Alkaloid analysis method based on LVSS-MSS-CDEKC and its application in quality control of Yangxin tablets

Abstract

The present application relates to the alkaloid analysis method based on LVSS-MSS-CDEKC and the application in the quality control of Yangxinshi tablets. The present application combines two enrichment methods of LVSS and MSS to construct a two-step stacking strategy, analyzes the cationic alkaloid components in Yangxinshi tablets based on the capillary electrokinetic chromatography separation mode modified by cyclodextrin, and realizes the content determination research of different batches of Yangxinshi tablet samples. In order to obtain the best application performance, the related experimental parameters involved in the LVSS-MSS-CDEKC method are optimized, and a series of methodological verification results show that the method has good accuracy and stability. In summary, the LVSS-MSS-CDEKC method established in the present application is accurate, stable and reliable, which systematically determines a variety of alkaloid components in Yangxinshi tablets, and provides certain reference value for the quality evaluation and efficacy mechanism research of Yangxinshi tablets.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of alkaloid analysis methods, and particularly relates to an alkaloid analysis method based on large-volume sample stacking-micellar to solvent stacking-cyclodextrin electrokinetic chromatography (LVSS-MSS-CDEKC) and application of the analysis method to quality control of Yangxin Shi tablets. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the background information forms a prior art that is already known in any form.

[0003] Cardiovascular diseases (CVDs) are a general term for heart and peripheral vascular diseases, which seriously threaten human life and health, and have the characteristics of high morbidity, high disability rate and high mortality. After investigation, it is found that the number of patients with cardiovascular diseases in China is increasing year by year, and the patient population is no longer limited to the elderly, but gradually developing towards a younger trend. Yangxin Shi tablets are a traditional Chinese medicine preparation for treating symptoms such as coronary heart disease, angina pectoris, myocardial infarction, hyperlipidemia or hyperglycemia in clinical practice, which is composed of 13 kinds of traditional Chinese medicines such as Astragalus, Ginseng, Salvia miltiorrhiza, Codonopsis, Pueraria, Epimedium, Hawthorn, Rehmannia, Angelica, Coptis, Corydalis (fried), Ganoderma and Licorice (fried). The chemical composition of traditional Chinese medicine preparation is complex and diverse, and the clinical effect is the result of the synergistic effect of multiple chemical components. However, only the content standard of Astragaloside A is specified in the 2020 edition of Chinese Pharmacopoeia, which seems to be unable to meet the requirements of effectively controlling the quality of Yangxin Shi tablets and exploring the pharmacological mechanism. Therefore, some researchers have established an effective analysis method based on HPLC-DAD technology, which has successfully realized the simultaneous determination of multiple chemical components. However, the analysis time is long, the efficiency is low, and the target components are mainly concentrated in flavonoids and phenolic acid compounds. Zhu et al. used HPLC-ESI-Q-TOF-MS to realize the simultaneous determination of flavonoids, phenolic acids, alkaloids and other components, but lacked quantitative analysis. In addition, the alkaloid components in Yangxin Shi tablets have been predicted to be one of the active components for treating cardiovascular diseases. Based on this, it is urgent to establish an efficient and rapid analysis method to carry out systematic research on the alkaloid components in Yangxin Shi tablets.

[0004] Capillary electrophoresis (CE) has been widely used in medicine, food and chemical industry due to its simple operation, fast analysis time and low consumption of organic solvents. In recent years, various online sample pre-concentration techniques have been developed, which do not require additional equipment and to some extent solve the problem of low sensitivity of traditional electrophoretic separation mode. Large volume sample stacking (LVSS) refers to the introduction of a longer sample solution to maximize sample loading, thereby improving the enrichment efficiency. These two methods have been proven to be efficient even in complex sample analysis. Micellar to solvent stacking (MSS) is a method for enriching cationic or anionic components, the main mechanism is based on the effective electrophoretic mobility inversion of charged analytes at the boundary rich in organic solvent, the whole process mainly involves the migration, release and accumulation of analytes. When LVSS is coupled with MSS, the ion components dispersed in the sample region will be refocused during the MSS process, thereby improving the stacking efficiency and separation performance to some extent. SUMMARY

[0005] The present application combines large volume sample stacking (LVSS) and micellar to solvent stacking (MSS) to construct a two-step stacking strategy, which is based on the capillary electrophoretic separation mode modified by cyclodextrin (CDEKC) to analyze the cationic alkaloid components in Yangxin tablets. The method can simultaneously detect multiple alkaloid components, and has the advantages of rapidness, sensitivity and good repeatability, and has good industrial application prospect in the quality control of Yangxin tablets.

