CN114778264A

CN114778264A - Method for simultaneously accumulating, enriching and separating multiple arsenic forms in natural medicine on line based on micelle-solvent accumulation

Info

Publication number: CN114778264A
Application number: CN202210481619.3A
Authority: CN
Inventors: 曹君; 余亚玲
Original assignee: Hangzhou Normal University
Current assignee: Hangzhou Normal University
Priority date: 2022-05-05
Filing date: 2022-05-05
Publication date: 2022-07-22

Abstract

The invention discloses a method for simultaneously accumulating, enriching and separating various arsenic forms in natural medicines on line based on micelle-solvent accumulation. Two-step stacking capillary zone electrophoresis CZE of As (v), As (iii), MMA and DMA was performed efficiently and sensitively by electrokinetic injection and micelle-to-solvent stacking of MSS in co-electroosmotic flow co-EOF using zwitterionic surfactant PAPS. HDMB coats fused silica capillaries to meet the condition of the same EOF direction between anions and capillaries. The background solution is borax buffer solution. Specifically, micelles and target analytes are injected and stacked through a field enhanced sample under negative pressure; methanol was injected, with the analyte scanned being carried by the micelle to the MSS boundary. The invention utilizes the coating capillary electrophoresis from micelle to solvent accumulation method to detect arsenic in natural medicines, realizes the rapid detection of arsenic form and improves the sensitivity of on-line enrichment of capillary electrophoresis samples.

Description

Method for simultaneously accumulating, enriching and separating multiple arsenic forms in natural medicine on line based on micelle-solvent accumulation

Technical Field

The invention belongs to the field of separation and enrichment of arsenic forms in natural medicines, and relates to a method for enriching arsenic on line by using a capillary electrophoresis technology, in particular to a method for simultaneously accumulating, enriching and separating various arsenic forms in natural medicines on line based on micelle-solvent accumulation.

Background

Arsenic (As) is a natural element in the earth's crust, contains over 50 compounds with different toxicities, and is widely distributed in the atmosphere, water, and ground. Due to industrial pollutionPenetrating into the living aspect, people are exposed to high concentrations of inorganic arsenic, which is the most toxic species. Chronic arsenic poisoning that causes cardiovascular disease, neurological disease, diabetes or skin bladder and lung cancer can be caused by chronic exposure to inorganic arsenic, primarily by drinking contaminated water, eating foods processed with such water, and irrigating foods with arsenic-rich water. Arsenic is classified by the World Health Organization (WHO) as one of 10 chemicals that pose significant public health problems due to its serious impact on the human body. The newest arsenic index of the world health organization for drinking water is 0.01mg/L, when the dosage exceeds 6X 10^-4At mg/L, there is a carcinogenic risk. However, the establishment of this index value is only temporary due to the measurement difficulties and the practical difficulties of removing arsenic from drinking water. Although arsenic is extremely harmful to humans, it is used as an ingredient in certain pesticide and poultry feed formulations to prevent pests and promote growth. Therefore, continuous research on the accurate detection technology of arsenic content is an extremely important and profound research topic.

Arsenic exists in both inorganic (arsenite and arsenate) and organic (arsenite, arsine, monomethylarsine and dimethylarsinic acid) forms, and different kinds of arsenic have different toxicities, with inorganic arsenic compounds being more toxic than organic arsenic compounds (such as those found in seafood). Inductively coupled plasma mass spectrometry (ICP-MS) and hydride generation atomic fluorescence spectrometry (HG-AFS) are by far the most commonly used combination techniques for the separation and analysis of arsenic species. In addition, other important analytical methods have been developed to detect trace amounts of arsenic in actual samples, such as high performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS), ICP optical emission spectrometry (ICP-OES), and quadrupole time-of-flight mass spectrometry (ESI-qTOF-MS). Capillary Electrophoresis (CE) has strong separation performance, but its sensitivity is reduced by the short optical path and small sample size of the capillary. To overcome these limitations, various in-line sample pre-enrichment techniques in CE are effective methods for arsenic species enrichment, including field-enhanced sample injection (FESI) and transient isotachophoresis (t-ITP). In addition, many techniques combined with CE, such as CE and hydride generation electro-thermal atomic absorption spectroscopy (CE-HG-ETAAS), CE-ICP-MS, and CE and electrospray mass spectrometry (CE-ESI-MS), are also ideal methods to address these limitations. However, the direct detection of arsenic by capillary electrophoresis has the advantages of high efficiency, simplicity and convenience, and has great attraction. Therefore, the application of a new high-sensitivity pre-enrichment method to arsenic detection remains an interesting research topic.

