Method for simultaneously and quantitatively analyzing various components in Yang-warming Xiaozheng decoction by combining dispersive solid-phase extraction (MCDS)-sweeping-MEKC

Abstract

The invention belongs to the field of medicine detection, particularly belongs to the field of traditional Chinese medicine prescription detection, and more particularly relates to a method for simultaneously and quantitatively analyzing various components in yang-warming Xiaozheng decoction through dispersive solid-phase extraction combined with MCDS-sweeping-MEKC. According to the method, hydroxypropyl-beta-cyclodextrin is creatively applied to dispersive solid-phase extraction as an eluent for the first time, and is combined with an MCDS-sweeping-MEKC online enrichment technology, so that an off-line enriched sample is directly subjected to subsequent analysis without being volatilized and redissolved, the operation process is greatly reduced, the sensitivity of sample analysis is further improved, and the method is suitable for large-scale industrial production. The method is simple and convenient to operate, good in reproducibility, short in analysis time, high in separation efficiency and small in organic solvent dosage, the requirements of green chemistry are better met, and meanwhile the maximum enrichment multiple of target analytes can be 208 times.

Description

Technical Field

[0001] The invention belongs to the field of drug detection, in particular to the field of traditional Chinese medicine prescription detection, and more specifically relates to a method for simultaneously quantitatively analyzing multiple types of components in Wenyang Xiaozheng Decoction by using dispersed solid phase extraction combined with MCDS-sweeping-MEKC. Background Art

[0002] Wenyangxiaozheng Decoction is a Chinese patent medicine prescription composed of Astragalus, Codonopsis, Curcuma, Chuanxiong, Cistanche, Epimedium and Peach Kernel. Astragalus and Codonopsis nourish the spleen; Epimedium and Cistanche warm and nourish the kidney yang; Chuanxiong is a blood Qi medicine, pungent and warm, activating blood circulation and qi; Peach Kernel activates blood circulation and removes blood stasis, moistens the intestines and relieves constipation; Curcuma breaks blood circulation and promotes qi circulation, eliminates symptoms and removes blood stasis. As a classic prescription for the treatment of chronic kidney disease, this prescription treats both the symptoms and the root cause, attacks and supplements at the same time, replenishes the deficiency without leaving the evil, and activates blood circulation without hurting the body. It has achieved good results in the treatment of kidney diseases. In particular, this prescription also shows effective improvement effects for renal fibrosis, chronic renal failure and its complications such as renal anemia, secondary hyperparathyroidism, cardiovascular function damage, etc.

[0003] However, as a traditional decoction prescription, Wenyang Xiaozheng Decoction has no clear quality inspection method, which makes it difficult to ensure the stability of drug efficacy and consistency of treatment effect in clinical application. There is also a lack of clear and effective monitoring methods for the metabolism of Wenyang Xiaozheng Decoction after medication. Summary of the invention

[0004] Based on this, the present invention aims to provide a method for simultaneously quantitatively analyzing multiple types of components in Wenyang Xiaozheng Decoction, so as to ensure the effectiveness and consistency of compound drugs in clinical use, and also lay the foundation for the further development of Wenyang Xiaozheng Decoction and clinical drug exploration.

[0005] In order to achieve the above-mentioned purpose of the invention, the present invention discloses a method for simultaneously quantitatively analyzing multiple types of components in Wenyang Xiaozheng Decoction by using dispersed solid phase extraction combined with MCDS-sweeping-MEKC, the method comprising the following steps: S1: Use cyclodextrin-assisted dispersed solid phase microextraction to treat the sample to be tested, and extract the target substance to be detected into the extraction solution; S2: Using a capillary electrophoresis instrument, cyclodextrin is used as the sample matrix, and the analyte to be tested is enriched online by combining with the sweeping agent in the micellar electrokinetic chromatography background buffer system, and the capillary electrophoresis spectrum of the sample solution is obtained by detection at a detection wavelength of 205 nm; S3: preparing a mixed standard solution of codonopsis pilosula, echinacoside, verbascoside, and verbascoside isoflavones, and performing detection according to the capillary electrophoresis detection conditions in S2 to obtain a capillary electrophoresis spectrum of the mixed standard solution; S4: Using the capillary electrophoresis spectrum of the sample solution obtained in S2 and the capillary electrophoresis spectrum obtained in S3, the qualitative and / or quantitative results of dangshen glycoside, echinacoside, verbascoside, and verbascoside isoflavones in Wenyang Xiaozheng Decoction are obtained.