[0006] In the first aspect of the present application, an alkaloid analysis method based on LVSS-MSS-CDEKC is provided, which is used for simultaneously detecting epiberberine, dehydrovilline, jatrorrhizine, coptisine and berberine in a to-be-tested sample. The LVSS-MSS-CDEKC electrophoretic operation mode is as follows: the running buffer solution is filled in the capillary column, the SDS micellar solution and the to-be-tested sample are injected under a certain pressure, and finally the methanol solution is introduced at the front end of the capillary column, and a separation voltage is applied to separate the alkaloids in the to-be-tested sample.

[0007] In the detection method, the pretreated capillary column needs to be filled with running buffer solution to adjust the internal environment, and then a section of micelle solution, a certain length of sample solution and a section of organic solvent are introduced in succession. In the process of electrophoretic separation, the electroosmotic flow (EOF) has a certain interference effect on the effective mobility of the above alkaloids. In view of this problem, the buffer solution in the method is prepared in an acidic phosphate solution to control the strength of the EOF. When a positive voltage is applied at both ends of the capillary, the negatively charged SDS micelles migrate to the anode end of the electrode, and the positively charged cationic components in the sample area migrate to the cathode end of the electrode. Subsequently, the cationic components are swept into a band with the micelles and migrate to the anode end of the electrode with the micelles. The micelles have different critical micelle concentrations in different solution matrices. When the SDS micelles carrying the to-be-detected components migrate to the organic solvent area, the critical micelle concentration of SDS increases, and the micelles break, so that the cationic components are released. Subsequently, the cationic components migrate to the cathode end of the electrode under the driving of the positive voltage until the detection window.

[0008] The detection of the above-mentioned solvent system in the capillary is of great significance for the enrichment and separation of alkaloid components in Yangxin tablets. The analysis method provided in the first aspect of the present application can effectively realize the simultaneous detection of the five alkaloid components, and the enrichment efficiency is increased by 12-15 times compared with ordinary electrophoretic separation.

[0009] Preferably, the running buffer is a phosphate buffer with a pH of 2-3, and the buffer further includes a certain concentration of hydroxypropyl-β-cyclodextrin (HP-β-CD); the phosphate is an acid phosphate, including but not limited to one or more of sodium dihydrogen phosphate (NaH2PO4), ammonium dihydrogen phosphate (NH4H2PO4), and sodium monohydrogen phosphate (Na2HPO4).

[0010] In one embodiment of the present application, the phosphate is NaH2PO4, and the concentration is 20-40 mM, preferably 25-35 mM, and further preferably 27 mM, 28 mM, 29 mM, 30 mM, 31 mM or 32 mM.

[0011] In addition, the concentration of the HP-β-CD is 10-20 mg / mL, preferably 13-17 mg / mL, and more preferably 14 mg / mL, 15 mg / mL or 16 mg / mL.

[0012] Preferably, the capillary column is an uncoated quartz capillary column, and the effective length should reach more than 40 cm, and the inner diameter is 45-66 μm. In a specific embodiment of the present application, the length of the capillary is 50 cm, the inner diameter is 50 μm, and the effective length is 41.5 cm.

[0013] Preferably, the capillary column is pretreated before the analysis, the steps are as follows: the capillary is activated by washing with alkali and water, and the capillary is washed with running buffer solution before electrophoresis.

[0014] Further, the alkali is 0.08-0.12M NaOH solution.

[0015] Further, the washing time of the alkali and water is 1-3min, and the washing time of the buffer solution is 2-4min.

[0016] Preferably, the SDS micelle solution is phosphate buffer solution containing a certain concentration of SDS, the type and concentration of phosphate in the phosphate buffer solution are the same as those in the running buffer solution, and the concentration of SDS is 15-25mM, and further preferably 18-22mM.

[0017] Preferably, the injection pressure of the SDS micelle solution is 45-55mbar, and the injection time is 4-6s.

[0018] Preferably, the sample to be tested is extracted with methanol solution as the solvent, and the concentration of the methanol solution can be adjusted according to the dissolution effect of the sample to be tested, and the feasible concentration is 50-99%.

[0019] Preferably, the injection pressure of the sample to be tested is 45-55mbar, and the injection time is 25-35s.

[0020] Preferably, the concentration of the methanol solution injected into the front end of the capillary is 45-55%, the injection pressure is 45-55mbar, and the injection time is 4-6s.

[0021] Preferably, the separation voltage is 23-27kV.

[0022] Preferably, the analysis process of the alkaloid sample further comprises detecting the alkaloid in the separated sample by using a detector, and the detector includes but is not limited to ultraviolet detector, visible spectrophotometric detector, laser-induced fluorescence detector, and electrochemical detector; the most commonly used detector is ultraviolet detector, the detection temperature is 22-27℃, and the detection wavelength is 220-240nm.

[0023] In the second aspect of the present application, the analysis method in the first aspect is applied to the quality control of Yangxin tablets.

