A method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis.

Abstract

This invention discloses a method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis, belonging to the field of biochemical analysis technology. The invention includes the following steps: First, prepare borate stock solution, cyclodextrin phosphate buffer, DES background buffer solution, and mixed standards for later use; employ a capillary electrophoresis system equipped with a diode array detector, using uncoated fused silica capillary tubes with an inner diameter of 75 μm and an effective length of 60 cm; pre-treat the capillary tubes, perform separation and detection, obtain the electrophoretic peak diagram of the sample, extract the peak area values ​​of each peak, calculate the migration time and concentration of each component, and achieve the separation and quantification of eight flavonoid glycosides. The buffer solution used in this invention can perfectly separate flavonoid glycosides that cannot be separated by HPLC due to structural similarity; all eight flavonoid glycosides elute within approximately 12 minutes with symmetrical and well-separated peaks.

Description

Technical Field

[0001] This invention belongs to the field of biochemical analysis technology, specifically relating to a method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis. Background Technology

[0002] High-performance liquid chromatography (HPLC) is a commonly used method for separating flavonoids. Due to its high resolution, stability, and applicability, HPLC plays a vital role in compound separation. It can effectively separate compounds with complex structures and different polarities, and is particularly suitable for the analysis of multiple components in complex samples. However, flavonoids often exhibit subtle structural differences that can lead to significant variations in biological activity. Because of their high chemical similarity, HPLC can sometimes struggle to achieve sufficient resolution. Especially when dealing with flavonoids with very similar polarities, HPLC separation may not be as effective as expected, and may even lead to peak overlap or decreased separation efficiency. Despite its many advantages, HPLC often faces certain limitations when dealing with compounds with similar polarities or structures.

[0003] Therefore, given the potential limitations of HPLC in separating structurally similar flavonoids, developing a separation technique to complement HPLC is crucial. One possible solution is the introduction of capillary zone electrophoresis (CZE). Compared to HPLC, CZE offers unique advantages in separating compounds with similar polarity. Based on the differences in electrophoretic mobility of molecules in an electric field, CZE provides not only higher resolution but also the ability to separate multiple components in a shorter time. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis, which is efficient, sensitive and environmentally friendly.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis includes the following steps:

[0007] 1) Prepare buffer solutions: Prepare borate stock solution, cyclodextrin phosphate buffer, and DES background buffer solution;

[0008] 2) Preparation of mixed standards: Dissolve the standards of eight flavonoid glycosides and the mixed sample in dimethyl sulfoxide aqueous solution to prepare stock solutions of individual standards and working solutions of mixed standards;

[0009] 3) Setting capillary detection conditions: The SCIEX P / ACE MDQ Plus capillary electrophoresis system was used, equipped with a diode array detector. The capillary used was an uncoated fused silica capillary with an inner diameter of 75 μm and an effective length of 60 cm.

[0010] 4) Capillary pretreatment;

[0011] 5) Quantitative analysis: The sample is separated and detected according to the detection conditions in step 3) to obtain the electrophoresis peak diagram of the sample to be tested, the peak area value of each peak is extracted, and the migration time and concentration of each component are calculated by the peak area integration method to achieve the separation and quantification of eight flavonoid glycosides.

[0012] The eight flavonoid glycosides are isoquercitrin, hyperoside, meadowsweet, quercetin-7-O-glucoside, quercetin, luteolin, vitexin, and isovitexin.

[0013] Furthermore, in step 1), the pH of the borate stock solution is 8.95-9.45.

[0014] Further, in step 1), the cyclodextrin is selected from one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, and 2-hydroxypropyl-β-cyclodextrin.

[0015] Further, in step 1), the hydrogen bond donor in the DES background buffer solution is selected from one of choline chloride, betaine, and proline, and the hydrogen bond acceptor is selected from one of lactic acid, D-sorbitol, glucose, and urea. The molar ratio of hydrogen bond donor to hydrogen bond acceptor is 1~3:1.

