese medicine compound formulas depends on the synergistic effect of the active ingredients in each herb. However, safflower and astragalus root contain numerous active ingredients, and their content is easily affected by various factors such as the origin of the herbs, harvest season, processing technology, and formulation process. If these key active ingredients cannot be accurately identified and quantitatively controlled, quality differences between batches can easily occur, thus affecting the stability of clinical efficacy. Therefore, addressing the shortcomings in the current quality control of Yishiqing granules by establishing a quality detection method based on characteristic spectroscopy to achieve comprehensive monitoring of its intrinsic material basis is of significant practical importance and application value for improving product quality standards, ensuring safe and effective clinical use, and promoting the industrial upgrading and market competitiveness of the product. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide a characteristic spectrum for detecting the material basis of Yishiqing granules, its construction method and application, so as to solve the technical problem that the existing detection methods for Yishiqing granules are difficult to fully reflect the complex chemical components of the preparation, thereby ensuring the uniformity and stability of quality.

[0007] To achieve the above objectives, the present invention employs the following technical solution: The first aspect of this invention discloses a method for constructing a characteristic spectrum for detecting the material basis of Yishiqing granules, comprising the following steps: S1. Weigh different batches of Yishiqing granules, add extraction solvent, sonicate and then evaporate to dryness to obtain the test solution; S2. Using hydroxysafflower yellow A, rutin, kaempferol-3-O-rutinoside, astragaloside, astragaloside and kaempferol as reference standards, prepare single reference solutions containing 51 μg hydroxysafflower yellow A, 49 μg rutin, 10 μg kaempferol-3-O-rutinoside, 45 μg astragaloside, 51 μg astragaloside and 40 μg kaempferol per 1 mL. S3. Inject the test solution obtained in S1 and the reference solution obtained in S2 into the high performance liquid chromatograph for chromatographic analysis and record the corresponding chromatograms. S4. Perform similarity analysis on the chromatograms obtained in S3 to confirm the reliability of the results; S5. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain the total ion chromatogram; based on the total ion chromatogram and the mass spectrometry results of the chemical components, and combined with the chromatogram of the single reference solution obtained in S3, determine the chemical components of each peak in the characteristic spectrum to obtain the characteristic spectrum of Yishiqing granules.

[0008] Preferably, in S1, the preparation method of the Yishiqing granule test solution is as follows: weigh 2.5 g of different batches of Yishiqing granules, add 25 mL of water, sonicate for 45 min, evaporate to dryness, filter, and obtain the Yishiqing granule test solution.

[0009] Preferably, in S3, the liquid chromatography conditions are as follows: column type Hedera ODS-2-C18, 4.6×250 mm×5µm; diode array detector, detection wavelength of 200 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; column temperature: 30℃.

[0010] Preferably, in S3, the gradient elution procedure is as follows: .

[0012] Preferably, in S5, the high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300℃, auxiliary gas temperature 300℃, full scan mode, and mass-to-charge ratio scan range m / z of 100. 1500.

[0013] Preferably, in S5, based on the total ion chromatogram and the mass spectrometry results of each chemical component, combined with the reference chromatogram obtained in S3, the chemical components of each peak in the chromatogram of the test sample are determined as follows: Peak 7 is hydroxysafflower yellow A, retention time 28.620 min; Peak 14 is rutin, retention time 39.693 min; Peak 17 is kaempferol-3-O-rutin, retention time 44.577 min; Peak 18 is astragaloside, retention time 46.837 min; Peak 21 is astragaloside, retention time 49.190 min; Peak 31 is kaempferol, retention time 68.297 min.

[0014] Preferably, after obtaining the characteristic spectrum of Yishiqing granules, an attribution analysis is performed on the characteristic spectrum results of Yishiqing granules. Hydroxysafflower yellow A, rutin, and kaempferol-3-O-rutin are derived from safflower, while astragaloside, astragaloside, and kaempferol are derived from astragalus.

