Method for capillary electrophoresis quantitative analysis of active ingredients of wubishen pill

Abstract

The application discloses a capillary electrophoresis quantitative analysis method of active ingredients of Wuobi Shanyao pills. The method comprises the following steps: pretreating and purifying the powder of the test sample Wuobi Shanyao pills by MSPD-SPE, and quantitatively determining the active ingredients of the test sample, i.e., loganin, geniposidic acid, trilobatin and verbascoside by CE-UV. The MSPD with high extraction efficiency, the SPE with strong purification capacity and the CE with less consumption of organic solvents are combined for the first time, and are used for green, efficient and accurate content determination of the four components (loganin, geniposidic acid, trilobatin and verbascoside) in Wuobi Shanyao pills. In the sample preparation process and in the CE separation and determination, the consumption of organic solvents is far less than the amount consumed in the prior art.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical analysis, and relates to a capillary electrophoresis quantitative analysis method for active ingredients of Wubishiwan pill. BACKGROUND

[0002] Wubishiwan pill is a kind of pill, which is brown water pills, with light fragrance, sweet and slightly bitter taste. Wubishiwan pill has the effects of invigorating the spleen and kidney, and is mainly used for treating kidney qi deficiency, dizziness, tinnitus, waist soreness, cold limbs, frequent urination, incontinence, or leucorrhea, pale tongue, and soft pulse. It can be used for tonifying deficiency, invigorating the body, eliminating heat and soothing the nerves, and digesting food and breaking accumulation. The prescription of Wubishiwan pill is: Shanyao 300g, Shudihuang 100g, Duzhong (fry with ginger juice) 300g, Ruzhizong 400g, Shanzhuyu (steamed) 100g, Fuling 100g, Tusizi 300g, Bazhitian 100g, Zexie 100g, Niuxi 100g, Wuyi (steamed) 150g, Chishiri (calcined) 100g. Among them, the active ingredient in the prescription of Shanzhuyu is Loganin, which has the effects of promoting macrophage phagocytosis, delaying aging, and good anticancer and radiation protection. The active ingredient in the prescription of Duzhong is Geniposidic Acid, which has the effects of preventing the decrease of sexual function, preventing cancer and tumor caused by X-rays, antioxidant and anti-aging, etc. As the active ingredients in the prescription of Ruzhizong, Pinosylvin and Verbascoside have the effects of antioxidant, nerve protection, improving learning and memory, anti-inflammatory, anti-tumor, liver protection, and immune regulation, etc. The 2020 edition of Chinese Pharmacopoeia only stipulates that the total content of Pinosylvin and Verbascoside should not be less than 2.5mg / g, and does not clearly stipulate the content limit of single component. Therefore, in order to better control the quality of Wubishiwan pill, a method for simultaneously determining the content of more active ingredients in Wubishiwan pill needs to be established. However, the original quality standard of the preparation is collected in the Ninth Part of the Standard of Traditional Chinese Medicine, which only has physical and chemical identification, and no content detection method. Wang Xinyu et al. disclosed a method for simultaneously determining the content of three active ingredients in Wubishiwan pill by reverse phase high performance liquid chromatography, and established a method for determining the content of adenosine, loganin and schisandrin in Wubishiwan pill by reverse phase high performance liquid chromatography. However, a large amount of organic solvent is used in sample preparation and separation and determination by HPLC.

[0003] In modern analytical separation technology, capillary electrophoresis (CE) has the characteristics of convenient operation, high efficiency, fast analysis speed, and low solvent consumption for measuring analytes, which is a replacement measurement method to meet the requirements of modern green chemistry. Therefore, it has become an effective tool for analyzing complex matrices such as biological samples and traditional Chinese medicinal materials. However, due to the small sample volume injected into capillary electrophoresis and the small detection light path of the ultraviolet detector, the detection sensitivity is low, which limits its wide application. At present, offline and / or online enrichment methods are used to improve the sensitivity of CE without changing the instrument for capillary electrophoresis. The offline enrichment method is also called sample pretreatment method. At present, there are many extraction methods that can be coupled with CE. The most commonly used solid sample extraction techniques include microwave-assisted extraction, ultrasonic-assisted extraction, supercritical fluid extraction and reflux extraction method, etc. However, some of these methods often require a large amount of organic solvent and expensive equipment, and sometimes it is difficult to achieve satisfactory extraction efficiency.

[0004] Matrix solid phase dispersion technology (MSPD) was first proposed by Barker et al. in 1989. This technology has the advantages of high extraction efficiency, low solvent and sample consumption, and does not require expensive instruments, and has been widely used in the fields of traditional Chinese medicine, environment and food, etc. However, for extremely complex traditional Chinese medicine samples, MSPD is not sufficient to purify them. Usually, additional purification steps are needed to obtain a chromatogram with almost no interference, and to accurately quantify the target analytes.

[0005] SPE (solid phase extraction) is a commonly used enrichment and purification technique, which is usually used in combination with MSPD for purifying extracts for subsequent instrumental analysis. At present, online MSPD-SPE technology and MSPD-SPE co-column containing double adsorbents have been successfully applied to complex matrices such as fish, seaweed, sludge samples, honey and coffee. Although the MSPD chromatographic column containing two different types of adsorbents shows high flexibility and selectivity, the MSPD extract containing high concentration of organic solvent is often directly penetrated from the column without being retained by the adsorbent when loaded onto the SPE column, resulting in the loss of target analytes. Therefore, when MSPD is used in combination with SPE, it is important to select the appropriate method according to the physical and chemical properties of the analytes to reduce unnecessary loss. SUMMARY

[0006] The purpose of the present application is to provide a capillary electrophoresis quantitative analysis method for the active ingredients of Wuobishanyao pills.