[0006] Preferably, the sweeping agent is sodium dodecyl sulfate.

[0007] Preferably, step S1 specifically includes the following steps: S1-1: first, the Wenyang Xiaozheng Decoction is concentrated to dryness, and then dissolved in pure water to prepare a Wenyang Xiaozheng Decoction crude extract, an adsorbent is added, the adsorbent and the Wenyang Xiaozheng Decoction crude extract are vortexed and adsorbed, centrifuged, and the supernatant is removed; S1-2: Then add hydroxypropyl-β-cyclodextrin solution to the adsorbent and vortex to elute; S1-3: After elution, filter the supernatant through an organic filter head.

[0008] Further preferably, the adsorbent is arbitrarily selected from cross-linked polyvinylpyrrolidone (PVPP), colloidal silicon dioxide, florisil, silica gel, C 18 Or any one of MCM-48. Among them, the most preferred adsorbent is cross-linked polyvinylpyrrolidone (PVPP).

[0009] Preferably, the mixing ratio of the adsorbent to the crude extract of Wenyang Xiaozheng Decoction is 1:1.

[0010] Preferably, the adsorbent and the crude extract of Wenyang Xiaozheng Decoction are vortexed for 1-2.5 min, preferably 1.5 min.

[0011] Preferably, the vortex elution time is 1-3 min, preferably 2 min.

[0012] In a preferred technical solution, the amount of hydroxypropyl-β-cyclodextrin added is 1000 μL.

[0013] Preferably, in step S2, the capillary electrophoresis instrument may select one or more of the following electrophoresis conditions: (1) The background buffer solution is a mixture of 55 mM sodium tetraborate (borax) solution, 50-100 mM sodium dodecyl sulfate (SDS) solution, and 0-30% (v / v) methanol; (2) The cyclodextrin is α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin, wherein the β-cyclodextrin is hydroxypropyl-β-cyclodextrin (HP- β -CD) or hydroxyethyl-β-cyclodextrin (HE- β -CD); (3) The cyclodextrin concentration is below 150 mM; (4) The injection condition is to inject the sample solution at a pressure of 50 mBar for 100 s; (5) The separation voltage is +25KV and the separation temperature is 25℃.

[0014] Preferably, the capillary electrophoresis analysis conditions are: 55 mmol sodium tetraborate solution, 75 mmol sodium dodecyl sulfate solution (SDS) and 25% (v / v) methanol as background buffer solution, 125 mmol hydroxypropyl-β-cyclodextrin (HP- β -CD) was used as the sample matrix, and the sample solution was injected at a pressure of 50 mbar for 100 s, with a separation voltage of +25 KV and a temperature of 25 °C.

[0015] Furthermore, the present invention also discloses a quantitative analysis and detection method for multiple types of compounds including phenylethanoid glycosides, flavonoids, and saponins, which is specifically: Prepare a mixed standard solution of dangshen glycoside, echinacoside, verbascoside, and verbascoside isoflavones in a concentration range of 1-100 μg / mL, detect according to the electrophoresis analysis conditions of step S2, perform parallel determination three times, draw a capillary electrophoresis spectrum of the obtained mixed standard solution, and draw standard curves of dangshen glycoside, echinacoside, verbascoside, and verbascoside respectively with the peak area of ​​each standard in the obtained spectrum as the ordinate and the concentration of the standard in the mixed standard solution as the abscissa, thereby completing the construction of the standard curve; The peak areas of dangshenyl glycoside, echinacoside, verbascoside and verbascoside isoflavone in the capillary electrophoresis spectrum of the sample solution were substituted into the standard curve, and then the contents of dangshenyl glycoside, echinacoside, verbascoside and verbascoside isoflavone in the sample were calculated.