[0024] The main application mode of the above-mentioned analysis method is as follows: based on the analysis method of the first aspect, a plurality of batches of Yangxin Shisan tablets are analyzed to obtain a spectrum containing information of the alkaloids, and data processing is performed to obtain a control fingerprint based on the alkaloids; the sample to be tested Yangxin Shisan tablets are analyzed according to the method of the first aspect to obtain a sample fingerprint, and similarity comparison is performed with the control fingerprint, so as to realize quality control of the sample to be tested Yangxin Shisan tablets.

[0025] In a third aspect of the present application, a detection method for the content of alkaloids in Yangxin Shisan tablets is provided, which comprises the step of analyzing the Yangxin Shisan tablets by the analysis method of the first aspect.

[0026] Preferably, the detection method further comprises the preparation of a control sample and a test sample. The control sample is a mixed solution of standard samples of table berberine, dehydrocorydaline, jatrorrhizine, coptisine and berberine, and the test sample is a methanol extract of Yangxin Shisan tablets.

[0027] In the above-mentioned control sample solution, the concentrations of the standard samples of table berberine, dehydrocorydaline, jatrorrhizine, coptisine and berberine can be routinely adjusted according to the actual content of alkaloids in the test sample, the detection limit of the detection platform and other factors, and the five kinds of alkaloids can be clearly distinguished in the spectrum. In one embodiment verified by the present application, the mixed solution of the standard samples comprises 5 μg / mL of table berberine, 20 μg / mL of dehydrocorydaline, 5 μg / mL of jatrorrhizine, 10 μg / mL of coptisine and 20 μg / mL of berberine, and is diluted with water to obtain a series of concentrations, i.e. the control sample.

[0028] The specific preparation method of the test sample solution is as follows: the Yangxin Shisan tablets to be tested are removed from the coating and ground into powder, the drug powder is accurately weighed and added into a methanol solution for ultrasonic extraction, and after cooling, the sample extract is obtained by constant volume. The above-mentioned ultrasonic extraction and constant volume process are routine operations in the art, and the specific parameters can be adjusted according to the drug solution. If necessary, the technical personnel can concentrate the above-mentioned sample extract to improve the injection concentration, and the concentration method is, for example, nitrogen blowing and redissolving. In one embodiment verified by the present application, the drug concentration in the test sample of Yangxin Shisan tablets is 2-3 mg / mL.

[0029] The beneficial effects of one or more of the above technical solutions are as follows:

[0030] The application provides an analysis method suitable for various alkaloid components, and realizes more than 10 times of enrichment effect based on two enrichment strategies of LVSS and MSS. Since the matrix is relatively complex, it is difficult to detect flavonoids, alkaloids and phenolic acid components in Yangxin tablets, but alkaloids as the main active components of the above drugs are of great significance for quality control. The application has good accuracy and stability, and has important significance for the quality evaluation of Yangxin tablets. BRIEF DESCRIPTION OF DRAWINGS

[0031] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The embodiments of these drawings illustrate the preferred embodiment of the application and, together with the description, serve to explain the principles of the application.

[0032] Figure 1 It is the LVSS-MSS-CDEKC method mechanism diagram described in Example 1;

[0033] Wherein, SB: sweep set boundary, MSSB: micelle to solvent stacking boundary;

[0034] Figure 2 It is the influence of each parameter on the peak area of five kinds of alkaloid components described in Example 1;

[0035] Wherein, (A) SDS concentration; (B) MeOH ratio; (C) sample injection time;

[0036] Figure 3 It is the influence of each parameter on the migration time of five kinds of alkaloid components described in Example 1;

[0037] Wherein, (A) buffer concentration; (B) buffer pH; (C) HP-β-CD concentration; (D) voltage; (E) temperature;

[0038] Wherein, detection wavelength: 230 nm; sample concentration: 2.5 mg / mL;

[0039] Figure 4 It is the chromatogram of standard mixed solution (lower waveform line) and Yangxin tablet sample solution (upper waveform line) under the separation mode of LVSS-MSS-CDEKC described in Example 1;

[0040] Wherein, each chromatographic peak is as follows, 1: epiberberine, 2: dehydrovijainine, 3: jatrorrhizine, 4: coptisine, 5: berberine;

[0041] Figure 5Electropherogram of Yangxinshipian sample solution in typical LVSS-MSS-CDEKC mode (upper wave line, sample concentration: 2.5 mg / mL) and in common CDEKC mode (lower wave line, sample concentration: 10 mg / mL) as described in Example 1;

[0042] In which, each chromatographic peak is attributed as follows, 1: table berberine, 2: dehydrogenated corydalis alkaloids, 3: jatrorrhizine, 4: coptisine, 5: berberine. DETAILED DESCRIPTION

[0043] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0044] It is also important to note that the terms "including", "comprising", and / or "having" as used herein are specifically intended to be open-ended and also to mean including, but not limited to. As used herein, the singular forms "a", "an" and / or "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Further, to the extent that any term is defined by a singular or plural form of a noun or pronoun, the plural form also encompasses the singular form, and vice versa, unless the context clearly dictates otherwise.