[0007] The purpose of the present application can be achieved by the following technical solutions:

[0008] The application discloses a capillary electrophoresis quantitative analysis method for active ingredients of Wuobi Shanyao pills, and comprises the following steps: pretreating and quantitatively determining active ingredients of the Wuobi Shanyao pills, such as lognansyringin, geniposidic acid, trilobatin and verbascoside, by means of MSPD-SPE and CE-UV; wherein, the molecular sieve SBA-3, the molecular sieve KIT-6 or the molecular sieve MCM-48 is used as the adsorbent, and the low alcohol, the low alcohol solution, the ethyl acetate or the acetonitrile is used as the eluent 1 when the sample is extracted by the MSPD method; the HLB is used as the filler, and the low alcohol, the low alcohol solution, the ethyl acetate or the acetonitrile is used as the eluent 2 when the extraction liquid of the MSPD is purified by the SPE.

[0009] As a preferred embodiment of the application, the method for extracting the sample by the MSPD method is as follows: the adsorbent and the Wuobi Shanyao pill powder are respectively precisely weighed, added into a marbled mortar, mixed uniformly, ground, and then the mixture is transferred into a small column with a sieve plate at both ends, then the eluent 1 is added, the mixture is eluted by applying pressure, the eluent 1 flowing out is connected with a centrifuge tube, and then dried in a solvent evaporation instrument, finally, the sample solution is prepared by redissolving with 1-10% (v / v) low alcohol for subsequent use of the SPE.

[0010] As a further preferred embodiment of the application, the mass ratio of the adsorbent to the Wuobi Shanyao pill powder in the MSPD method is 1:0.5-4, preferably 1:1-2, and further preferably 1:1; the grinding time is 90-180s, preferably 120-150s, and further preferably 150s.

[0011] As a further preferred embodiment of the application, the eluent 1 used in the MSPD is ethanol, ethyl acetate, acetonitrile, 50% (v / v) methanol solution or methanol, and preferably 50% (v / v) methanol solution; the mass-volume ratio of the Wuobi Shanyao pill powder to the eluent 1 is 1mg:8-25μL, preferably 1mg:15-20μL, and further preferably 1mg:16.7μL.

[0012] As a preferred embodiment of the application, the purification step of the extraction liquid of the MSPD by the SPE is as follows:

[0013] (1) activating the SPE column loaded with the HLB filler;

[0014] (2) sample loading: transferring the sample solution prepared by the MSPD method into the activated SPE column, pushing the liquid out by using a pressure device, and controlling the flow rate to be 0.5-1.5mL / min, preferably 1mL / min;

[0015] (3) washing: washing the SPE column after sample loading with pure water to remove impurities;

[0016] (4) elution: eluting the SPE column loaded with the sample by using the eluent 2;

[0017] (5) Finally, the eluted solution was evaporated to dryness, reconstituted with 40-60% (v / v) lower alcohol, and the reconstituted solution was filtered through a 0.22 μm microfiltration membrane and used for subsequent CE analysis.

[0018] As a further preferred embodiment of the present application, 3-5 mL of methanol and 3-5 mL of pure water were used respectively to activate the SPE column; the sample solution prepared by the MSPD method was loaded at a volume of 500 μL, the eluent 2 was selected from methanol, and the volume ratio of the sample solution to the eluent 2 was 1:1-2.5, preferably 1:1.8-2.2, and further preferably 1:2; and the lower alcohol was selected from methanol.

[0019] As a preferred embodiment of the present application, the quantitative analysis of the target analyte in the solution purified by MSPD-SPE was determined by CE-UV, which included:

[0020] (1) Preconditioning of the uncoated fused quartz capillary column: 0.1 M sodium hydroxide, pure water, and background buffer solution BGS were used respectively to flush for 3-8 min;

[0021] (2) Injection procedure: injection at a pressure of 50 mbar for 5 s;

[0022] (3) Running conditions: running buffer: same as the background buffer solution BGS; detection wavelength: 245 nm; separation voltage: +20 KV; temperature: 25-28 °C.

[0023] As a further preferred embodiment of the present application, the background buffer solution BGS was a solution containing 60-75 mM of sodium tetraborate and 15-30% (v / v) of methanol.

[0024] As a further preferred embodiment of the present application, the background buffer solution BGS was a solution containing 65 mM of sodium tetraborate and 20% (v / v) of methanol.

[0025] As a further preferred embodiment of the present application, the capillary electrophoresis quantitative analysis method of the active ingredients of the Wuwei Shanyaji pill comprises the following steps:

[0026] S1: MSPD method for extracting a sample:

[0027] (1) 60 mg of adsorbent MCM-48 and 60 mg of Wuwei Shanyaji pill powder were precisely weighed respectively and added to a marver, mixed uniformly, and ground for 150 s;

[0028] (2) After the grinding was completed, the mixture was transferred to a small column with a sieve plate plugged at both ends, 1 mL of 50% (v / v) methanol eluent 1 was then added, the mixture was eluted by applying pressure, and the eluent flowing out was collected with a centrifuge tube;

[0029] (3) The eluate from the centrifuge tube in the previous step was placed in a solvent evaporation instrument to evaporate the solvent, and finally 500 μL of 5% (v / v) methanol was used to reconstitute the sample solution for subsequent SPE use;

[0030] S2: Purification of the MSPD extraction solution by SPE to remove impurities:

[0031] (1) Activate the SPE column loaded with HLB filler: activate the column with 3 mL of methanol and 3 mL of pure water, respectively;

[0032] (2) Sample loading: transfer the 500 μL of sample solution prepared by the MSPD method to the activated SPE column using a pipette, and use a pressure device to push the liquid out, controlling the flow rate at 1 mL / min;

[0033] (3) Elution: elute the SPE column after sample loading with 1 mL of pure water to remove impurities;

[0034] (4) Elution: elute the SPE column loaded with the sample with 1 mL of methanol;

[0035] (5) Finally, evaporate the eluted solution, reconstitute it with 200 μL of 50% (v / v) methanol, and filter the reconstituted solution through a 0.22 μm microporous filter for subsequent CE analysis;

[0036] S3: CE:-UV analysis of the target analyte in the purified solution:

[0037] (1) Precondition the capillary column: flush with 0.1 M sodium hydroxide, pure water, and background buffer solution BGS for 5 min each; the BGS is a 65 mM sodium tetraborate and 20% (v / v) methanol aqueous solution;

[0038] (2) Sample injection procedure: inject at a pressure of 50 mbar for 5 s;

[0039] (3) Running conditions: running buffer: 65 mM sodium tetraborate and 20% (v / v) methanol aqueous solution; detection wavelength: 245 nm; separation voltage: +20 KV; temperature: 25°C.