Typical methods for online preconcentration of CE are FESI, t-ITP and dynamic pH ligation. In addition to the above enrichment techniques, micelle-solvent stacking (MSS) is one of the on-line sample concentration techniques that has emerged in recent years. In addition, transient capture, focusing of analytes by micelle collapse (AFMC), and micelle-cyclodextrin stacking (MCDS) are advantageous for similarly increasing detection sensitivity. MSS was first proposed by Joselito p.guirino in 2009 to achieve reversal of the effective electrophoretic migration direction of organic cations by using micelles in sample solutions and organic solvents in background solutions (BGS). The focusing effect is achieved by reversing the effective electrophoretic mobility of the target analyte at the boundary region between the micelle and the organic solvent-modified BGS. The excellent detection sensitivity of MSS has attracted much attention and is continuously innovated on this basis. Innovations are mainly focused on the type of micelles and the location of the organic solvents and micelles. In general, most previous studies have focused on the use of cationic surfactants for the detection and isolation of anionic analytes in MSS. There are generally three models in MSS: the BGS comprises a one-step stacking of an organic solvent and a mixed sample in a micelle, a two-step stacking of the organic solvent, the sample and the micelle injection in the BGS, and a three-step stacking of the organic solvent, an analyte and the micelle injection, and is respectively used for detecting various compounds. Selecting an injection model between electrokinetic injection and hydrodynamic injection may also significantly improve performance, particularly in achieving a minimum detection limit. In light of the foregoing, it is desirable to develop a more sensitive and efficient MSS technology.

The present invention establishes a new two-step stacking model for detecting four arsenic species in coated Capillary Zone Electrophoresis (CZE). The capillary walls were coated with Hypromellose (HDMB), an important step in reversing electroosmotic flow. Zwitterionic surfactant 3- (N, N-dimethyl palmitylamino) propane sulfonate (PAPS) is used as a micelle, mixed with a target analyte and used as a sample solution, and is introduced through negative voltage electrokinetic injection, and then the sample solution is independently injected to promote the formation of MSS boundaries, so that a key step of sample accumulation is realized. Various optimization conditions were studied, including micelle type and concentration, methanol percentage, borax buffer concentration and injection time, to improve stacking efficiency and resolution. The method is used for measuring arsenic in the kelp.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for simultaneously accumulating, enriching and separating various arsenic forms in natural medicines on line based on micelle-solvent accumulation, which is faster, more efficient and more sensitive than the previous enrichment method and is suitable for detecting arsenic in natural medicines with complex matrixes.

The method is realized by adopting the following technical scheme:

step (1), preparation of target analyte sample:

cleaning natural medicine, drying, pulverizing, sieving, and mixing with HNO₃Mixing, ultrasonic treating at 40-45 deg.C for 0.5-1h, shaking at room temperature for 1.5-2h, centrifuging at high speed for 10-15min, and collecting supernatant. Regulating the supernatant to be neutral by NaOH to obtain a target analyte sample;

preferably, the natural drug refers to kelp;

preferably, the natural drug is mixed with HNO₃The mass-to-volume ratio of (A) is 200 mg: 5 mL;

preferably, HNO₃Is 2% (w/v, mg/mL);

preferably, the rotation speed of the centrifugation is 4000-;

step (2), enrichment and separation of target analyte samples:

2-1, activated capillary column

Fused silica capillaries were coated with 1% (w/v, g/mL) of Hypromellose (HDMB) for 20-25 minutes to meet the same EOF orientation condition between the anion and capillary.