[0015] In a second aspect, the present invention discloses the characteristic spectrum of the Yishiqing granules obtained by the above-described construction method.

[0016] A third aspect of the present invention discloses the application of the above-described construction method in the quality testing of Yishiqing granules and its namesake drugs.

[0017] In a fourth aspect, the present invention discloses a method for detecting the material basis of Yishiqing granules. The method utilizes the characteristic spectrum of the above-mentioned Yishiqing granules to detect the quality of Yishiqing granules or its equivalent medicine, using one or more of hydroxysaffron yellow A, rutin, kaempferol-3-O-rutinoside, astragaloside, astragaloside and kaempferol as quality markers.

[0018] Preferably, based on the material basis of Yishiqing granules or its equivalent formula, the detected components include: hydroxysafflower yellow A, rutin, kaempferol-3-O-rutinoside, astragaloside, astragaloside and kaempferol.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for constructing a characteristic chromatogram for detecting the material basis of Yishiqing granules. Using hydroxysaffron yellow A, rutin, kaempferol-3-O-rutinoside, astragaloside, astragaloside, and kaempferol as reference standards, and combining high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HDMS), six characteristic chemical components were successfully identified, achieving comprehensive quality control of Yishiqing granules. By identifying more characteristic peaks and utilizing high-resolution mass spectrometry, multiple active ingredients related to efficacy were successfully identified, achieving a more comprehensive coverage of the chemical composition of the formulation, enabling quality evaluation to move from single-indicator control to holistic quality control. The characteristic chromatogram obtained by this method identified 31 common peaks, of which 6 characteristic peaks were clearly identified, significantly improving the application effect of the chromatogram. This characteristic chromatogram has significant specificity and can comprehensively reflect the chemical composition of the formulation, providing a rapid, accurate, and comprehensive detection method for the quality assessment of traditional Chinese medicine compound preparations. The chromatogram obtained by this invention has a stable baseline and good peak shape, providing technical support for the improvement of formulation quality standards. By examining the stability, repeatability, and precision of retention time and peak area, the results showed that the RSD values ​​were all below 3%, confirming that the method is simple to operate, has good reproducibility, reliable data, and high precision, making it suitable for the quality control of Yishiqing granules. Furthermore, based on systematic analysis of multiple batches of samples, the quality evaluation system established in this invention is more comprehensive and objective, capable of accurately assessing the quality uniformity and stability between different batches of products, ensuring that the chemical composition of each batch is highly consistent with clinically effective batches, thereby guaranteeing the reliability of clinical efficacy and medication safety from the source. This technological breakthrough not only lays a solid foundation for improving the quality standards of Yishiqing granules but also provides a referable technical path for the modernization of quality control of traditional Chinese medicine, which is of positive significance for promoting high-quality development of the industry.

[0020] Furthermore, by systematically optimizing chromatographic analysis conditions, including key parameters such as extraction solvent selection, extraction method evaluation, mobile phase composition, column temperature control, and flow rate adjustment, an octadecylsilane-bonded silica column was adopted, and gradient elution was performed using acetonitrile-phosphoric acid aqueous solution as the mobile phase. This significantly improved analytical efficiency, effectively overcame the bottleneck of long running time in existing methods, significantly reduced the time and labor costs of quality control, improved the separation efficiency of various active ingredients, and enabled the characteristic chromatograms to present richer characteristic peak information. Attached Figure Description