[0045] In order to enable persons skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples.

[0046] 1. Materials and instruments

[0047] 1.1 Standard

[0048] Table berberine, dehydrogenated corydalis alkaloids, jatrorrhizine, coptisine and berberine were purchased from Chengdu Desit Biotech Co., Ltd. (purity > 98%).

[0049] 1.2 Reagents

[0050] Chromatographic grade methanol was purchased from Fisher Company, USA; test water was ultrapure water; sodium dodecyl sulfate (SDS) and hydroxypropyl-β-cyclodextrin (HP-β-CD) were purchased from Beijing Solabio Biotech Co., Ltd.; sodium dihydrogen phosphate (NaH2PO4), sodium hydroxide (NaOH) and phosphoric acid (85%) were purchased from Tianjin Comin Biotech Co., Ltd. The buffer solution was prepared in ultrapure water and the pH value was adjusted by phosphoric acid. All solutions were filtered through a 0.22 μm filter membrane before sample injection and analysis.

[0051] 1.3 Medicinal materials

[0052] Ten batches of Yangxin Shisan tablets were purchased from Shanghai Pharmaceutical Group Qingdao Guofeng Pharmaceutical Co., Ltd. and were stored in the Institute of Chinese Medicine Research of Tianjin University of Chinese Medicine after being reviewed by Professor Chang Yanxu of Tianjin University of Chinese Medicine.

[0053] 1.4 Instruments

[0054] Agilent 7100 capillary electrophoresis instrument: Agilent, USA

[0055] Uncoated quartz capillary column: Hebei Yongnian Ruihong Chromatography Device Co., Ltd.

[0056] Mill-Q IQ 7005 ultrapure water preparation instrument: Millipore

[0057] KQ-250E ultrasonic cleaner: Kunshan Ultrasonic Instruments Co., Ltd.

[0058] BP121S one-thousandth balance: Sartorius, Germany

[0059] XW-80A vortex mixer: Shanghai Luxi Analytical Instrument Factory

[0060] pH meter: Mettler-Toledo Instruments (Shanghai) Co., Ltd.

[0061] Ultrafiltration membrane: Tianjin Linghang Experimental Equipment Co., Ltd.

[0062] 2. Experimental Methods

[0063] 2.1 Preparation of solutions

[0064] 2.1.1 Preparation of standard solution and quality control solution

[0065] An appropriate amount of epiberberine, dehydrocoptisine, jatrorrhizine, berberine and berberine reference substances were accurately weighed and dissolved in methanol to prepare a standard stock solution with a concentration of 1 mg / mL. An appropriate amount of single standard solution was dried by nitrogen flow and then reconstituted with water to obtain a mixed standard solution containing 5 μg / mL epiberberine, 20 μg / mL dehydrocoptisine, 5 μg / mL jatrorrhizine, 10 μg / mL berberine and 20 μg / mL berberine. The mixed standard solution was gradually diluted with water to obtain a series of concentration points for investigating the linearity of the test components. The mixed standard solution with appropriate concentration was diluted with water to three concentration levels of low, medium and high as quality control solutions for studying the stability and accuracy of the established method. The above solutions were stored in a 4°C refrigerator for future use.

[0066] 2.1.2 Preparation of test sample solution

[0067] Take one tablet of Yangxinshi tablet, remove the coating and grind into powder in a mortar. Precisely weigh 1.0 g of the sample powder into a conical flask with a stopper, and precisely add 20 mL of 70% methanol with a pipette. Weigh the conical flask with the sample. Ultrasonically extract the conical flask with the sample at room temperature for 30 min (360 W). Weigh the conical flask again after cooling to room temperature, and make up the weight loss with 70% methanol. Centrifuge the sample extract at a speed of 13400 r / min for 10 min. Filter the supernatant extract through a 0.22 um microporous filter membrane, and store in a 4°C refrigerator for standby. Take an appropriate amount of sample extract, dry it with a nitrogen stream, and then dissolve it with water to prepare a sample solution of 10 mg / mL for traditional CDEKC analysis, and a sample solution of 2.5 mg / mL for LVSS-MSS-CDEKC analysis.