[0016] Further, in step 2), the preparation method of the single standard stock solution is as follows: weigh appropriate amounts of isoquercitrin, hyperoside, meadowsweet, quercetin-7-O-glucoside, quercetin, luteolin, vitexin and isovitexin standards respectively, dissolve them in 25% DMSO aqueous solution to prepare a stock solution with a concentration of 50 μg / mL, and store at 4℃ in the dark.

[0017] Further, in step 2), the method for preparing the mixed standard working solution is as follows: take an appropriate amount of each individual standard stock solution, dilute it with 25% DMSO aqueous solution, prepare a mixed standard working solution with a concentration of 20 μg / mL, filter it through a 0.22 μm cellulose acetate membrane and set it aside for later use.

[0018] Furthermore, in step 3), the injection conditions are: 0.5 psi for 5 seconds; electric field strength of 15 kV, electric field direction from capillary inlet to outlet; during separation, capillary temperature is maintained at 25°C, and sample storage chamber temperature is set to 10°C.

[0019] Furthermore, in step 3), the detection wavelength is 360 nm; the separation time is 25 min; and the background buffer solution is replaced after every three analyses.

[0020] Further, in step 4), capillary pretreatment: before each sample loading, rinse with 200mM NaOH solution and deionized water at 50psi pressure for 5 min respectively, and then run with buffer at 20kV for 4 min to complete the pretreatment; before each sample separation program, rinse with 200mM NaOH solution, deionized water and buffer at 50psi pressure for 1 min respectively, and dip both ends of the capillary in deionized water once before and after sample injection.

[0021] Furthermore, the R-values ​​of the standard curves for the eight flavonoid glycosides... 2 The limit of detection (LOD) was 0.996-0.999; the limit of quantitation (LOQ) was 0.207-0.581 μg / mL; and the limit of quantitation (LOQ) was 0.689-1.938 μg / mL.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The present invention uses an optimized modifier as a buffer for capillary electrophoresis, which can perfectly separate flavonoid glycosides that cannot be separated by HPLC due to their similar structures. All eight flavonoid glycosides elute at around 12 min with symmetrical peaks and good separation. Moreover, compared with HPLC, the mobile phase of capillary electrophoresis is borate-based, and only 2 mL of mobile phase is needed for a single injection. The mobile phase is more environmentally friendly and requires less volume.

[0024] (2) The present invention relies on the ultra-high resolution of capillary electrophoresis and the optimized modifier as a buffer for capillary electrophoresis, which can be used as a means to detect, separate or discover new flavonoid glycosides. Attached Figure Description

[0025] Figure 1 This is a graph showing the peak elution of the mixed standard of this application in borate buffer under six different pH conditions;

[0026] Figure 2 This is a graph showing the peak elution of the mixed standards of this application in six different cyclodextrin borate buffer solutions;

[0027] Figure 3 This is a graph showing the peak assignments of the mixed standards in Me-β-cyclodextrin borate buffer of this application;

[0028] Figure 4 This is a graph showing the peak elution of the mixed standards of this application in six different DES borate buffer solutions;

[0029] Figure 5 This is a peak elution diagram of the blank solvent in this application;

[0030] Figure 6 This is a standard curve of the eight single standard reference stock solutions of this application in Me-β-cyclodextrin borate buffer;

[0031] Figure 7 This is a standard curve diagram of the eight flavonoid glycosides in this application. Detailed Implementation

[0032] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0033] The purity of the flavonoid glycosides used in the following examples was ≥98% by HPLC, all of which were analytical standards. Isoquercetin (S33219), quercetin (B20526), ​​luteolin (B20887), vitexin (B20875), and isovitexin (B50649) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; hyperoside (Q109801) was purchased from Aladdin Reagent (Shanghai) Co., Ltd.; meadowsweet (A1344) and quercetin-7-O-glucoside (491-50-9) were purchased from Chengdu Mansite Biotechnology Co., Ltd. and Wuhan Kostan Biotechnology Co., Ltd., respectively.