[0040] The uncoated fused silica capillary column can have an inner diameter of 75 μm, 100 μm; a length of 40-60 cm, preferably a total length of 50 cm, an effective length of 41.5 cm, and an inner diameter of 50 μm.

[0041] The logp of the four analytes (logania, geniposidic acid, echinacoside and verbascoside) is less than 3 (i.e. relatively large polarity) by querying the website https: / / scifinder.cas.org. Therefore, according to the size of the polarity, the type of the SPE column is selected according to the flow chart shown in the figure. Figure 1 Since the analytes are not ionized in the MSPD extract (50% (v / v) methanol), a hydrophilic-lipophilic filler, HLB, is selected for the SPE purification process. The filler is composed of a hydrophobic divinylbenzene structure and a hydrophilic N-vinylpyrrolidone structure, which has strong adsorption to low molecular weight and polar compounds. When the Rouyi Shanyao pill is made into a water pill, refined honey is added. The main component of the refined honey is sugar compounds. In order to reduce the interference of the matrix during analysis and affect the quantitative analysis of the target analyte, the sugar compounds are removed by eluting the SPE column with pure water after loading.

[0042] Advantages:

[0043] The present application combines MSPD with high extraction efficiency, SPE with strong purification ability and CE with less consumption of organic solvents for the first time to determine the content of the four components (logania, geniposidic acid, echinacoside and verbascoside) in Rouyi Shanyao pill in a green, efficient and accurate manner. Only 60 mg of Rouyi Shanyao pill sample and 3.625 mL of organic solvent are used in the sample preparation process (MSPD-SPE process), and the amount of organic solvent used in the CE separation and determination is 400 μL, which is much less than the amount of organic solvent consumed in the HPLC separation and determination.

[0044] The green potential of the developed MSPD-SPE-CE method is evaluated using the Analytical GREEnness Metric Approach. Compared with the ultrasonic extraction method recorded in the 2020 edition of the Chinese Pharmacopoeia, the method has the advantages of less sample and organic solvent consumption.

[0045] The present application method can simultaneously determine the four components (logania, geniposidic acid, echinacoside and verbascoside) in Rouyi Shanyao pill, with high accuracy, precision and good reproducibility, and is green and environmentally friendly. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 Selection of SPE column type according to the physicochemical properties of the analytes

[0047] Figure 2Adsorbent type optimization of MSPD (silica, Florisil, alumina, C18, SBA-3, KIT-6, MCM-48); conditions: 65 mM borate + 20% (v / v) methanol; injection time: 5 s; wavelength: 245 nm; sample matrix: 50% (v / v) methanol; peaks: 1 - loganin, 2 - geniposidic acid, 3 - echinacoside, 4 - verbascoside.

[0048] Figure 3 Sample to adsorbent mass ratio optimization (2:1 ; 1 :1 ; 1 :2; 1 :3; 1 :4); conditions: 65 mM borate + 20% (v / v) methanol; injection time: 5 s; wavelength: 245 nm; sample matrix: 50% (v / v) methanol; peaks: 1 - loganin, 2 - geniposidic acid, 3 - echinacoside, 4 - verbascoside Figure 4 Grinding time optimization (90 s, 120 s, 150 s, 180 s, 210 s); conditions: 65 mM borate + 20% (v / v) methanol; injection time: 5 s; wavelength: 245 nm; sample matrix: 50% (v / v) methanol; peaks: 1 - loganin, 2 - geniposidic acid, 3 - echinacoside, 4 - verbascoside

[0049] Figure 5 Eluent type optimization (acetonitrile, ethyl acetate, absolute ethanol, methanol and 50% (v / v) methanol); conditions: 65 mM borate + 20% (v / v) methanol; injection time: 5 s; wavelength: 245 nm; sample matrix: 50% (v / v) methanol; peaks: 1 - loganin, 2 - geniposidic acid, 3 - echinacoside, 4 - verbascoside

[0050] Figure 6 Eluent volume optimization (250 μL, 500 μL, 1000 μL, 1500 μL); conditions: 65 mM borate + 20% (v / v) methanol; injection time: 5 s; wavelength: 245 nm; sample matrix: 50% (v / v) methanol; peaks: 1 - loganin, 2 - geniposidic acid, 3 - echinacoside, 4 - verbascoside

[0051] Figure 7 Eluent volume optimization (500 μL, 1000 μL, 1500 μL, 2000 μL, 2500 μL); conditions: 65 mM borate + 20% (v / v) methanol; injection time: 5 s; wavelength: 245 nm; sample matrix: 50% (v / v) methanol; peaks: 1 - loganin, 2 - geniposidic acid, 3 - echinacoside, 4 - verbascoside

[0052] Figure 8Optimization of BGS concentration: Electropherograms of four analytes at different concentrations (20, 35, 50, 65, 80 mM) (A). Effect of sodium borate concentration (B) on the separation of analytes. BGS: 65 mM sodium borate and 20% (v / v) methanol. Injection time: 5 s; separation conditions: +20 kV, 25 °C. Peaks: 1 - loganin, 2 - geniposidic acid, 3 - echinacoside, 4 - verbascoside.

[0053] Figure 9 Optimization of methanol content in BGS: Electropherograms of four analytes at different amounts of methanol addition (5%, 10%, 15%, 20%, 25%) (A). Effect of different amounts of methanol addition in BGS (B) on the separation of analytes. BGS: 65 mM sodium borate and 20% (v / v) methanol. Injection time: 5 s; separation conditions: +20 kV, 25 °C. Peaks: 1 - loganin, 2 - geniposidic acid, 3 - echinacoside, 4 - verbascoside.

[0054] Figure 10 Comparison of extraction efficiency of MSPD and UAE methods (A); comparison of electropherograms obtained by UAE-CE, MSPD-CE and MSPD-SPE-CE methods (B).