[0068] 2.2 Instrument conditions

[0069] All instrument operations and data analysis during the experiment were performed on the Agilent ChemStation work platform. A total length of 50 cm, an inner diameter of 50 um, and an effective length of 41.5 cm of an uncoated quartz capillary column were used to achieve electrophoretic separation and enrichment. A new capillary column was activated before use by flushing with 1M NaOH, 0.1M NaOH and water for 30 min (935 mbar) in sequence. During operation, 0.1M NaOH was used to flush for 2 min, deionized water was used to flush for 2 min, and running buffer solution was used to flush for 3 min before each analysis. After the last analysis of each day, the capillary column was flushed with 1M NaOH, 0.1M NaOH and water for 10 min respectively. In order to ensure good reproducibility of the analysis results during the experiment, new buffer solution was needed after each two runs.

[0070] 2.3 Traditional CDEKC mode running conditions

[0071] In the traditional CDEKC method, the composition of the running buffer solution was 30mM NaH2PO4 solution (pH=2.5) containing 15mg / mL HP-β-CD. First, the running buffer solution was filled into the entire capillary column, and then the sample solution was injected for 5s at 50mbar. The separation voltage was 25kV, the detection temperature was 25°C, and the detection wavelength was 230nm.

[0072] 2.4 LVSS-MSS-CDEKC mode running conditions

[0073] In the LVSS-MSS-CDEKC process, the composition of running buffer solution was 30 mM NaH2PO4 solution (pH = 2.5) containing 15 mg / mL HP-β-CD. The micellar solution was prepared by dissolving 20 mM SDS in 30 mM NaH2PO4 solution. The running buffer solution was first filled into the whole capillary column, then the SDS micellar solution was injected at 50 mbar for 5 s, followed by the sample solution at 50 mbar for 30 s, and finally 50% methanol was introduced into the front end of the capillary column at 50 mbar for 5 s. The separation voltage was 25 kV, the detection temperature was 25 °C, and the detection wavelength was 230 nm.

[0074] 2.5 Methodology Investigation

[0075] To verify the accuracy and reliability of the established LVSS-MSS-CDEKC method, the linearity, precision, 24 h stability and sample recovery rate of five components, i.e., epiberberine, dehydrovilline, jatrorrhizine, berberine and berberine, were investigated based on the method. The low, medium and high concentration quality control samples of the five alkaloid components were analyzed for one day and three consecutive days to investigate the intra-day and inter-day precision. The quality control samples analyzed at room temperature were placed for 24 h to investigate the 24 h stability of the tested components. According to 3 times the signal-to-noise ratio (S / N) and 10 times the signal-to-noise ratio, the limit of quantification (LOQ) and the limit of detection (LOD) of the five components in the LVSS-MSS-CDEKC method were calculated. In addition, the recovery rate of the five alkaloid components was achieved by adding the same concentration of mixed standard solution to the sample with known concentration, and the value was expressed as a percentage (%), and the calculation formula was: recovery rate (%) = (measured value - amount of tested component in sample) / standard addition amount x 100%.

[0076] 2.6 Data Analysis

[0077] Compared with the traditional pressure injection CDEKC mode, the enrichment factor (EF) of the tested component was calculated as follows: EF = C1 x A2 / C2 x A1.

[0078] Wherein C1 represents the sample concentration in the CDEKC mode, C2 represents the sample concentration in the LVSS-MSS-CDEKC mode, A1 represents the peak area of the tested component in the CDEKC mode, and A2 represents the peak area of the tested component in the LVSS-MSS-CDEKC mode.

[0079] 3 Results and Discussion

[0080] 3.1 Establishment of LVSS-MSS-CDEKC Method

[0081] Figure 1The main mechanism of LVSS-MSS-CDEKC method was described. In order to obtain the best enrichment efficiency without affecting the resolution between the components, the related influencing factors in the LVSS-MSS-CDEKC process were investigated, including the concentration and pH value of buffer solution, the concentration of micelles, the concentration of organic solvent, sample injection volume, separation voltage and temperature, etc.

[0082] 3.2 Optimization of LVSS-MSS-CDEKC method

[0083] In order to obtain the best enrichment efficiency without loss of resolution, the related parameters involved in LVSS-MSS-CDEKC were studied respectively. The main factors affecting the enrichment efficiency include the concentration of SDS, sample injection time and the proportion of organic solvent, while the concentration and pH of buffer salt solution, the addition amount of HP-β-CD, and the separation voltage and temperature have greater influence on the resolution of the components to be tested.

[0084] 3.2.1 Concentration of SDS

[0085] SDS is a commonly used anionic surfactant in capillary electrochromatography, which can electrostatically bind cationic compounds with its negative charge on the surface. SDS micelles can sweep the dispersed components to be tested in the sample solution zone into a narrow band, so that the analyte can obtain high peak area even at low concentration level. To determine the optimal concentration of micelle solution, 10 mM, 20 mM and 30 mM SDS were investigated respectively ( Figure 2 A). With the increase of SDS concentration, it can be observed that the peak area of the five alkaloids is improved. However, when the concentration of SDS increases to 30 mM, the peak area of each component no longer shows an upward trend, which indicates that 20 mM of SDS concentration is sufficient for sweeping the sample components. In addition, the migration time of the components to be tested is also constantly prolonged with the increase of SDS concentration. Therefore, the optimal concentration of SDS micelle solution is determined to be 20 mM.