[0034] All other reagents were of analytical grade unless otherwise specified. Sodium tetraborate decahydrate, anhydrous sodium dihydrogen phosphate, choline chloride, betaine, proline, lactic acid, sorbitol, glucose, and urea were purchased from Sinopharm Chemical Reagent Co., Ltd.; α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, and 2-hydroxypropyl-β-cyclodextrin were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Acetonitrile, acetic acid, sodium hydroxide, and dimethyl sulfoxide were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0035] Example 1

[0036] 1) Preparation of buffer solution

[0037] Preparation of borate stock solution: 9.53 g of sodium tetraborate decahydrate and 1.2 g of sodium dihydrogen phosphate were placed in a 1 L volumetric flask and diluted to 1 L with deionized water, resulting in a final borate stock solution containing 25 mM sodium tetraborate decahydrate and 10 mM sodium dihydrogen phosphate. 50 mL of the borate stock solution was then transferred to separate glass bottles, and the pH of the borate stock solution was adjusted to 8.95, 9.04, 9.15, 9.23, 9.34, and 9.45 using 1 M sodium hydroxide solution. The solutions were then stored at 4°C until use.

[0038] Preparation of cyclodextrin phosphate background buffer: Weigh 0.2432g α-cyclodextrin, 0.2837g β-cyclodextrin, 0.3243g γ-cyclodextrin, 0.3258g methyl-β-cyclodextrin, 0.3853g carboxymethyl-β-cyclodextrin, and 0.3854g 2-hydroxypropyl-β-cyclodextrin into 50ml volumetric flasks respectively, and standardize to 50mL with the above borate stock solution to prepare phosphate background buffer containing various cyclodextrins. The final buffer solution contains 5mM of borate stock solution of each cyclodextrin. After preparation, store at 4℃ for later use.

[0039] DES solution preparation: Hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs) in different molar ratios were mixed with water in 250 mL Erlenmeyer flasks and placed in a water bath at 80 °C. The mixture was stirred at 150 rpm until a homogeneous and transparent liquid was formed. The parameters of each raw material are shown in Table 1 below.

[0040] Table 1. Preparation ratios of the six DES solutions

[0041]

[0042] Preparation of DES background buffer solution: Take 1 mL of each of the six prepared DES solutions and place them in a 100 mL volumetric flask. Then, use the above-mentioned borate stock solution to make up to 100 mL, so that the volume ratio of DES in the final background buffer solution is 1%. The prepared buffer solutions are also stored at 4℃ until use.

[0043] 2) Solution preparation

[0044] Blank solvent: 25% (v / v) DMSO solution. Preparation method: Take 250 mL of DMSO and place it in a 1 L volumetric flask, then standardize it to 1 L with deionized water and store it in a refrigerator at 4 °C until use.

[0045] Reference solution:

[0046] Single standard reference stock solution: Weigh appropriate amounts of eight flavonoid glycosides standards, namely isoquercitrin, hyperoside, meadowsweet, quercetin-7-O-glucoside, quercetin, luteolin, vitexin and isovitexin, and dissolve them in 25% DMSO aqueous solution to prepare a stock solution with a concentration of 50 μg / mL. Store at 4°C in the dark.

[0047] Mixed standard working solution: Transfer an appropriate amount of each single standard stock solution, dilute with 25% DMSO aqueous solution to prepare a mixed standard working solution with a concentration of 40 μg / mL, filter through a 0.22 μm cellulose acetate membrane and set aside for use.

[0048] 3) Set capillary parameters: injection conditions are 0.5 psi for 5 seconds; electric field strength is 15 kV, and the electric field direction is from capillary inlet to outlet; during separation, the capillary temperature is maintained at 25℃, and the sample storage chamber temperature is set to 10℃; the detection wavelength is 360 nm, the separation time is 25 min, and the background buffer solution is replaced after every three analyses.