[0016] The present invention uses the four main active ingredients of codonopsis pilosula glycoside, echinacoside, verbascoside and verbascoside isoflavones as the target objects to be detected for the quality detection of the Wenyang Xiaozheng prescription, which can better characterize the quality of the Wenyang Xiaozheng prescription.

[0017] Compared with qualitative and quantitative analysis methods such as thin layer chromatography, high performance liquid chromatography, and gas chromatography, capillary electrophoresis technology is widely used in the analysis of food, medicine, and the environment because of its advantages of high separation efficiency, fast analysis speed, short analysis time, and low solvent consumption. However, due to its own injection limitations and the interference caused by complex matrices, capillary electrophoresis has the problem of low sensitivity when analyzing complex matrices.

[0018] However, codonopsis glycoside is a neutral analyte and is not easily ionized, while echinacoside, verbascoside and verbascoside isoflavone are weakly acidic substances and can be ionized in an alkaline buffer system, which makes it difficult to qualitatively and quantitatively detect the four main active ingredients of codonopsis glycoside, echinacoside, verbascoside and verbascoside using a micellar electrokinetic chromatography based on capillary electrophoresis. This is because micellar electrokinetic chromatography forms micelles by adding surfactants to the capillary electrophoresis buffer, and these micelles can selectively wrap the analyte, thereby changing the migration rate and distribution behavior of the analyte and achieving separation, but this method is suitable for analyzing neutral substances and charged substances, and cannot be applied to non-neutral and uncharged substances in the present invention.

[0019] In the present invention, the inventor creatively proposes an online enrichment method, so that online enrichment can provide analyte sensitivity, thereby improving the accuracy and sensitivity of detection. In the present invention, hydroxypropyl-β-cyclodextrin is used as a sample matrix, combined with a sweeping agent SDS, and a MCDS-sweeping-MEKC strategy is developed to enrich the analytes to be tested, thereby overcoming the problems existing in the detection of the analytes to be tested, such as codonopsis pilosula, echinacea glycoside, verbascoside, and verbascos isoflavones in micellar electrokinetic chromatography, and realizing the application of micellar electrokinetic chromatograph in the quality detection of Wenyang Xiaozheng Decoction.

[0020] At the same time, the present invention creatively applies hydroxypropyl-β-cyclodextrin as an eluent in dispersed solid phase extraction for the first time, and combines it with the MCDS-sweeping-MEKC online enrichment technology, so that the samples enriched offline can be directly subjected to subsequent analysis without evaporation and redissolution, which greatly reduces the operation process and further improves the sensitivity of sample analysis.

[0021] Compared with the prior art, the technical solution disclosed in the present invention has the following beneficial effects: 1. The present invention combines the dispersed solid phase extraction offline pre-concentration technology MCDS-sweeping-MEKC capillary online technology for the first time in the capillary micellar electrokinetic chromatography mode, and successfully applies it to the separation and detection of phenylethanoid glycosides, flavonoids, and saponin compounds (echinacoside, verbascoside, verbascoside isoflavones, and codonopsis glycoside) in the Wenyang Xiaozheng prescription.

[0022] 2. This invention is the first to combine HP- β -CD was used as an eluent in dispersive solid phase microextraction (DMSPE) and was successfully applied to the extraction of Wenyang Xiaozheng prescription.

[0023] 3. The method of the present invention is easy to operate, has good reproducibility, short analysis time, high separation efficiency, and low organic solvent usage, which is more in line with the requirements of green chemistry. At the same time, the enrichment multiple of the target analyte can be up to 208 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the structural formula of codonopsis pilosula, echinacoside, verbascoside and verbascoside isoflavones.

[0025] Figure 2 Schematic diagram of the effect of adsorbent type on DMSPE extraction effect.