[0086] 3.2.2 Proportion of organic solvent

[0087] When the micelles carrying the components to be tested migrate to the organic solvent region, whether the micelles can be broken into monomers and release the analyte depends on the concentration of organic solvent, so the concentration of organic solvent plays an important role in the enrichment of analyte. Methanol is a commonly used organic solvent in MSS method, so the effects of different concentrations of methanol (10%, 30%, 50%, 70%, 100%) on the analyte were investigated respectively ( Figure 2B) When 10% methanol was introduced into the front end of the capillary, only one component, berberine, could be detected with a low response value, which indicated that the micellar solution was hardly broken and the alkaloids were carried out of the capillary by the micelles under the driving of the positive voltage. When the concentration of methanol was increased to 30%, all the components could be detected with a higher response value, but the dehydrovilline and jatrorrhizine could not be separated well. With the further increase of the concentration (50-100%), it was observed that the response value of each component did not increase significantly and the resolution was improved, while the migration time of the components was prolonged with the increase of the methanol concentration. Based on the above analysis, 50% methanol was selected for the LVSS-MSS-CDEKC method.

[0088] 3.2.3 Injection time

[0089] During the running of the LVSS-MSS-CDEKC, the sample amount introduced into the capillary is also an important factor affecting the enrichment efficiency. Generally, the more the sample amount introduced into the capillary, the more the loading amount of the components to be detected, that is, the higher the enrichment efficiency. Therefore, different injection times (20 s, 30 s, 40 s) were investigated in this experiment. Figure 2 C) With the increase of the injection time of the sample to 40 s, the peak area of the sample was increased, but the migration time was also prolonged. Although a longer injection time of the sample could obtain a higher enrichment efficiency, a too long sample matrix solution could lead to the decrease of the resolution of the analytes. When the sample was injected for 30 s, the components to be detected could be enriched and separated well, but when the time was increased to 40 s, the dehydrovilline and jatrorrhizine could not be separated effectively. In order to obtain the best resolution and separation efficiency, the best injection time of the sample was 30 s.

[0090] 3.2.4 Concentration of phosphate

[0091] The buffer solution can not only maintain the pH of the solution between the two electrodes to be basically unchanged during the electrophoresis, but also has a certain conductivity which is conducive to the migration of the components to be detected. The phosphate buffer solution can provide a relatively stable ionic environment and pH buffering capacity, and is a kind of buffer solution widely used in electrophoresis analysis [28-29] . Under the premise of ensuring that the phosphate solution has a certain buffering capacity, the higher the concentration of the phosphate is, the more the Joule heat is generated, thereby reducing the resolution of the components to be detected. Based on this, under the condition of pH 2.5 and 15 mg / mL HP-β-CD, the effects of different concentrations of NaH2PO4 (10 mM, 30 mM, 50 mM) on the electrophoretic separation behavior of the components to be detected were investigated. From Figure 3As observed in Figure A, the migration time of the analyte gradually increased with increasing NaH₂PO₄ concentration, exhibiting poor resolution at 50 mM. This may be because higher phosphate concentrations generate more Joule heat, affecting the solution viscosity and thus altering the time it takes for the sample to reach the detection window. When the phosphate concentration is below 30 mM, the analyte exhibits poor peak shape, possibly because the 10 mM NaH₂PO₄ solution lacks sufficient buffering capacity. Therefore, a phosphate concentration of 30 mM was chosen for further experimental research.

[0092] 3.2.5 The role of pH

[0093] To effectively control the intensity of EOF, the running buffer solution was prepared in an acidic environment. Using 30 mM NaH₂PO₄ and 15 mg / mL HP-β-CD as initial conditions, the effect of these conditions on the electrophoretic separation of analytes was investigated at pH 2, pH 2.5, and pH 3. With changes in the pH of the buffer solution, the peak area of ​​the analytes was not significantly affected, but the separation efficiency was affected to some extent. Figure 3 As shown in Figure B, the migration time of each component gradually shortens with increasing pH, but this shortening of migration time is often accompanied by a decrease in separation. At pH 3, dehydrocorydaline exhibits a poor peak shape and cannot be effectively separated from purpuric acid. Furthermore, a higher current value was observed during electrophoresis with a buffer solution at pH 2, which may be due to the increased Joule heating generated at higher acidity. Considering all these factors, the optimal pH for the operating buffer solution was ultimately determined to be 2.5.