[0049] 4) Capillary pretreatment: Before each sample loading, rinse with 200mM NaOH solution and deionized water at 50psi pressure for 5min, and then run with the test buffer from step (1) at 20kV for 4min to complete the pretreatment; before each sample separation program, rinse with 200mM NaOH solution, deionized water and the test buffer from step (1) at 50psi pressure for 1min, and dip both ends of the capillary in deionized water once before and after sample loading to prevent sample contamination.

[0050] 5) Detection and Analysis

[0051] Six different pH borate stock solutions were used as buffers to separate the mixed standard working solution by electrophoresis. Each pH value was measured in triplicate, and the electrophoretic patterns, migration times, and peak widths of each component were recorded. Results are shown below. Figure 1 .

[0052] Depend on Figure 1 It can be seen that at pH 8.95, the elution times of the eight flavonoids were distributed between approximately 8.5 min and 13 min. Except for luteolin (8.771 min), which eluted first and showed a slightly delayed peak shape, the peaks of the other compounds were basically symmetrical. Quercetin-7-O-glucoside eluted next (10.112 min), with a resolution of 5.228 between these two compounds. Isovitexin (11.079 min) and meadowsweet (11.216 min) subsequently eluted. Compared to the perfect separation of the first two compounds, the resolution of this pair of flavonoid molecules was only 0.830. Although it did not reach the baseline separation level, it was still possible to separate them from the target flavonoids. Figure 1 Two distinct peaks were observed. The same pattern was observed for the pair of compounds quercetin (12.804 min) and hyperoside (13.012 min), with a resolution of 0.958, very close to complete separation. However, vitexin and isoquercetin exhibited very similar retention times, 12.021 min and 12.096 min respectively, failing to show effective separation in the figure, with a resolution of only 0.630.

[0053] As the pH of the buffer solution increased, the migration times of all target compounds showed a significant increasing trend. Under high pH conditions, the differences in charge density among the eight structural analogs caused by small differences in dissociation constants (pKa) were further amplified. When the pH of the background buffer solution increased from 8.95 to 9.23, the three pairs of substances that were originally poorly separated—violin and meadowsweet, vitexin and isoquercitrin, and quercetin and hyperoside—achieved baseline separation. At pH 9.04, the separation rates increased from 0.830, 0.630, and 0.958 to 1.194, 1.000, and 0.981, respectively; subsequently, at pH 9.15, they further increased to 1.813, 1.069, and 1.046, and finally reached 2.689, 2.581, and 1.229 at pH 9.23. When the pH exceeds 9.45, the system current increases due to changes in the ionic strength and conductivity of the buffer solution, and the Joule heating effect leads to peak broadening, resulting in a slight decrease in separation efficiency.

[0054] However, from Figure 1 A clear reversal of the migration order of vitexin and isoquercitrin was observed as the pH increased from 8.95 to 9.04, followed by a second reversal around pH 9.34-9.45. This confirms the differential regulatory effect of pH on the dissociation behavior of flavonoids with different structures. While this characteristic verifies the differences in dissociation kinetics among different structural derivatives, this high sensitivity presents a double-edged sword in routine analysis and quality control applications. Even small fluctuations in the background electrolyte pH can significantly alter migration times and even cause shifts in peak order, severely impacting the accuracy and reproducibility of qualitative results. Therefore, establishing a robust and highly buffered reference background electrolyte is essential for constructing a reliable standardized fingerprint library.

[0055] Six different cyclodextrin borate buffers were used as buffers for electrophoretic separation of the mixed standard working solution. Each buffer was measured in triplicate, and the relevant data were recorded. Results are shown below. Figure 2-3 .