[0026] Figure 3 Schematic diagram of the effect of the ratio of sample to adsorbent on the DMSPE extraction effect.

[0027] Figure 4 Schematic diagram of the effect of vortex adsorption time on DMSPE extraction effect.

[0028] Figure 5 Schematic diagram of the effect of vortex elution time on DMSPE extraction effect.

[0029] Figure 6 Schematic diagram of the effect of elution volume on DMSPE extraction efficiency.

[0030] Figure 7 Schematic diagram of the effect of borax concentration in the background buffer on the CE online stacking effect.

[0031] Figure 8 Schematic diagram of the effect of methanol content in the background buffer on CE online stacking effect.

[0032] Fig. 9 Schematic diagram of the effect of SDS in the background buffer on the online stacking effect of CE.

[0033] Fig.10 Schematic diagram of the effect of cyclodextrin types in the sample matrix on CE online stacking effect.

[0034] Fig.11 HP- β -Schematic diagram of the effect of CD concentration on CE online stacking effect.

[0035] Fig.12 Chromatograms of mixed standard solution (A) and real sample spiked serum (B).

[0036] Fig.13 The present invention is a schematic diagram of the process of the quality detection method of Wenyang Xiaozheng Decoction disclosed in the present invention. DETAILED DESCRIPTION

[0037] The present invention is further described below by means of specific embodiments, but the protection scope of the present invention is not limited thereto.

[0038] In the following examples, standard products of codonopsis pilosula, echinacoside, verbascoside and verbascoside isoflavones were purchased from Chengdu Mansite Biotechnology Co., Ltd. (structural formula see Figure 1 ); sodium hydroxide and sodium tetraborate were obtained from Tianjin Yongda Chemical Reagent Co., Ltd. and Shanghai Lingfeng Chemical Reagent Co., Ltd., respectively; α-CD was purchased from Shanghai MacLean Reagent Co., Ltd.; γ-CD and HE- β -CD was purchased from Shanghai Myrel Biochemical Technology Co., Ltd.; HP- β -CD was provided by Zibo Qianhui Biotechnology Co., Ltd.; purified water was provided by Hangzhou Wahaha Group Co., Ltd.; SDS was purchased from Xilong Chemical Co., Ltd.; PVPP was purchased from Shanghai Wokai Pharmaceutical Co., Ltd.; MCM-48, C 18 , Florisil, and silica gel were purchased from Nanjing Jicang Nanotechnology Co., Ltd., Shanghai Bozhi Biotechnology Co., Ltd., Shanghai Sigma-Aldrich Trading Co., Ltd., and Chengdu Cologne Chemicals Co., Ltd., respectively.

[0039] The specific steps of the preparation method of the reference solution are as follows: accurately weigh appropriate amounts of dangshen glycoside, echinacoside, verbascoside and verbascos isoflavone reference substances, and prepare a stock solution with a concentration of 1 mg / mL with methanol. Store in a refrigerator at 4°C for subsequent CE analysis.

[0040] The instrument used in the embodiment of the present invention is an Agilent capillary electrophoresis instrument (Agilent CE 7100, Agilent Technologies, Waldbronn, Germany) equipped with a UV detector. The electrophoresis experiment was performed using a fused silica capillary with an inner diameter of 50 μm provided by Yongnian Ruifeng Chromatographic Equipment Co., Ltd. The total length and effective length of the capillary were 50 cm and 42 cm, respectively. The detection wavelength was 205 nm. Example 1

[0041] 1 mL of 5 µg / mL rat spiked serum was mixed with 15 mg PVPP and vortexed for 1.5 min. The supernatant was removed and 1 mL of 125 mM HP- β -CD was eluted for 2 min and the supernatant was collected.

[0042] Pretreatment of the capillary: before use, the new capillary column was activated by flushing with 1 mol / L NaOH for 20 min, 0.1 mol / L NaOH for 10 min, and pure water for 10 min; before daily injection, it was flushed with 0.1 mol / L NaOH, pure water, and background buffer for 10 min respectively; between every two sample analysis runs, it was flushed with 0.1 mol / L NaOH, pure water, and background buffer for 3 min to maintain the reproducibility of the analysis, and the background buffer should be replaced in time after every two runs.