[0055] Figure 11 AGREE pictograms of three extraction methods; UAE (A), MSPD (B), MSPD-SPE (C) Figure 12 Application of MSPD-SPE-CE-UV in real samples. Conditions: 65 mM borax + 20% (v / v) methanol; injection time: 5 s; wavelength: 245 nm; sample matrix: 50% (v / v) methanol; peaks: 1 - loganin, 2 - geniposidic acid, 3 - echinacoside, 4 - verbascoside DETAILED DESCRIPTION

[0056] The method for quantitative analysis of active ingredients in Wubishenyaji pills by capillary electrophoresis comprises the following steps:

[0057] S1: extract the sample by the MSPD method:

[0058] (1) precisely weigh 60 mg of adsorbent MCM-48 and 60 mg of Wubishenyaji pill powder, respectively, and add them to a marver mortar, mix them uniformly, and grind for 150 s;

[0059] (2) after the grinding is completed, transfer the mixture to a small column with a sieve plate plugged at both ends, then add 1 mL of 50% (v / v) methanol eluent 1, elute the mixture by applying pressure, and collect the eluent flowing out with a centrifuge tube;

[0060] (3) The eluate from the centrifuge tube in the last step was placed in a solvent evaporation instrument to evaporate the solvent, and finally 500 μL of 5% (v / v) methanol was used to reconstitute the sample solution for subsequent SPE use;

[0061] S2: Purification of the extract from MSPD by SPE to remove impurities:

[0062] (1) The SPE column loaded with HLB filler was activated: 3 mL of methanol and 3 mL of pure water were used to activate the column, respectively;

[0063] (2) Sample loading: 500 μL of the sample solution prepared by the MSPD method was transferred to the activated SPE column by a pipette, and the liquid was pushed out using a pressure device, with a flow rate of 1 mL / min;

[0064] (3) Elution: 1 mL of pure water was used to elute the SPE column after sample loading to remove impurities;

[0065] (4) Elution: 1 mL of methanol was used to elute the SPE column loaded with the sample;

[0066] (5) Finally, the eluted solution was evaporated, 200 μL of 50% (v / v) methanol was used to reconstitute it, and the reconstituted solution was filtered through a 0.22 μm microporous filter for subsequent CE analysis;

[0067] S3: Analysis of the target analyte in the purified solution by CE-UV:

[0068] (1) Preconditioning of the capillary column: 0.1 M sodium hydroxide, pure water, and background buffer solution BGS were used to flush the column for 5 min, respectively; the BGS is a water solution containing 65 mM sodium tetraborate and 20% (v / v) methanol;

[0069] (2) Sample injection procedure: injection for 5 s at a pressure of 50 mbar;

[0070] (3) Running conditions: running buffer: water solution containing 65 mM sodium tetraborate and 20% (v / v) methanol; detection wavelength: 245 nm; separation voltage: +20 KV; temperature: 25°C.

[0071] Example 1

[0072] (I) Optimization of sample pretreatment related parameters

[0073] 1) Optimization of adsorbent type

[0074] In the process of dispersion, the analyte can be slightly combined with the adsorbent, which is conducive to the more efficient and convenient extraction of the target by the eluent. However, different types of adsorbents have different effects on the adsorption and desorption of target components due to their different properties. Therefore, the effects of bonded silica adsorbent C18, normal phase adsorbents (silica gel, Florisil and alumina) and three types of molecular sieves (SBA-3, KIT-6 and MCM-48) on the MSPD extraction effect were investigated. In the experiment, the amount of sample and adsorbent was fixed at 60 mg, and other operations were consistent with the steps of the above-mentioned MSPD-SPE method.

[0075] The results are shown in Figure 2 The spectrum and peak area chart show that when traditional adsorbents alumina and Florisil are used as adsorbents, no peak of geniposidic acid is found, which may be because the adsorption of traditional adsorbents alumina and Florisil on geniposidic acid is very strong, and it cannot be eluted by the eluent. The adsorption effect of molecular sieve MCM-48 on the four analytes is greater than that of the four traditional adsorbents and the other two molecular sieves SBA-3 and KIT-6. The main reason may be that SBA-3 has small micropores in the mesoporous pore wall, and the structure of the four analytes is not a straight-chain alkane, so it cannot enter the micropores well. Although molecular sieve KIT-6 has a unique three-dimensional cubic pore channel, its pore volume (0.5 cm 3 / g) is smaller than that of MCM-48 (1.1 cm 3 / g), so its loading capacity for the four analytes is poor, and the adsorption capacity is lower than that of MCM-48. For molecular sieve MCM-48, it not only has two independent mirror-symmetric three-dimensional spiral pore channel network structures, but also has a large pore volume, so the analyte can be well loaded in the pore size, making the adsorption effect better than other adsorbents. Therefore, molecular sieve MCM-48 is selected for further study.

[0076] 2) Optimization of the mass ratio of sample to adsorbent

[0077] In the whole MSPD process, the mass ratio of sample to adsorbent directly determines the degree of contact between target compounds and adsorbent, and is a key factor affecting the efficiency of sample pre-concentration. Therefore, while keeping the sample amount at 60 mg, the amount of MCM-48 adsorbent was changed to 1:1, 1:2, 1:3, 1:4 and 2:1, respectively, and the extraction efficiency of target components was investigated.

[0078] The results are shown in Figure 3As shown, it was found that the extraction efficiency of target analytes could be improved to some extent by increasing the amount of adsorbent. When the mass ratio of sample to adsorbent was 1:1, the peak areas of two target analytes reached the maximum value, and the extraction efficiency was optimal. When the amount of adsorbent was further increased, the interaction between sample and adsorbent was too strong, and the analytes were difficult to be eluted from the pores of the molecular sieve, resulting in a decrease in extraction efficiency. Therefore, the mass ratio of sample to adsorbent was selected as 1:1 in the subsequent experiments.

[0079] 3) Optimization of grinding time

[0080] Grinding is the most important step in the entire MSPD process. This step destroys the structure of biological tissues and the sample itself through friction and shear force, greatly increasing the contact area between sample components and adsorbent, making them completely dispersed on the surface of the adsorbent, thereby enhancing the extraction efficiency. In the experiment, the amount of sample and adsorbent MCM-48 was fixed at 60 mg, and the effect of different grinding times (90 s, 120 s, 150 s, 180 s, 210 s) on extraction efficiency was optimized.