[0056] Depend on Figure 2-3It was found that although α-CD and γ-CD could elute all eight compounds, the overall separation effect was poor. The chromatographic peaks generally exhibited asymmetric peak shapes, severe tailing, and peak broadening. In contrast, the experimental results for β-CD showed an extremely short migration time (8.75 min), with only seven major chromatographic peaks observed. Because CM-β-CD carries a negative charge in the alkaline buffer, it migrates towards the anode under the influence of an electric field, opposite to the direction of electroosmotic flow pointing towards the cathode. This countercurrent effect significantly prolonged the migration time of the analytes (up to 14.5 min). Furthermore, within the 12.5-13.5 min range, four key chromatographic peaks showed severe overlap and compression. As neutral derivatives, Me-β-CD and 2HP-β-CD significantly improved the resolution, achieving complete separation of all eight compounds, with moderate migration times (both 12 min). However, careful observation revealed that some chromatographic peaks of 2HP-β-CD lacked sharpness and appeared slightly blunt. After screening, Me-β-CD was determined to be the optimal additive.

[0057] Six different buffer solutions containing 1% of each type of DES were used as background buffers for electrophoretic separation of the mixed standard working solution. Each buffer solution was measured in triplicate, and the relevant data were recorded. Results are shown below. Figure 4 .

[0058] To investigate whether a non-charge mechanism based on "solventization environment regulation" could achieve similar separation results, or serve as an alternative to CD, the experiment subsequently examined the effects of adding 1% (v / v) of each of the six DESs individually. The experimental results are as follows... Figure 4 The results showed that relying solely on DES to adjust the solubilization microenvironment failed to effectively improve separation. Notably, most DES systems resulted in the disappearance of chromatographic peaks. While adding Bet-Sor background buffer allowed for peak elution, the effect was far less pronounced than with Me-β-CD. This is due to the non-specific adsorption of DES components onto the capillary wall or the formation of overly stable complexes that prevented elution. This comparative result indicates that for structurally very similar flavonoid derivatives in this application, a single "solventization effect" is insufficient to provide adequate selectivity, and its separation potential is far lower than the stereorecognition mechanism of cyclodextrin. Therefore, a borate stock solution containing 5 mM Me-β-CD was ultimately determined to be the optimal additive. The blank solubilization chromatogram is shown below. Figure 5 As shown, the standard curves of eight single-standard reference stock solutions in Me-β-cyclodextrin borate buffer are as follows. Figure 6 As shown.

[0059] Example 2

[0060] 1. Linear relationship

[0061] Buffer solution preparation:

[0062] Borate stock solution: Place 9.53 g of sodium tetraborate decahydrate and 1.2 g of sodium dihydrogen phosphate in a 1 L volumetric flask, and dilute to 1 L with deionized water to make the final borate stock solution contain 25 mM sodium tetraborate decahydrate and 10 mM sodium dihydrogen phosphate; take 50 mL of the borate stock solution in a glass bottle, adjust the pH of the borate stock solution to 9.23 with 1 M sodium hydroxide solution, and store at 4 °C for later use.

[0063] Cyclodextrin phosphate background buffer: Weigh 0.3258 g of methyl-β-cyclodextrin into a 50 ml volumetric flask and titrate to 50 mL with the above borate stock solution to prepare a phosphate background buffer containing methyl-β-cyclodextrin, so that the final buffer contains 5 mM of methyl-β-cyclodextrin borate stock solution. After preparation, store at 4 °C for later use.

[0064] Preparation of mixed standard products:

[0065] Single standard stock solution: Weigh appropriate amounts of eight flavonoid glycosides standards, namely isoquercitrin, hyperoside, meadowsweet, quercetin-7-O-glucoside, quercetin, luteolin, vitexin and isovitexin, and dissolve them in 25% DMSO aqueous solution to prepare a stock solution with a concentration of 50 μg / mL. Store at 4°C in the dark.

[0066] Mixed standard working solutions: Take appropriate amounts of each stock solution and dilute with 25% DMSO aqueous solution to prepare mixed standard working solutions with concentrations of 2.5, 5, 10, 20, and 40 μg / mL. Filter the solutions through a 0.22 μm cellulose acetate membrane for later use.