[0043] The electrophoresis analysis conditions were as follows: 55 mM sodium tetraborate solution, 75 mM sodium dodecyl sulfate solution (SDS) and 25% (v / v) methanol as background buffer solution, 125 mM hydroxypropyl- β -Cyclodextrin (HP- β -CD) was used as the sample matrix and the sample solution was injected at a pressure of 50 mbar for 100 s. After the injection, a separation voltage of +25 kV was applied, the detection temperature was 25 °C, and the detection wavelength was 205 nm.

[0044] According to the results, the contents of echinacoside, verbascoside and verbascoside isoflavones in the sample determined by the method disclosed in the present invention were 9.93±0.03, 4.59±0.02 and 0.46±0.01 µg / mg, respectively.

[0045] Process such as Fig.13 shown. Example 2

[0046] According to the method in Example 1, PVPP was replaced by colloidal silica, florisil, silica gel, C 18 and MCM-48, and investigated the effects of different adsorbents. The experimental results are shown in Figure 2 .

[0047] It can be seen that compared with the other five adsorbent materials, PVPP has the best enrichment effect on the target components.

[0048] PVPP has a highly physically cross-linked three-dimensional network structure and contains many functional groups that can form hydrogen bonds. We found that the three-dimensional network structure of PVPP and the functional groups that can form extremely strong hydrogen bonds with the target analytes can interact with the target analytes.

[0049] Therefore, PVPP is the preferred adsorbent material. Example 3

[0050] According to the method in Example 1, only the mixing volume ratio of the adsorbent and the Wenyang Xiaozheng Decoction crude extract was adjusted, and the adsorbent was added to the crude extract in a ratio of 1:2, 1:1, 2:1 or 3:1. Figure 3 As shown. The results show that when the ratio of sample to dispersant is 1:1, the highest extraction efficiency of the three analytes is observed. It may be that the content of the adsorbent is too low so that the analytes cannot be completely adsorbed at a ratio of 2:1. As the adsorbent in the system gradually increases (1:2 and 1:3), the target analytes are adsorbed by excess adsorbent, and the increase in the binding capacity between the adsorbent and the target analyte makes it difficult to elute the target analyte, resulting in a decrease in the extraction efficiency. Therefore, in subsequent experiments, the ratio of sample to adsorbent is 1:1. Example 4

[0051] According to the method in Example 1, only the time of vortex elution and vortex adsorption was adjusted. We set the time of unselected elution to 1 min, 1.5 min, 2 min, 2.5 min, and the time of vortex elution to analyze the effect of different adsorption-elution times on the results. The results are as follows. Figure 4 and Figure 5 It can be seen that when the vortex adsorption time is 1.5 min and the vortex elution time is 2 min, the extraction efficiency of the target analyte reaches the highest. Example 5

[0052] According to the method of Example 1, only the eluent HP- β -CD dosage was 500 μL, 1000 μL, 1500 μL, and 2000 μL. Figure 6 As shown, it can be seen that the three target analytes are all in the HP- β The maximum extraction efficiency was achieved when the amount of -CD was 1000 µL. When the eluent volume was 500 µL, the eluent failed to fully elute the target analytes adsorbed on PVPP, resulting in low extraction efficiency. When the eluent amount was 1500 or 2000 µL, the increase in the amount of eluent caused the sample to be diluted and the extraction efficiency to decrease. Therefore, the amount of cyclodextrin was selected to be 1000 µL. Example 6

[0053] A suitable buffer system can effectively improve the separation efficiency and detection sensitivity of the analyte. According to the method disclosed in Example 1, only the sodium tetraborate solution was changed. The results are as follows Figure 7As shown, it can be seen that the separation efficiency can be improved by increasing the concentration of borax. When the borax concentration is set to 55 mM, the separation effect of the four analytes is the best. As the borax concentration increases, the high-concentration buffer solution produces a stronger ζ potential, higher conductivity and working current, resulting in a Joule heating effect. Therefore, 55 mM borax was selected for subsequent studies. Example 7