[0081] The results are shown in Figure 4 With the extension of grinding time, the peak areas of four target analytes showed a trend of first increasing and then decreasing. When the grinding time was 150 s, the peak areas of four analytes reached the highest value, and the adsorption equilibrium between sample and adsorbent was achieved. If the grinding time was further extended, the extraction efficiency would be reduced due to the over-strong adsorption. Therefore, 150 s was selected as the grinding time for the subsequent experiments.

[0082] 4) Optimization of eluent type

[0083] MSPD is based on the relative polarity between eluent, target components and adsorbent to achieve extraction and separation. Therefore, selecting the appropriate eluent is helpful for the full extraction of target components in the sample. In this experiment, the effects of acetonitrile, ethyl acetate, anhydrous ethanol, methanol and 50% (v / v) methanol on the extraction efficiency of two alkaloid components were investigated.

[0084] From the spectrum and peak area results Figure 5As can be seen in Figure 1, acetonitrile and ethyl acetate are not suitable for the elution of the four analytes. Since the solubility of the four analytes in these two solvents is very small, the analytes cannot be eluted from the molecular sieve MCM-48 (the interaction between the eluent acetonitrile or ethyl acetate and the analytes is smaller than the interaction between MCM-48 and the analytes). The solubility of the four analytes in anhydrous ethanol is lower than that in methanol or aqueous methanol, so the elution effect is weaker. The solubility of the four analytes in aqueous methanol is stronger than that in pure methanol, so the extraction effect is stronger than that of pure methanol, and the eluent is consistent with the extraction solvent used in the pharmacopoeia extraction method. Therefore, 50% (v / v) methanol is finally selected as the eluent for subsequent experiments.

[0085] 5) Eluent volume optimization

[0086] On the basis of a fixed sample-to-sorbent mass ratio of 1:1, a grinding time of 150 s, and 50% (v / v) methanol as the eluent, the effect of eluent volume (250 μL, 500 μL, 1000 μL, 1500 μL) on the elution of the analytes in the MSPD process was investigated. As can be seen in Figure 2, Figure 6 , but as the eluent volume increases, the peak area of the target components also gradually increases. When the eluent volume reaches 1000 μL, the components are fully eluted, and saturation is achieved, so that even if the volume of the eluent is further increased, the elution effect of the analytes will not increase. Therefore, the final volume is determined to be 1000 μL.

[0087] 6) Eluent volume optimization in the SPE process

[0088] After the parameters of the MSPD process are optimized (i.e., the sample-to-sorbent mass is 60 mg, the grinding time is 150 s, and 1000 μL of 50% (v / v) methanol is used to elute the homogenized mixture), in order to use a smaller amount of methanol to achieve the best elution effect, the optimization of the eluent volume in the SPE process (500 μL, 1000 μL, 1500 μL, 2000 μL, and 2500 μL) is investigated in this experiment. The results are shown in Figure 3, Figure 7 , as the eluent volume continues to increase, the peak area of the analytes also increases. When the eluent volume reaches 1000 μL, the elution effect is optimal, and the analytes are fully eluted. Subsequent increases in the volume of the eluent do not have an effect on the elution effect, so 1000 μL is finally selected as the volume of the eluent in the SPE process.

[0089] (II) Optimization of Related Parameters for Determination by Capillary Electrophoresis

[0090] 1) Optimization of borax concentration in BGS

[0091] Borate salts are reported to complex with polyol compounds such as glycosides, phenolic acids and flavonoids, causing a change in the charge ratio of the analytes, and are therefore widely used in BGS for capillary zone electrophoresis / micellar electrokinetic capillary chromatography to achieve selective separation. In the present invention, since the four analytes (loganin, geniposidic acid, echinacoside and verbascoside) all belong to glycoside compounds containing 1,2 or 1,3-diol, borax was chosen as the BGS for subsequent research. Changes in the concentration of borate salts result in differences in complexing strength, thus causing specific changes in the charge-to-mass ratio of the analytes. A suitable borate salt concentration can also achieve satisfactory separation efficiency and migration time. Therefore, the present study tested the effect of borate salt concentration in the range of 20-80 mM on the separation between adjacent peaks. As shown in Figures 8A and 8B, increasing the concentration of borax can improve separation efficiency. When the borax concentration is increased to 65 mM, the separation of the four analytes reaches an optimal state (separation efficiency between adjacent peaks >1.5). However, further addition of salt leads to an increase in the Joule effect, resulting in poor peak shape. Therefore, a 65 mM borax solution was chosen for subsequent research. Figure 8

[0092] 2) Optimization of the methanol content in the BGS

[0093] The addition of methanol not only increases the solubility of the analytes, but also improves the separation efficiency. Therefore, the effect of the methanol content in the BGS in the range of 5% to 25% (v / v) was studied (Figures 9A and 9B). It can be seen that as the concentration of methanol in the BGS increases, the separation efficiency of the four analytes gradually improves. However, when the amount of methanol is 25% (v / v), the migration time of the analytes is also gradually extended to 80 min (not shown in the figure), and as the EOF value and the conductivity of the system decrease, the peak value of the analytes becomes wide. Considering the resolution and analysis time, 20% (v / v) was chosen as the amount of methanol added to the BGS. Figure 9

[0094] Example 2

[0095] 1) Ultrasonic extraction method recorded in the 2020 edition of the Chinese Pharmacopoeia (modified part of the content, consistent with the material ratio of MSPD extraction method, i.e. the ratio of material to liquid is 0.3 g:5 mL — to investigate the extraction efficiency of MSPD and ultrasonic extraction method)

[0096] First, 10 g of Dioscorea opposita Thunb. pills were ground into powder. Then, 0.3 g of the sample powder was accurately weighed and then transferred to a 50 mL conical flask. After that, the sample was extracted with 5 mL of a methanol / water mixture (50:50, v / v) in an ultrasonic bath for 30 minutes, and then transferred to a centrifuge tube and centrifuged at a speed of 13,000 rpm for 5 minutes. Finally, the supernatant was filtered through a 0.22 μm filter membrane for subsequent CE analysis. ​​

[0097] 2) MSPD method

[0098] Precisely weigh 60 mg of adsorbent MCM-48 and 60 mg of Wuyi Shanyao pill powder respectively, add them into the agate mortar, mix them well, and grind for 150 seconds. After grinding, transfer the mixture to a small column with a sieve plate at both ends. Then add 1 mL of 50% (v / v) methanol, elute the mixture by applying pressure, and collect the eluent with a 2 mL centrifuge tube. Place the centrifuge tube in a solvent evaporation instrument to evaporate the solvent. Finally, reconstitute the eluted solution with 200 μL of 50% (v / v) methanol to prepare a sample solution for subsequent CE analysis.