[0067] The mixed standard working solutions prepared above with concentration gradients of 2.5, 5, 10, 20, and 40 μg / mL were analyzed. A standard curve was plotted with the concentration of each compound on the x-axis and the corresponding peak area on the y-axis, and linear regression analysis was performed. The results are shown in Table 2 and [Table data missing]. Figure 7 .

[0068] Table 2. Linear relationships and ranges of eight flavonoid glycosides.

[0069]

[0070] From Table 2 and Figure 7 It can be seen that within the concentration range (2.5-40 μg / mL), all eight flavonoids exhibited excellent linearity, with the coefficients of determination of the regression equations for each component ranging from 0.996 to 0.999. This indicates that the method has good sensitivity and proves that the quantitative results at this resolution are accurate and reliable, meeting the requirements of practical analysis.

[0071] 2. Limit of Detection and Limit of Quantification

[0072] Buffer solution preparation: This refers to the buffer solution under the linear relationship term;

[0073] Preparation of mixed standard products:

[0074] Single standard stock solution: that is, the buffer solution under the linear relationship term;

[0075] Mixed standard working solution: Take an appropriate amount of each stock solution and dilute it with 25% DMSO aqueous solution to prepare a mixed standard working solution with a concentration of 20 μg / mL. Filter the solution through a 0.22 μm cellulose acetate membrane for later use.

[0076] The mixed standard working solution was tested with a signal-to-noise ratio (S / N) of 3 as the limit of detection (LOD) and an S / N of 10 as the limit of quantitation (LOQ). The results are shown in Table 3.

[0077] Table 3. Detection and Quantification Limits for Eight Flavonoid Glycosides

[0078]

[0079] As shown in Table 3, the LOD values ​​of the eight flavonoid compounds ranged from 0.207 to 0.581 μg / mL, the detection limits of all compounds were below 0.1 μg / mL, the LOQ values ​​ranged from 0.689 to 0.1938 μg / mL, and the average quantitation limit was 0.189 μg / mL, which is significantly lower than the actual analytical concentration of the target components in conventional samples. Moreover, all quantitation limits were at a low level, indicating that this method can still achieve accurate quantification of the target compounds at trace concentrations. The quantitative results are reliable and highly accurate, and can meet the needs of quantitative analysis of flavonoid glycosides in actual samples.

[0080] 3. Resolution

[0081] Buffer solution preparation: This refers to the buffer solution under the linear relationship term;

[0082] Preparation of mixed standards: This is the buffer solution under the linear relationship term.

[0083] Place the mixed standard working solution into the sample rack, start the capillary electrophoresis system according to the method in Example 1, perform separation and detection, record the electrophoretic pattern, and the results are shown in Table 4.

[0084] Table 4. Resolution results of eight flavonoid glycosides

[0085]

[0086] As shown in Table 4, the resolution R of each adjacent component is between 1.027 and 6.061, which all meet the requirements for quantitative analysis (R≥1.0). Among them, most components are completely separated (R≥1.5), and the separation effect is excellent.

[0087] 4. Repeatability

[0088] Three mixed standard samples with concentrations of 5.12, 11.99, and 19.99 μg / mL were prepared and added to a 20 μg / mL standard mixture, and analyzed three times. The precision, repeatability, and accuracy of the method were evaluated through spiked experiments at three concentration levels. The results are shown in Table 5. The calculation formulas include:

[0089] Relative recovery rate (%) =

[0090] Precision (RSD%) =

[0091] Accuracy (%) =

[0092] Table 5. Repeatability results of the separation method

[0093]

[0094]

[0095] Table 5 shows that the intra-day relative standard deviation (RSD) of precision remained below 3.06%, indicating excellent repeatability of the instrument response. Method accuracy, expressed as relative error, ranged from 0.01% to 4.16%, fully within the acceptable range for analytical methods. The recoveries (RR) of spiked samples ranged from 95.84% to 101.29%, indicating efficient sample preparation procedures and no significant matrix effects interfering with the analysis. In summary, these results demonstrate that this method exhibits satisfactory reliability and accuracy for the qualitative and quantitative analysis of flavonoids in real samples.