[0021] Figure 1 The chromatograms are of the test solution of Yishiqing granules of the present invention; wherein, chromatogram (A) and 3D diagram (B); Figure 2 The chromatograms of hydroxysaffron yellow A of the present invention are shown below; wherein, the chromatogram of hydroxysaffron yellow A standard is shown in (A), the 3D image of hydroxysaffron yellow A standard is shown in (B), and the UV spectrum of hydroxysaffron yellow A is shown in (C). Figure 3 The chromatogram (A), 3D image (B), and UV spectrum (C) of the rutin standard of the present invention are shown. Figure 4 The chromatogram (A), 3D chromatogram (B), and UV spectrum (C) of the kaempferol-3-O-rutinoside standard of the present invention are shown below. Figure 5 The chromatogram (A), 3D image (B), and UV spectrum (C) of the astragalin standard of the present invention are shown. Figure 6 The following are the chromatograms (A), 3D images (B), and UV spectra (C) of the astragaloside standard of the present invention. Figure 7 The chromatogram (A), 3D chromatogram (B), and UV spectrum (C) of the kaempferol standard of the present invention are shown. Figure 8 This is the total ion chromatogram of negative ions in the mass spectrometry of the Yishiqing particles of the present invention; Figure 9 This is the total positive ion chromatogram of the Yishiqing particles according to the present invention. Figure 10 This is the mass spectrum of hydroxysafflower yellow pigment A of the present invention; Figure 11 This is the mass spectrum of rutin in this invention; Figure 12 This is the mass spectrum of kaempferol-3-O-rutin glycoside of the present invention; Figure 13 This is the mass spectrum of astragaloside according to the present invention; Figure 14 This is the mass spectrum of Astragalus complanatus glycosides of the present invention; Figure 15 This is the mass spectrum of kaempferol in this invention; Figure 16 The characteristic spectra of 15 batches of Yishiqing granules test samples of the present invention are shown below; Figure 17 This is a chromatogram obtained by optimizing the mobile phase composition under chromatographic conditions according to the present invention; Figure 18 This is a chromatogram obtained by optimizing the extraction method during the preparation of the test sample solution according to the present invention; Figure 19 This is a chromatogram obtained by optimizing the extraction solvent during the preparation of the test sample solution according to the present invention; Figure 20 This is a chromatogram obtained by optimizing column temperature under chromatographic conditions according to the present invention; Figure 21 This is a chromatogram obtained by optimizing the flow rate under chromatographic conditions according to the present invention; Figure 22 This is a chromatogram obtained by optimizing the feed-to-liquid ratio under chromatographic conditions according to the present invention; Figure 23 This is a chromatogram obtained by optimizing the elution procedure under chromatographic conditions according to the present invention. Detailed Implementation

[0022] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0023] This invention provides a method for constructing a characteristic spectrum for detecting the material basis of Yishiqing granules, comprising the following steps: S1. Preparation of test solution: Weigh 2.5 g of Yishiqing granules from different batches, add 25 mL of extraction solvent, seal tightly, weigh, sonicate and then extract by rotary evaporation to obtain test solution; S2. Preparation of single reference solution: Accurately weigh each reference standard and add it to an aqueous solution to prepare a single reference solution containing 51 μg hydroxysafflower yellow A, 49 μg rutin, 10 μg kaempferol-3-O-rutinoside, 45 μg astragaloside, 51 μg astragaloside, and 40 μg kaempferol per 1 mL. S3. Inject the test solution obtained in S1 and the reference solution obtained in S2 into the high-performance liquid chromatograph (HPLC) for chromatographic analysis. The chromatographic conditions are as follows: column type: Hedera ODS-2-C18 (4.6 × 250 mm × 5 µm); mobile phase: acetonitrile (A) - 0.05% phosphoric acid aqueous solution (B); diode array detector: detection wavelength: 200 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; column temperature: 30℃; gradient elution; and record the corresponding chromatograms. S4. Import the chromatograms obtained in S3 into the Chinese medicine chromatographic fingerprint chromatogram similarity evaluation system, select the chromatographic peaks that are present in the chromatograms of different batches of Yishiqing granules as common peaks, and perform similarity analysis on the chromatograms of the test sample solution after data import, multi-point correction and data matching to confirm the reliability of the results. S5. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain the total ion chromatogram; based on the total ion chromatogram and the mass spectrometry results of the chemical components, and combined with the chromatogram of the single reference solution obtained in S3, determine the chemical components of each peak in the characteristic spectrum to obtain the characteristic spectrum of Yishiqing granules.