[0099] 3) MSPD-SPE method:

[0100] a) MSPD process

[0101] Precisely weigh 60 mg of adsorbent and 60 mg of Wuyi Shanyao pill powder respectively, add them into the agate mortar, mix them well, and grind for 150 seconds. After grinding, transfer the mixture to a small column with a sieve plate at both ends. Then add 1 mL of 50% (v / v) methanol, elute the mixture by applying pressure, and collect the eluent with a 2 mL centrifuge tube. Place the centrifuge tube in a solvent evaporation instrument to evaporate the solvent. Finally, reconstitute the eluted solution with 500 μL of 5% (v / v) methanol to prepare a sample solution for subsequent SPE use.

[0102] b) SPE process

[0103] • Activate the SPE column loaded with HLB filler: activate the column with 3 mL of methanol and 3 mL of pure water respectively.

[0104] • Sample loading: transfer the above-mentioned 500 μL of sample solution prepared by the MSPD method to the activated SPE column using a pipette, and then use a pressure device to push the liquid out.

[0105] • Elution: elute the column after sample loading with 1 mL of pure water to remove impurities.

[0106] • Elution: elute the SPE column loaded with the sample with 1 mL of methanol.

[0107] • Finally, evaporate the eluted solution, reconstitute it with 200 μL of 50% (v / v) methanol, filter the reconstituted solution through a 0.22 μm microporous filter, and then use it for subsequent CE analysis.

[0108] 4) Preparation of spiked samples (for calculating the recovery rate of spiking)

[0109] The recovery rate was calculated by three spiked levels, i.e. the amount of standard added was 80%, 100% and 120% of the content of the four target analytes (sinoacutine, geniposidic acid, echinacoside and verbascoside) in the Wuwei Shanyang pills. The four standards were added to the samples according to the following standards, and the spiked samples were treated by the pretreatment method of MSPD-SPE. Finally, the treated samples were determined by CE.

[0110] Low spiked level (the amount added was 80% of the content of the analytes in the original sample): the amount of sinoacutine standard added was 0.28 mg / g, the amount of geniposidic acid added was 0.75 mg / g, the amount of echinacoside added was 6.83 mg / g, and the amount of verbascoside added was 2.00 mg / g.

[0111] Medium spiked level (the amount added was 100% of the content of the analytes in the original sample): the amount of sinoacutine standard added was 0.35 mg / g, the amount of geniposidic acid added was 0.93 mg / g, the amount of echinacoside added was 8.33 mg / g, and the amount of verbascoside added was 2.83 mg / g.

[0112] High spiked level (the amount added was 120% of the content of the analytes in the original sample): the amount of sinoacutine standard added was 0.42 mg / g, the amount of geniposidic acid added was 1.12 mg / g, the amount of echinacoside added was 10.00 mg / g, and the amount of verbascoside added was 3.33 mg / g.

[0113] 5) Preparation of mixed standard solution

[0114] An appropriate amount of sinoacutine, geniposidic acid, echinacoside and verbascoside was precisely weighed and added to an appropriate amount of 50% (v / v) methanol to prepare a 1 mg / mL stock solution. Then 100 μL of each of the four analyte stock solutions was taken and added to 600 μL of 50% (v / v) methanol to prepare 1 mL of a 100 μg / mL mixed standard solution.

[0115] In order to further evaluate the effectiveness of the method, the UAE, MSPD and MSPD-SPE methods were compared. First, the extraction efficiency of the four analytes by UAE and MSPD was investigated when the solid-liquid ratio was 0.3 g:5 mL. Figure 10 A shows that the yield of the four analytes extracted by the MSPD method from the Wuwei Shanyang pills was higher than that by the UAE method. Therefore, MSPD was selected as the extraction method for the subsequent Wuwei Shanyang pills to extract more analytes. However, as shown in B, the extraction efficiency of the four analytes by MSPD-SPE was higher than that by MSPD. Figure 10As shown in Figure B, MSPD was not enough to purify the traditional Chinese medicine due to the high content of honey in the Fuyan-yisheng pill, which led to the existence of matrix interference, affecting the accurate determination of trichosanthin and verbascoside. Therefore, it is necessary to further purify the MSPD extract with SPE to reduce the matrix interference. Considering the risk of sample loss in multi-step offline sample pretreatment, the loss of the four analytes before and after the use of SPE was further studied to eliminate the influence on quantitative determination. The recovery rate was 91.7-104.2% through methodological validation, indicating that the loss during the pretreatment process was small, and the determination result was accurate.

[0116] The current trend of analytical methods mainly focuses on the application of sample preparation methods, so the pretreatment method tends to be green, safe and efficient. In this study, the green potential of the three methods was evaluated by the analytical greenness evaluation method (AGREE). According to the 12 principles of green analytical chemistry, the evaluation results were converted into a unified 0-1 scale, and the final results calculated according to the significance principle were output in the form of pictograms, with the total score and the color in the middle. The derived AGREE results are shown in Figure Figure 11 As can be seen, the scores of the four analytes extracted from the sample by MSPD-SPE method are comparable to those of MSPD, but higher than those of UAE. It is confirmed that the method is simple to operate, consumes less sample and organic reagent, has less environmental pollution, can be applied under environmental conditions, does not require special laboratory equipment / instruments, and has good green potential.