[0096] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis, characterized in that: Includes the following steps: 1) Prepare buffer solutions: Prepare borate stock solution, cyclodextrin phosphate buffer, and DES background buffer solution; 2) Preparation of mixed standards: Dissolve the standards of eight flavonoid glycosides and the mixed sample in dimethyl sulfoxide aqueous solution to prepare stock solutions of individual standards and working solutions of mixed standards; 3) Setting capillary detection conditions: The SCIEX P / ACE MDQ Plus capillary electrophoresis system was used, equipped with a diode array detector. The capillary used was an uncoated fused silica capillary with an inner diameter of 75 μm and an effective length of 60 cm. 4) Capillary pretreatment; 5) Quantitative analysis: The sample is separated and detected according to the detection conditions in step 3) to obtain the electrophoresis peak diagram of the sample to be tested, the peak area value of each peak is extracted, and the migration time and concentration of each component are calculated by the peak area integration method to achieve the separation and quantification of eight flavonoid glycosides. The eight flavonoid glycosides are isoquercitrin, hyperoside, meadowsweet, quercetin-7-O-glucoside, quercetin, luteolin, vitexin, and isovitexin.

2. The method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis according to claim 1, characterized in that: In step 1), the pH of the borate stock solution is 8.95-9.

45.

3. The method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis according to claim 1, characterized in that: In step 1), the cyclodextrin is selected from one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, and 2-hydroxypropyl-β-cyclodextrin.

4. The method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis according to claim 1, characterized in that: In step 1), the hydrogen bond donor in the DES background buffer solution is selected from one of choline chloride, betaine, and proline, and the hydrogen bond acceptor is selected from one of lactic acid, D-sorbitol, glucose, and urea. The molar ratio of hydrogen bond donor to hydrogen bond acceptor is 1~3:

1.

5. The method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis according to claim 1, characterized in that: In step 2), the preparation method for the stock solution of a single standard is as follows: weigh appropriate amounts of isoquercitrin, hyperoside, meadowsweet, quercetin-7-O-glucoside, quercetin, luteolin, vitexin and isovitexin standards, dissolve them in 25% DMSO aqueous solution to prepare a stock solution with a concentration of 50 μg / mL, and store at 4℃ in the dark.

6. The method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis according to claim 1, characterized in that: In step 2), the preparation method of the mixed standard working solution is as follows: take an appropriate amount of each single standard stock solution, dilute it with 25% DMSO aqueous solution to prepare a mixed standard working solution with a concentration of 20 μg / mL, filter it through a 0.22 μm cellulose acetate membrane and set it aside for later use.

7. The method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis according to claim 1, characterized in that: In step 3), the injection conditions are: 0.5 psi for 5 seconds; electric field strength is 15 kV, and the electric field direction is from capillary inlet to outlet; during the separation process, the capillary temperature is maintained at 25℃, and the sample storage chamber temperature is set to 10℃.

8. The method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis according to claim 1, characterized in that: In step 3), the detection wavelength is 360 nm; the separation time is 25 min; and the background buffer solution is replaced after every three analyses.

9. The method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis according to claim 1, characterized in that: In step 4), capillary pretreatment: Before each sample loading, rinse with 200mM NaOH solution and deionized water at 50psi pressure for 5 minutes each, and then run with buffer at 20kV for 4 minutes to complete the pretreatment; before each sample separation program, rinse with 200mM NaOH solution, deionized water and buffer at 50psi pressure for 1 minute each, and dip both ends of the capillary in deionized water once before and after sample injection.

10. The method for detecting and separating eight structurally similar flavonoid glycosides using capillary electrophoresis according to claim 1, characterized in that: The R-values ​​of the standard curves for the eight flavonoid glycosides are... 2 The limit of detection (LOD) was 0.996-0.999; the limit of quantitation (LOQ) was 0.207-0.581 μg / mL; and the limit of quantitation (LOQ) was 0.689-1.938 μg / mL.

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