[0024] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0025] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0026] 1. Instruments The instruments used in this invention are shown in Table 1.

[0027] Table 1. Instruments used in this invention

[0028] 2. Medicines and Reagents The 15 batches of Yishiqing granules used in this invention were all purchased from the market and produced and provided by Xi'an Afanggong Pharmaceutical Co., Ltd.; the reference standards: hydroxysaffron yellow A (batch number: 111637, purity: 98%) and rutin (batch number: 100080, purity: 98%) were purchased from the China National Institutes for Food and Drug Control; kaempferol-3-O-rutinoside (batch number: 110708-200505, purity: 98%) and astragaloside (batch number: PS010127, purity: 98%) were purchased from Chengdu Pusi Biotechnology Co., Ltd.; astragaloside (batch number: DSTDS001502, purity: 98%) was purchased from Chengdu Desite Biotechnology Co., Ltd.; and kaempferol (batch number: AF20100214, purity: 98%) was purchased from Chengdu Efa Biotechnology Co., Ltd. The reagents used in this invention are shown in Table 2.

[0029] Table 2 Reagents used in this invention

[0030] Example 1 I. A method for constructing a characteristic spectrum for detecting the material basis of Yishiqing granules, comprising the following steps: S1. Preparation of test solution: Accurately weigh 2.5 g of 15 batches of Yishiqing granules, place them in an Erlenmeyer flask, add 25 mL of water, seal tightly, weigh, sonicate for 45 min, weigh again, make up the weight loss with water, evaporate to dryness, add 5 mL of water to dissolve, filter with a 0.45 µm microporous membrane to obtain the Yishiqing granules test solution.

[0031] S2. Preparation of reference solution: Accurately weigh hydroxysaffron yellow A, rutin, kaempferol-3-O-rutinoside, astragaloside, astragaloside and kaempferol respectively, place them in a stoppered conical flask, add water to prepare a single reference solution containing 51 μg hydroxysaffron yellow A, 49 μg rutin, 10 μg kaempferol-3-O-rutinoside, 45 μg astragaloside, 51 μg astragaloside and 40 μg kaempferol per 1 mL.

[0032] S3. Accurately pipette 10 µL each of the test solution obtained in S1 and the reference solution in S2, and inject them into the high-performance liquid chromatograph for chromatographic analysis. Record the corresponding chromatograms. Figure 1 ; Figures 2-7 ); The liquid chromatography conditions were as follows: column: Hedera ODS-2-C18 (4.6 × 250 mm × 5 µm) column; detector: diode array detector, detection wavelength: 200 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; column temperature: 30℃; mobile phase: acetonitrile-0.05% phosphoric acid aqueous solution, gradient elution, the elution program is shown in Table 3. Table 3 Elution Procedure

[0033] S4. Import the chromatograms obtained in S3 into the Chinese medicine chromatographic fingerprint chromatogram similarity evaluation system, select the chromatographic peaks that are present in all chromatograms of 15 batches of Yishiqing granules as common peaks, and perform similarity analysis on the chromatograms of the test solution after data import, multi-point correction and data matching (Table 4).

[0034] Table 4. Similarity between each batch of Yishiqing granules and the common chromatographic peak mode.

[0035] S5. To determine the chemical composition in the characteristic spectrum, the above-mentioned test solution was subjected to high-resolution mass spectrometry analysis. The high-resolution mass spectrometry detection conditions were: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300℃, auxiliary gas temperature 300℃, full scan mode, and mass-to-charge ratio scan range m / z of 100. 1500, to obtain the total ion chromatogram ( Figure 8 and Figure 9 Import the total ion chromatogram data into Xcalibur software, enter the QualBrowser interface, and perform data analysis based on the peaks of chemical components in the total ion chromatogram and the chromatogram of the test sample obtained in S3 to obtain the mass spectrometry results of each chemical component. Figures 10-15 ).