[0054] According to the method disclosed in Example 1, only the amount of methanol added was changed, and the influence on the separation effect was analyzed in the percentage range of 0% to 30% (v / v). The results are as follows: Figure 8 As shown. It can be seen that with the increase of methanol concentration, the separation of the target analytes gradually improves. However, when the amount of methanol is 30% (v / v), the migration time of the analytes gradually prolongs, and the peak of the analytes becomes broader. Therefore, 25% (v / v) is the preferred addition amount of methanol. Example 8

[0055] The SDS concentration in the background buffer solution can significantly affect the enrichment performance of the analyte by changing the amount of analyte partitioned into the micelles, affecting the electroosmotic flow, conductivity and distribution coefficient of the BGE. In order to evaluate the effect of SDS concentration on the accumulation of target analytes, experiments were conducted by adding different SDS concentrations of 50-100 mM to the background buffer solution. According to the method disclosed in Example 1, keeping other conditions unchanged, the results are shown in Fig. 9 It can be seen that the peak area of ​​the analyte decreases with the increase of SDS concentration. When the SDS concentration is 75 mM, the separation and enrichment effect of the analyte is the best, so 75 mM is selected as the optimal concentration. Example 9

[0056] According to the method disclosed in Example 1, α-CD, γ-CD, hydroxyethyl- β -CD (HE- β -CD) and hydroxypropyl- β -CD (HP- β -CD) were used to analyze the effect of the type of cyclodextrin on the separation and stacking efficiency of the target analytes. The results are shown in Fig.10 As shown. Fig.10 It can be seen that when HP- β -CD showed the highest peak area of ​​the analyte and also achieved satisfactory resolution. We speculate that HP- β -CD formed a stronger interaction force with SDS micelles, which narrowed the stacking area. Example 10

[0057] The concentration of cyclodextrin (CD) in the sample solution is an important factor for enhancing and separating target analytes in MCDS-sweeping MEKC analysis. In this example, we followed the method in Example 1 and only changed the CD concentration from 0 mM to 150 mM to explore the effect of CD concentration on analyte separation and enrichment. The results are shown in Figure 2. Fig.11 As shown. When the CD concentration increases from 0 mM to 125 mM, the migration time of the target analyte is shortened and the peak gradually becomes narrower. However, by further increasing the CD concentration from 125 mM to 150 mM, the band of the target analyte is scanned narrower, but the peak intensity and separation are slightly reduced. This may be due to the strong interaction between the excess CD and the analyte, which leads to a weakened binding interaction between the analyte and the SDS micelle, resulting in a decrease in the peak area. Therefore, 125 mM was selected as the optimal concentration of CD. Embodiment 11

[0058] Take appropriate amount of 1 mg / mL standard solution respectively, accurately prepare mixed standard solutions of 1-100 μg / mL codonopsis glycoside, echinacoside, verbascoside, and verbascoside isoflavone (1.0, 2.5, 5, 10, 12.5, 25, 50, 100 μg / mL), perform electrophoresis analysis based on online enrichment method under the detection conditions described in Example 1, and measure the capillary electrophoresis spectrum of the mixed standard solution three times in parallel. Draw standard curves of codonopsis glycoside, echinacoside, verbascoside, and verbascoside respectively with the peak area of ​​each standard in the spectrum as the ordinate and the concentration of the mixed standard solution as the abscissa to complete the construction of the standard curve. The results are shown in Table 1, and all four analytes show good linear relationships (r>0.9984). The 5 μg / mL mixed standard solution was injected 6 times in one day to evaluate the intra-day precision, and the inter-day precision was evaluated by injecting 3 times a day for 3 consecutive days. Finally, the calculated relative standard deviation (RSD) of the peak area was lower than 2.98%, indicating that the method had good reproducibility.