[0117] Methodological validation of Example 3

[0118] In order to verify the effectiveness of the developed MSPD-SPE-CE-UV, the following series of experiments were carried out under the optimal conditions. The analysis results are shown in Table 1. Egenitine was prepared at concentrations of 50 μg / mL, 60 μg / mL, 100 μg / mL, 200 μg / mL and 300 μg / mL in 50% (v / v) methanol; Geniposidic acid was prepared at concentrations of 60 μg / mL, 100 μg / mL, 200 μg / mL, 250 μg / mL and 300 μg / mL; Trichosanthin was prepared at concentrations of 100 μg / mL, 200 μg / mL, 300 μg / mL, 1000 μg / mL and 4000 μg / mL; and Verbascoside was prepared at concentrations of 60 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL and 1000 μg / mL, and determined in triplicate under the optimal conditions. The peak area of the analyte was taken as the ordinate, and the concentration was taken as the abscissa, to obtain the standard curve of the four analytes. As can be seen from the data in the table, the peak areas of the four analytes have good linearity (R 2>0.9983). The LOD (limit of detection) and LOQ (limit of quantitation) of the analytes were calculated based on signal-to-noise ratios of 3 and 10. The LODs for loganin, geniposide, echinacoside, and verbascoside were all 25 μg / mL; except for loganin, whose LOQ was 40 μg / mL, the LOQs for geniposide, echinacoside, and verbascoside were 50 μg / mL. Intra-day precision was evaluated by injecting a 100 μg / mL mixed standard solution six times consecutively within one day, and inter-day precision was evaluated by injecting three times daily for three consecutive days. The calculated peak area RSDs were all less than 2.84%, indicating good reproducibility of the method.

[0119] Table 1 Methodological Investigation

[0120]

[0121] α y: peak area (mAU); x: analyte concentration (μg / mL).

[0122] β is calculated based on the signal-to-noise ratio (S / N) being equal to 3.

[0123] γ is calculated based on a signal-to-noise ratio (S / N) of 10.

[0124] Example 4: Analysis of Actual Samples

[0125] The optimal MSPD-SPE-CE-UV method was used to analyze actual samples of Wubi Yam Pills, verifying the feasibility of using this method for determining the content of four components in this traditional Chinese medicine. Figure 12 As can be seen, the two-step pretreatment method effectively avoids interference from other impurity peaks, making the quantitative analysis of analytes more accurate and effective. The contents of the four analytes in the sample were calculated based on the standard curve, and the data are shown in Table 2. The data in the table show that the total content of echinacoside and verbascoside is 11.35 mg / g, far exceeding the pharmacopoeia requirement (the content of echinacoside and verbascoside in each traditional Chinese medicine preparation should not be less than 2.5 mg).

[0126] To verify the interference of the sample matrix and the accuracy of the developed method, different amounts (spiking amounts of 80%, 100%, and 120% of the content of the four target analytes in the sample) of reference standards were added to the sample to investigate the spiked recovery rate. The spiked recovery rate was calculated as R% = (m spiked -m real ) / m added ×100% (m spiked : Analyte content after spiking; m real : The content of the analyte in the actual sample; m addedThe amount of the added standard sample). The specific data are shown in Table 3. As can be seen from the data in the table, the standard addition recovery rates of the four analytes are in an acceptable range, i.e. between 91.78% and 104.18% (RSD% is lower than 6.07%).

[0127] Table 2 Content of the four analytes in Wobishanyao pills

[0128]

[0129] Table 3 Standard addition recovery rate experiment of the four analytes in Wobishanyao pills

[0130]

[0131]

[0132] (m spiked : The amount of the analyte in the sample after standard addition; m real : The actual content of the analyte in the sample; m added : The standard addition amount)

[0133] Summary

[0134] 1) Advantages:

[0135] Compared with the traditional extraction method (the extraction method of Wobishanyao pills recorded in the 2020 edition of Chinese Pharmacopoeia), the extraction efficiency of the MSPD method adopted in the present application is higher than that of UAE. Before being combined with SPE, it can be found from the spectrum that the four active ingredients are all interfered by other components, thus making the quantitative determination inaccurate. After being combined with SPE, it is found that the interfering components around the four analytes are eliminated, making the quantitative results of the target analytes more accurate.

[0136] Moreover, in terms of sample and organic solvent consumption, only 60 mg of sample and 3.625 mL of organic solvent are used in the pretreatment process of the present application, and only 400 μL of organic solvent is used in the CE online separation determination. Compared with the HPLC method which needs a large amount of organic solvent for separation determination, the present method is more green and environmentally friendly under the premise of efficiently determining the four active ingredients in Wobishanyao pills, and meets the requirements of modern green chemistry.

Claims

1. A capillary electrophoretic quantitative analysis method for the active ingredients of Wubi Yam Pills, characterized in that, This includes pretreatment of the test sample, Wubi Yam Pill powder, by MSPD-SPE and quantitative determination of its active ingredients, loganin, genipin, echinacoside, and verbascoside, by CE-UV. in, When extracting samples using the MSPD method, molecular sieves SBA-3, KIT-6, or MCM-48 are used as adsorbents, and lower alcohols, lower alcohol solutions, ethyl acetate, or acetonitrile are used as eluent 1. When purifying the MSPD extract using SPE, HLB is used as the packing material, and lower alcohols, lower alcohol solutions, ethyl acetate, or acetonitrile are used as eluent 2. The MSPD method for extracting samples is as follows: accurately weigh the adsorbent and the powder of Wubi yam pills, add them to an agate mortar, mix them evenly, and grind them. After grinding, transfer the mixture to a small column with sieve plates at both ends, then add eluent 1, and elute the mixture by applying pressure. Collect the eluent 1 in a centrifuge tube, place it in a solvent evaporator to evaporate it, and finally redissolve it with 1-10% (v / v) lower alcohol to prepare a sample solution for subsequent SPE use. The mass ratio of adsorbent to Wubi yam pill powder is 1:0.5-4, and the grinding time is 90-180s. The purification steps of SPE on the MSPD extract are as follows: (1) Activate the SPE column loaded with HLB packing; (2) Sample loading: Transfer the sample solution prepared by the MSPD method to the activated SPE column, use a pressurizer to push the liquid out, and control the flow rate at 0.5-1.5 mL / min; (3) Rinsing: Rinse the SPE column after loading with pure water to remove impurities; (4) Elution: The SPE column loaded with the sample was eluted using eluent 2; (5) Finally, evaporate the eluted solution to dryness, redissolve it with 40-60% (v / v) lower alcohol, filter the redissolved solution through a 0.22 μm microporous membrane and use it for subsequent CE analysis; Quantitative analysis of target analytes in MSPD-SPE purified solutions using CE-UV includes: (1) Preconditioning of uncoated fused silica capillary columns: rinse with 0.1 M sodium hydroxide, pure water and background buffer solution BGS for 3-8 min each; (2) Injection procedure: Inject the sample for 5 seconds at a pressure of 50 mbar; (3) Operating conditions: Operating buffer: same as background buffer solution BGS; Detection wavelength: 245 nm; Separation voltage: +20 KV; Temperature: 25-28 ℃.