[0036] S6. Based on the total ion chromatogram and mass spectrometry results of each chemical component obtained in S5, and combined with the reference chromatogram obtained in S3, the chemical components of each peak in the chromatogram of the test sample were determined as follows: Peak 7 is hydroxysafflower yellow A, retention time 28.620 min; Peak 14 is rutin, retention time 39.693 min; Peak 17 is kaempferol-3-O-rutin, retention time 44.577 min; Peak 18 is astragaloside, retention time 46.837 min; Peak 21 is astragaloside, retention time 49.190 min; Peak 31 is kaempferol, retention time 68.297 min. The characteristic chromatogram of Yishiqing granules was obtained. Figure 16 The chemical composition of Yishiqing granules was then analyzed for attribution. The results showed that hydroxysafflower yellow A, rutin, and kaempferol-3-O-rutin were derived from safflower, while astragaloside, astragaloside, and kaempferol were derived from astragalus complanatus.

[0037] II. Methodological Examination 1. Precision test Take the test solution obtained in S1 and inject it in parallel 6 times under the chromatographic conditions in S3. The injection volume is 10 μL. Analyze the retention time and peak area and calculate the RSD value. The results are shown in Table 5. It can be seen that the RSD values ​​of retention time and peak area are both less than 3%, indicating that the instrument has good precision.

[0038] Table 5 Peak area and retention time in precision studies

[0039] 2. Stability test The test solution obtained in S1 was taken and analyzed at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h according to the chromatographic conditions in S3. The injection volume was 10 μL. The retention time and peak area of ​​the common peaks in the HPLC characteristic chromatogram of the sample were analyzed and the RSD value was calculated. The results are shown in Table 6. It can be seen that the RSD values ​​of retention time and peak area are both less than 3%, indicating that the Yishiqing granule test solution has good stability within 24 h.

[0040] Table 6 Peak area and retention time in stability studies

[0041] 3. Repeatability test Six samples of Yishiqing granules were taken and injected for analysis according to the chromatographic conditions in S3. The retention time and peak area of ​​the common peaks in the HPLC characteristic chromatograms of the samples were analyzed and the RSD value was calculated. The results are shown in Table 7. It can be seen that the RSD values ​​of retention time and peak area are both less than 3%, indicating that the method has good repeatability.

[0042] Table 7 Peak area and retention time in repeatability studies

[0043] Example 2 The purpose of this embodiment is to investigate the effect of different mobile phases (methanol-0.05% phosphoric acid, acetonitrile-0.1% phosphoric acid, acetonitrile-0.1% formic acid, acetonitrile-water, acetonitrile-0.1% acetic acid, acetonitrile-0.05% phosphoric acid) on the detection of Yishiqing granules in the test solution. The test solution was prepared using the method in Example 1. The detection results are as follows: Figure 17 As shown in the figure, it can be seen that the mobile phase of acetonitrile-0.05% phosphoric acid has a higher number of peaks and better extraction effect.

[0044] Example 3 The purpose of this embodiment is to investigate the effects of different extraction methods (ultrasound for 30 min, ultrasound for 45 min, ultrasound for 60 min, reflux for 30 min, reflux for 60 min, reflux for 90 min, and immersion) on the detection of Yishiqing granules test solution. The chromatographic conditions are the same as in Example 1, and the preparation conditions of the remaining test solutions are also the same as in Example 1. The detection results are as follows: Figure 18 Shown. Through Figure 3 It is known that the number of peaks and response values ​​are not significantly different when using ultrasonic and reflux extraction methods. However, ultrasonic extraction is more cost-effective and efficient than reflux extraction. Therefore, ultrasonic extraction is chosen as the extraction method. Figure 18 It can be seen that the peak response value and peak shape of ultrasound detection results after 45 min are better than those after 30 min and 60 min. Therefore, ultrasound for 45 min is selected as the best extraction method.