[0094] 3.2.6 Concentration of HP-β-CD

[0095] The hollow structure inside cyclodextrin allows other substances to be encapsulated within it. Based on the different binding forces that different compounds exhibit with cyclodextrin, cyclodextrin is often used as an additive in buffer solutions in electrophoretic analysis to achieve effective separation of analytes. HP-β-CD, derived from natural cyclodextrin, has better solubility. The effects of HP-β-CD concentrations of 10 mg / mL, 15 mg / mL, and 20 mg / mL on the separation efficiency of analytes were investigated. Figure 3 (C) The higher the concentration of cyclodextrin added, the longer the migration time of the analytes. When 10 mg / mL of HP-β-CD was added to the buffer solution, dehydrocorydaline, purslane, and berberine could not be effectively separated. When the concentration of HP-β-CD was increased to 15 mg / mL, each component achieved better peak shape and resolution. However, as the concentration was further increased to 20 mg / mL, the resolution of berberine decreased significantly. Therefore, adding 15 mg / mL to the buffer solution is the optimal choice.

[0096] 3.2.7 Separation voltage and temperature

[0097] Separation voltage and temperature can change the migration rate of the analytes, thus affecting the separation efficiency. The effects of different voltages (22 kV, 25 kV, 28 kV) and temperatures (20 °C, 25 °C, 30 °C) on the migration behavior of the five alkaloid components were investigated, respectively. From Figure 3 D and Figure 3 E, it can be seen that with the increase of separation voltage and temperature, the migration time of each component gradually shortens, accompanied by a decrease in separation efficiency. This indicates that high voltage and temperature can accelerate the migration rate of the analytes. Under the premise of ensuring good separation degree and shorter migration time of each component, 25 kV voltage and 25 °C temperature were finally selected as the optimal separation conditions.

[0098] 3.3 Methodology results

[0099] Table 1 lists the linear equations, linear ranges, LODs, LOQs, and recoveries of the five alkaloid components. The linear equation R 2 of the five components is greater than 0.996, indicating that they all show good linear relationships within the linear range. The LOD of the five components ranges from 0.20 to 0.5 pg / mL, the LOQ ranges from 0.70 to 1.6 pg / mL, and the recovery is within the range of 98.4-103%. Table 2 shows the precision and stability results of the analytes. The intra-day precision of the five alkaloid components is within the range of 0.74-3.79%, the inter-day precision is within the range of 2.55-3.99%, and the accuracy is within the range of 97.2-104%. In addition, the alkaloid components in the quality control sample have good stability within 24 h, with a retention rate within the range of 97.3-103%, and the RSD value is within 4.4%. Based on the above data analysis, the established LVSS-MSS-CDEKC method is stable and reliable, and can be applied to the determination and analysis of alkaloid components in Yangxin tablets.

[0100] Table 1 Linear equations, linear ranges, LODs, LOQs, and recoveries of the five alkaloid components (n = 6)

[0101]

[0102] Table 2 Intra-day, inter-day precision, accuracy, and 24 h stability of the five alkaloid components (n = 6)

[0103]

[0104]

[0105] 3.4 Application of LVSS-MSS-CDEKC method

[0106] The proposed LVSS-MSS-CDEKC method was successfully applied to the quantitative analysis of alkaloids in YXST. By comparing the electropherograms of YXST sample solution and mixed standard solution (Fig. 4), five alkaloids were finally identified. Figure 4 The content of alkaloids in different batches of YXST was investigated based on the proposed method. As shown in Table 3, the contents of epiberberine and jatrorrhizine in different batches were similar, and had lower content compared to other alkaloids, which were 0.698-0.871 μg / mg, 0.548-0.756 μg / mg, respectively. In addition, berberine had a higher content in different batches of YXST, which was 4.13-6.38 μg / mg. Although the content of five alkaloids in the same batch had a large difference, the content of the same alkaloid in different batches was roughly similar, indicating that the 10 batches of YXST samples had small batch-to-batch variability.

[0107] Table 3. The content of alkaloids in ten batches of YXST (μg / mg, n=3)

[0108]

[0109]

[0110] 3.5 Comparison with other analytical methods

[0111] The YXST sample was analyzed by LVSS-MSS-CDEKC method and common CDEKC separation mode, respectively, and the results were shown in Fig. 4. Figure 5 The peak area of five alkaloids measured by LVSS-MSS-CDEKC method was 12-15 times higher than that by common CDEKC mode.

[0112] In addition, some studies on the analysis of alkaloids in YXST were also summarized in Table 4. Compared with the methods listed in the table, the method established in this experiment had less consumption of organic solvents and shorter analysis time. At the same time, compared with HPLC or UPLC chromatographic technology, the method established in this experiment could realize the simultaneous determination of multiple alkaloids in YXST without additional expensive instruments. Based on this, the sample pretreatment method of LVSS-MSS-CDEKC method was simple, and the analysis was fast and sensitive, which could simultaneously determine multiple alkaloids in YXST, and provided a green and efficient analysis method for the quality evaluation of YXST.