[0059] Table 1 Linear range, detection limit, reproducibility and enrichment factor of the method

[0060] Enrichment factor = (peak area of ​​the analyte in this method / peak area of ​​the analyte in conventional injection) × dilution factor.

[0061] The conventional injection conditions are as follows: the background buffer solution contains 55 mM sodium tetraborate, 75 mM SDS and 25% methanol, and a mixed standard solution of 100 μg / mL codonopsis glycoside, echinacoside, verbascoside and verbascoside isoflavone is injected at a pressure of 50 mbar for 3 s. The calculated optimized online enrichment strategy makes the enrichment multiples of codonopsis glycoside, echinacoside, verbascoside and verbascoside isoflavone reach 106-208 times, which effectively improves the detection sensitivity of capillary electrophoresis for phenylethanoid glycosides, flavonoids and saponins in complex matrices. Example 12

[0062] In order to investigate the accuracy and sensitivity of the method, the optimized MCDS-sweeping-MEKC enrichment technique was used for the detection of dangshen glycoside, echinacoside, verbascoside and verbascoside isoflavones in spiked rat serum samples. Fig.12 As shown, (A) is the electrophoresis diagram of the 5 μg / mL mixed standard solution, and (B) is the electrophoresis diagram of the 5 μg / mL spiked rat serum. Fig.12 It can be seen that the sample peak separation is good. The feasibility of this method for the simultaneous determination of phenylethanoid glycosides, flavonoids and saponins was verified.

[0063] The above is a specific implementation 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 are also considered to be within the protection scope of the present invention.

Claims

1. Dispersive solid phase extraction combined with MCDS-sweeping-MEKC for simultaneous quantitative analysis of Wenyang Xiaozheng Decoction A multi-class component method characterized by, The method comprises the following steps: S1: Use cyclodextrin-assisted dispersed solid phase microextraction to treat the sample to be tested, and extract the target substance to be detected into the extraction solution; S2: Using a capillary electrophoresis instrument, cyclodextrin is used as a sample matrix, and the analyte to be tested is enriched online by combining with a scanning agent of micellar electrokinetic chromatography, and the capillary electrophoresis spectrum of the sample solution is obtained by detection at a detection wavelength of 205 nm; S3: preparing a mixed standard solution of codonopsis pilosula, echinacoside, verbascoside, and verbascoside isoflavones, and performing detection according to the capillary electrophoresis detection conditions in S2 to obtain a capillary electrophoresis spectrum of the mixed standard solution; S4: Using the capillary electrophoresis spectrum of the sample solution obtained in S2 and the capillary electrophoresis spectrum obtained in S3, the qualitative and / or quantitative results of dangshen glycoside, echinacoside, verbascoside, and verbascoside isoflavones in Wenyang Xiaozheng Decoction are obtained.

2. The method for simultaneous quantitative analysis of multiple components in Wenyang Xiaozheng Decoction by using dispersed solid phase extraction combined with MCDS-sweeping-MEKC according to claim 1, characterized in that: The sweeping agent is sodium lauryl sulfate.

3. The method for simultaneous quantitative analysis of multiple components in Wenyang Xiaozheng Decoction by using dispersed solid phase extraction combined with MCDS-sweeping-MEKC according to claim 1, characterized in that: Step S1 specifically includes the following steps: S1-1: First, Wenyang Xiaozheng Decoction is concentrated to dryness, and then dissolved in pure water to prepare a crude extract of Wenyang Xiaozheng Decoction, an adsorbent is added, and the adsorbent and the crude extract of Wenyang Xiaozheng Decoction are vortexed, centrifuged, and the supernatant is removed; S1-2: Then add hydroxypropyl-β-cyclodextrin solution to the adsorbent and vortex to elute; S1-3: The eluted solution is filtered through an organic filter head.

4. The method for simultaneous quantitative analysis of multiple components in Wenyang Xiaozheng Decoction by using dispersed solid phase extraction combined with MCDS-sweeping-MEKC according to claim 1, characterized in that: The adsorbent is arbitrarily selected from cross-linked polyvinylpyrrolidone (PVPP), colloidal silicon dioxide, florisil, silica gel, C 18 Or any one of MCM-48. Among them, the most preferred adsorbent is cross-linked polyvinylpyrrolidone (PVPP).