[0045] Example 4 The purpose of this embodiment is to investigate the effect of different extraction solvents (anhydrous ethanol, acetonitrile, methanol, water, 0.05% phosphoric acid) on the detection of Yishiqing granules test solution. The chromatographic conditions are the same as in Example 1, and the preparation conditions of the remaining test solutions are also the same as in Example 1. The detection results are as follows: Figure 19 As shown. (Through) Figure 19 It is known that the chromatogram of components obtained when water is used as the extraction solvent has the highest content and the best extraction effect, so water is chosen as the extraction solvent.

[0046] Example 5 The purpose of this embodiment is to investigate the effect of different column temperatures (25℃, 30℃, 35℃) on the detection of Yishiqing granules test solution. The test solution was prepared using the method of Example 1, and all other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figure 20 As shown. (Through) Figure 20 It can be seen that the separation effect of each component is better and more peaks are produced when the column temperature is 30℃. Therefore, 30℃ is selected as the column temperature condition.

[0047] Example 6 The purpose of this embodiment is to investigate the effect of different flow rates (0.6 mL / min, 0.8 mL / min, 1.0 mL / min) on the detection of Yishiqing granules test solution. The test solution was prepared using the method of Example 1, and all other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figure 21 As shown. (Through) Figure 21 It can be seen that the peak elution is optimal and the separation of each component is better when the flow rate is kept at 1.0 mL / min. Therefore, the flow rate of 1.0 mL / min was finally selected.

[0048] Example 7 The purpose of this embodiment is to investigate the effect of different solid-liquid ratios (0.04 g / mL, 0.06 g / mL, 0.08 g / mL, 0.10 g / mL, and 0.12 g / mL) on the detection of Yishiqing granules in the test solution. The test solution was prepared using the method in Example 1, and all other chromatographic conditions were the same as in Example 1. The detection results are as follows: Figure 22 As shown. (Through) Figure 22 As shown, the baseline was most stable and the separation effect of each component was better when the material-to-liquid ratio was 0.1 g / mL. Therefore, the material-to-liquid ratio of 0.1 g / mL was finally selected.

[0049] Example 8 The purpose of this embodiment is to investigate the effect of different elution programs on the detection of Yishiqing granules test solution. The test solution was prepared using the method of Example 1, and elution programs were set for each solution. All other chromatographic conditions were the same as in Example 1. Some elution programs are shown in Tables 3 and 8-11. Table 8 Elution Procedure 1

[0050] Table 9 Elution Procedure 2

[0051] Table 10 Elution Procedure 3

[0052] Table 11 Elution Procedure 4

[0053] Test results as follows Figure 23 As shown. (Through) Figure 23 As can be seen, the elution program in Table 3 has good separation, high peak height, and complete chromatographic information. Therefore, the elution program in Table 3 is selected as the optimal elution program.

[0054] The above experimental results show that the method for constructing characteristic spectra for detecting the material basis of Yishiqing granules provided by the present invention has the characteristics of good stability, high precision and good repeatability, and can comprehensively and objectively evaluate the quality of Yishiqing granules, providing quality assurance for clinical efficacy.