[0113] Table 4. Comparison of the method for the determination of alkaloids in YXST with the methods reported in the literature

[0114]

[0115]

[0116] 4Conclusion

[0117] Based on capillary electrokinetic chromatography modified by cyclodextrin, the LVSS-MSS-CDEKC two-step stacking strategy was constructed by combining LVSS and MSS two kinds of enrichment methods. Through a series of experimental studies, the optimal experimental conditions of the established method were determined. Compared with the conventional CDEKC mode, the LVSS-MSS-CDEKC method can realize more than 10 times enrichment factor, and based on the method, the simultaneous determination of 5 alkaloid components in different batches of Yangxin tablets was successfully realized. Through the methodological investigation, the results showed that the method had good stability, accuracy and reliability. In addition, compared with the existing research on the analysis of alkaloid components in Yangxin tablets, the results showed that the LVSS-MSS-CDEKC method required shorter analysis time and less organic reagent consumption. In summary, the LVSS-MSS-CDEKC method established in this study can realize the simultaneous determination of multiple alkaloid components in Yangxin tablets, and a quality evaluation system of alkaloid multi-component is constructed, which is a safe, fast, accurate and green analysis technology.

[0118] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for alkaloid analysis based on LVSS-MSS-CDEKC, characterized by, The analysis method is used for simultaneously detecting table berberine, dehydrogenation corydine, jatrorrhizine, coptisine and berberine in the sample to be detected, and the LVSS-MSS-CDEKC electrophoretic running mode is as follows: filling the capillary column with running buffer solution, injecting SDS micellar solution and the sample to be detected under a certain pressure, and finally introducing methanol solution at the front end of the capillary column, and applying a separation voltage to separate alkaloids in the sample to be detected; The capillary column is an uncoated quartz capillary column, and the effective length should be more than 40 cm, and the inner diameter is 45-66 μm; The SDS micellar solution is NaH2PO4 buffer solution containing 15-25 mM SDS; the injection pressure of the SDS micellar solution is 45-55 mbar, and the injection time is 4-6 s; The sample to be detected is extracted by using 50-99% methanol solution as a solvent; the injection pressure of the sample to be detected is 45-55 mbar, and the injection time is 25-35 s; The concentration of the methanol solution injected at the front end of the capillary is 45-55%, the injection pressure is 45-55 mbar, and the injection time is 4-6 s; and the separation voltage is 23-27 kV. The above-mentioned alkaloid sample analysis process further comprises detecting the alkaloids in the separated sample by using a detector, and the detector is an ultraviolet detector, the detection temperature is 22-27 ℃, and the detection wavelength is 220-240 nm.

2. The method of claim 1, wherein the method is based on LVSS-MSS-CDEKC. The running buffer is NaH2PO4 buffer solution, the concentration is 20-40 mM, the pH is 2-3, and the buffer solution further comprises 10-20 mg / mL hydroxypropyl-β-cyclodextrin.

3. The method of claim 1, wherein the alkaloid analysis is based on LVSS-MSS-CDEKC. Before the above-mentioned analysis running, the capillary column is pretreated, and the steps are as follows: sequentially flushing with 0.08-0.12 M NaOH solution and water for 1-3 min to activate the inner wall of the capillary, and further flushing the capillary with the running buffer solution for 2-4 min before electrophoretic running.

4. The use of the analysis method according to any one of claims 1-3 in the quality control of Yangxin Shu tablets, characterized in that, The application mode of the analysis method is as follows: based on the analysis method of any one of claims 1-3, a plurality of batches of Yangxin tablets are analyzed to obtain a spectrum containing information of the above-mentioned alkaloids, and through data processing, a control fingerprint based on alkaloids is obtained; the sample to be detected is analyzed according to the method of any one of claims 1-3 to obtain a sample fingerprint, and similarity comparison is performed with the control fingerprint, so that the quality control of the sample to be detected is realized.

5. A method for detecting the content of alkaloids in Yangxin Shihao tablets, characterized in that, The detection method comprises the steps of analyzing the Yangxin tablets by the analysis method of any one of claims 1-3.

6. The method for detecting the alkaloid content of Yangxin Shu according to claim 5, wherein the alkaloid content is detected by HPLC. The detection method further comprises the configuration of a control sample and a test sample, the control sample is a mixed solution of table berberine, dehydrogenation corydine, jatrorrhizine, coptisine and berberine standard, and the test sample is a methanol extract of the Yangxin tablets; The specific preparation method of the test sample solution is as follows: taking the Yangxin tablets to be detected, removing the coating and grinding into powder, accurately weighing the powder and adding methanol solution for ultrasonic extraction, and after cooling, the sample extract is obtained, and the sample extract is centrifuged and filtered to obtain the test sample solution.

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