2. The capillary electrophoretic quantitative analysis method for the active ingredients of Wubi Yam Pills according to claim 1, characterized in that, The mass ratio of adsorbent to Wubi yam pill powder is 1:1-2, and the grinding time is 120-150s.

3. The capillary electrophoretic quantitative analysis method for the active ingredients of Wubi Yam Pills according to claim 2, characterized in that, The mass ratio of adsorbent to Wubi yam pill powder is 1:1, and the grinding time is 150s.

4. The capillary electrophoretic quantitative analysis method for the active ingredients of Wubi Yam Pills according to claim 1, characterized in that, The eluent 1 used in MSPD is ethanol, ethyl acetate, acetonitrile, 50% (v / v) methanol solution or pure methanol, and the mass-volume ratio of Wubi Yam Pill powder to eluent 1 is 1 mg: 8-25 µL.

5. The capillary electrophoretic quantitative analysis method for the active ingredients of Wubi Yam Pills according to claim 1, characterized in that, The eluent 1 used in MSPD is a 50% (v / v) methanol solution; the mass-to-volume ratio of Wubi Yam Pill powder to eluent 1 is 1 mg: 15-20 µL.

6. The capillary electrophoretic quantitative analysis method for the active ingredients of Wubi Yam Pills according to claim 1, characterized in that, The mass-to-volume ratio of the powdered yam pills to eluent 1 was 1 mg: 16.7 µL.

7. The capillary electrophoretic quantitative analysis method for the active ingredients of Wubi Yam Pills according to claim 1, characterized in that, In the purification process of MSPD extract by SPE, step (2) involves loading the sample at a flow rate of 1 mL / min.

8. The capillary electrophoretic quantitative analysis method for the active ingredients of Wubi Yam Pills according to claim 1, characterized in that, The SPE column was activated with 3-5 mL of methanol and 3-5 mL of pure water, respectively; the sample solution prepared by the MSPD method was loaded with 500 μL; the eluent 2 was selected from methanol; the volume ratio of sample solution to eluent 2 was 1:1-2.5; and the lower alcohol used for redissolution was methanol.

9. The capillary electrophoretic quantitative analysis method for the active ingredients of Wubi Yam Pills according to claim 1, characterized in that, The volume ratio of sample solution to eluent 2 is 1:1.8-2.

2.

10. The capillary electrophoretic quantitative analysis method for the active ingredients of Wubi Yam Pills according to claim 1, characterized in that, The volume ratio of the sample solution to eluent 2 is 1:

2.

11. The capillary electrophoretic quantitative analysis method for the active ingredients of Wubi Yam Pills according to claim 1, characterized in that, The background buffer solution BGS is a solution containing 60-75 mM sodium tetraborate and 15-30% (v / v) methanol.

12. The capillary electrophoretic quantitative analysis method for the active ingredients of Wubi Yam Pills according to claim 1, characterized in that, The background buffer solution BGS is a solution containing 65 mM sodium tetraborate and 20% (v / v) methanol.

13. The capillary electrophoretic quantitative analysis method for the active ingredients of Wubi Yam Pills according to claim 1, characterized in that, Includes the following steps: S1: Sample extraction using MSPD method: (1) Weigh 60 mg of adsorbent MCM-48 and 60 mg of Wubi yam powder respectively, add them to an agate mortar, mix them evenly and grind for 150 s; (2) After grinding, the mixture is transferred to a small column with sieve plates at both ends, and then 1 mL of 50% (v / v) methanol eluent 1 is added. The mixture is eluted by applying pressure, and the eluent is collected in a centrifuge tube. (3) Place the eluent from the centrifuge tube in the previous step into a solvent evaporator to evaporate it, and finally redissolve it with 500 μL of 5% (v / v) methanol to prepare a sample solution for subsequent SPE use; S2: SPE is used to purify the MSPD extract to remove impurities. (1) Activate the SPE column loaded with HLB packing: Activate the column with 3 mL of methanol and 3 mL of pure water respectively; (2) Sample loading: Transfer 500 μL of the sample solution prepared by the MSPD method to the activated SPE column using a pipette, and use a pressurizer to push the liquid out, controlling the flow rate at 1 mL / min; (3) Rinsing: Rinse the SPE column after loading with 1 mL of pure water to remove impurities; (4) Elution: Elute the SPE column loaded with the sample using 1 mL of methanol; (5) Finally, the eluted solution was evaporated to dryness and redissolved with 200 μL of 50% (v / v) methanol. The redissolved solution was then filtered through a 0.22 μm microporous membrane for subsequent CE analysis. S3: CE-UV analysis and determination of target analytes in the purified solution: (1) Preconditioning of the capillary column: rinse with 0.1 M sodium hydroxide, pure water and background buffer solution BGS for 5 min each; BGS is an aqueous solution containing 65 mM sodium tetraborate and 20% (v / v) methanol. (2) Injection procedure: Inject the sample for 5 seconds at a pressure of 50 mbar; (3) Operating conditions: Operating buffer: an aqueous solution containing 65 mM sodium tetraborate and 20% (v / v) methanol; Detection wavelength: 245 nm; Separation voltage: +20 KV; Temperature: 25 ℃.

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