5. The method for simultaneous quantitative analysis of multiple components in Wenyang Xiaozheng Decoction by using dispersed solid phase extraction combined with MCDS-sweeping-MEKC according to claim 1, characterized in that: The mixing ratio of the adsorbent and the Wenyang Xiaozheng Decoction crude extract is 1:

1.

6. The method for simultaneous quantitative analysis of multiple components in Wenyang Xiaozheng Decoction by using dispersed solid phase extraction combined with MCDS-sweeping-MEKC according to claim 1, characterized in that: The adsorbent and the Wenyang Xiaozheng Decoction crude extract are vortexed for 1-2.5 min, preferably 1.5 min.

7. The method for simultaneous quantitative analysis of multiple components in Wenyang Xiaozheng Decoction by using dispersed solid phase extraction combined with MCDS-sweeping-MEKC according to claim 1, characterized in that: The vortex elution time is 1-3 min, preferably 2 min.

8. The method for simultaneous quantitative analysis of multiple components in Wenyang Xiaozheng Decoction by using dispersed solid phase extraction combined with MCDS-sweeping-MEKC according to claim 1, characterized in that: The amount of hydroxypropyl-β-cyclodextrin added is 1000 μL.

9. The method for simultaneous quantitative analysis of multiple components in Wenyang Xiaozheng Decoction by using dispersed solid phase extraction combined with MCDS-sweeping-MEKC according to claim 1, characterized in that: In step S2, the capillary electrophoresis instrument may select one or more of the following electrophoresis conditions: (1) The background buffer solution is a mixture of 55 mM sodium tetraborate solution, 50-100 mM sodium dodecyl sulfate solution (SDS), and 0-30% (v / v) methanol; (2) The cyclodextrin is α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin, wherein the β-cyclodextrin is hydroxypropyl-β-cyclodextrin (HP- β -CD) or hydroxyethyl-β-cyclodextrin (HE- β -CD); (3) The cyclodextrin concentration is below 150 mM; (3) The injection condition is to inject the sample solution at a pressure of 50 mBar for 100 s; (4) The separation voltage is +25KV and the separation temperature is 25℃; Further preferably, the capillary electrophoresis analysis conditions are: 55 mmol sodium tetraborate solution, 75 mmol sodium dodecyl sulfate solution (SDS) and 25% (v / v) methanol as background buffer solution, 125 mmol hydroxypropyl-β-cyclodextrin (HP- β -CD) was used as the sample matrix, and the sample solution was injected at a pressure of 50 mbar for 100 s, with a separation voltage of +25 KV and a temperature of 25 °C.

10. The method for simultaneous quantitative analysis of multiple components in Wenyang Xiaozheng Decoction by using dispersed solid phase extraction combined with MCDS-sweeping-MEKC according to claim 1, characterized in that: The specific quantitative detection method of phenylethanoid glycosides, flavonoids, saponins and other compounds is as follows: Prepare a mixed standard solution of dangshen glycoside, echinacoside, verbascoside, and verbascoside isoflavones in a concentration range of 1-100 μg / mL, detect according to the electrophoresis analysis conditions of step S2, perform parallel determination three times, draw a capillary electrophoresis spectrum of the mixed standard solution, and draw standard curves of dangshen glycoside, echinacoside, verbascoside, and verbascoside respectively with the peak area of ​​each standard in the obtained spectrum as the ordinate and the concentration of the standard in the mixed standard solution as the abscissa, thereby completing the construction of the standard curve; The peak areas of dangshenyl glycoside, echinacoside, verbascoside and verbascoside isoflavone in the capillary electrophoresis spectrum of the sample solution were substituted into the standard curve, and then the contents of dangshenyl glycoside, echinacoside, verbascoside and verbascoside isoflavone in the sample were calculated.

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