[0055] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for constructing a characteristic spectrum for detecting the material basis of Yishiqing granules, characterized in that, Includes the following steps: S1. Weigh different batches of Yishiqing granules, add extraction solvent, sonicate and then evaporate to dryness to obtain the test solution; S2. Using hydroxysafflower yellow A, rutin, kaempferol-3-O-rutinoside, astragaloside, astragaloside and kaempferol as reference standards, prepare single reference solutions containing 51 μg hydroxysafflower yellow A, 49 μg rutin, 10 μg kaempferol-3-O-rutinoside, 45 μg astragaloside, 51 μg astragaloside and 40 μg kaempferol per 1 mL. S3. Inject the test solution obtained in S1 and the reference solution obtained in S2 into the high performance liquid chromatograph for chromatographic analysis and record the corresponding chromatograms. S4. Perform similarity analysis on the chromatograms obtained in S3 to confirm the reliability of the results; S5. Perform high-resolution mass spectrometry analysis on the test solution obtained in S1 to obtain the total ion chromatogram; Based on the total ion chromatogram and the mass spectrometry results of the chemical components, combined with the chromatogram of the single reference solution obtained in S3, the chemical components of each peak in the characteristic spectrum were determined, and the characteristic spectrum of Yishiqing granules was obtained.

2. The method for constructing a characteristic spectrum for detecting the material basis of Yishiqing granules according to claim 1, characterized in that, In S1, the preparation method of the Yishiqing granules test solution is as follows: weigh 2.5 g of different batches of Yishiqing granules, add 25 mL of water, sonicate for 45 min, evaporate to dryness, filter, and obtain the Yishiqing granules test solution.

3. The method for constructing a characteristic spectrum for detecting the material basis of Yishiqing granules according to claim 1, characterized in that, In S3, the liquid chromatography conditions were as follows: column type Hedera ODS-2-C18, 4.6×250 mm×5 µm; diode array detector, detection wavelength 200 nm; flow rate: 1.0 mL / min; injection volume: 10 μL; column temperature: 30℃.

4. The method for constructing a characteristic spectrum for detecting the material basis of Yishiqing granules according to claim 1, characterized in that, In S3, the gradient elution procedure is as follows: 。 5. The method for constructing a characteristic spectrum for detecting the material basis of Yishiqing granules according to claim 1, characterized in that, In S5, the high-resolution mass spectrometry detection conditions are: electrospray ionization, spray voltage 3500 V, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, capillary temperature 300℃, auxiliary gas temperature 300℃, full scan mode, and mass-to-charge ratio scan range m / z of 100. 1500.

6. The method for constructing a characteristic spectrum for detecting the material basis of Yishiqing granules according to claim 1, characterized in that, In S5, based on the total ion chromatogram and the mass spectrometry results of each chemical component, combined with the reference chromatogram obtained in S3, the chemical components of each peak in the chromatogram of the test sample were determined as follows: Peak 7 is hydroxysafflower yellow A, retention time 28.620 min; Peak 14 is rutin, retention time 39.693 min; Peak 17 is kaempferol-3-O-rutin, retention time 44.577 min; Peak 18 is astragaloside, retention time 46.837 min; Peak 21 is astragaloside, retention time 49.190 min; Peak 31 is kaempferol, retention time 68.297 min.

7. The method for constructing a characteristic spectrum for detecting the material basis of Yishiqing granules according to claim 1, characterized in that, After obtaining the characteristic spectrum of Yishiqing granules, an attribution analysis was performed on the characteristic spectrum results of Yishiqing granules. Hydroxysafflower yellow A, rutin, and kaempferol-3-O-rutin were derived from safflower, while astragaloside, astragaloside, and kaempferol were derived from astragalus.

8. The characteristic spectrum of Yishiqing granules obtained by the construction method according to any one of claims 1 to 7.

9. A method for detecting the material basis of Yishiqing granules, characterized in that, The quality of Yishiqing granules or its equivalent medicine was detected using the characteristic spectrum of Yishiqing granules as described in claim 8, with one or more of hydroxysaffron yellow A, rutin, kaempferol-3-O-rutin, astragaloside, astragaloside and kaempferol as quality markers.

10. The method for detecting the material basis of Yishiqing granules according to claim 9, characterized in that, Based on the material basis of Yishiqing granules or its equivalent formula, the detected components include: hydroxysafflower yellow A, rutin, kaempferol-3-O-rutinoside, astragaloside, astragaloside and kaempferol.

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