2-2, washing with borax buffer solution for 5-10 minutes before feeding sample to the activated capillary column.

Preferably, the concentration of the borax buffer solution is 25-100mM, and is preferably 100 mM.

Preferably, the pH value of the borax buffer solution is 8.8-9.4, and more preferably 9.2.

2-3, in the capillary column, mixing the micelle and the target analyte to be used as a sample solution, injecting the sample solution into the stack for 30-240s under negative pressure, then injecting a methanol aqueous solution used as an organic solvent under the negative pressure, and bringing scanned analyte to MSS boundary by the micelle to realize sample accumulation and separation; wherein the micelle adopts 3- (N, N-dimethyl palmityl amino) propane sulfonate PAPS.

Preferably, the conditions for capillary electrophoresis are as follows: the detection wavelength is 192nm, the separation negative pressure is-20 kV, and the temperature is 20 ℃.

Preferably, the micelle concentration is 10-40mM, more preferably 20 mM.

Preferably, the injection time of the micelle is 180 s.

Preferably, the negative pressure for injecting the micelle is-10 kV.

Preferably, the injection parameters of the methanol water solution are-10 kV of injection power, 50mbar of injection pressure and 5s of injection time.

Preferably, the methanol aqueous solution has a volume content of 40-100% (v/v), preferably 60% (v/v).

The enrichment factors for the various arsenic forms are [ As (V), As (III), MMA and DMA ]1230, 840, 3820 and 1450, respectively. The detection limit (S/N ═ 3) ranged from 0.382 to 0.911 ng/mL. The capillary repeatability (% RSD, N is 3) migration time is 0.5-1.0%, and the peak area is 0.3-0.9%.

The invention has the advantages that:

(1) the invention provides an electric sample injection assisting MSS in a coating CE.

(2) The invention provides a zwitterionic surfactant 3- (N, N-dimethyl palmitylamino) propane sulfonate PAPS as a micelle solution in MSS, and then hydrabamine HDMB is used for reversing EOF.

(3) The present invention proposes enrichment factors for As (V), As (III), MMA and DMA 1230, 840, 3820 and 1450, respectively, compared to typical injections in CZE.

(4) The method utilizes the coating capillary electrophoresis from micelle to solvent accumulation to detect the arsenic in the natural medicine, not only realizes the rapid detection of the arsenic form, but also greatly improves the sensitivity of the online enrichment of the capillary electrophoresis sample. Compared with the traditional arsenic detection method, the method realizes the efficient detection and separation of typical arsenic forms.

Drawings

FIG. 1 is a diagram of the enrichment mechanism of the present invention.

FIG. 2 is the effect of micelle species (PAPS, CTAB, CTAC); wherein the CE condition is: micelle concentration in the sample solution is 10mM, analyte concentration is 10mg/L, and injection is carried out for 60s under 10 kV; MeOH (100%) was injected at 50mbar for 5 s; background solution: 100mM borax buffer.

FIG. 3 is the effect of PAPS concentration (0,5,10,20,30,40 mM); other CE conditions are the same as in fig. 2.

FIG. 4 is the effect of methanol content (40%, 60%, 80%, 100%); CE conditions: as in fig. 2.

FIG. 5 is the effect of borax concentration (25,50,75,100 mM); a: electrophoretogram, 1-As (V), 2-MMA,3-DMA,4-As (III); (B) apparent mobility, CE condition: the PAPS concentration in the sample solution is 20mM, the analyte concentration is 10mg/L, and the sample solution is injected for 60s under 10 kV; MeOH (60%) was injected at 50mbar for 5 s; background solution: 100mM borax buffer.

FIG. 6 is the effect of buffer pH (8.8,9.0,9.2, 9.4); other conditions are the same as in fig. 5.

FIG. 7 is the effect of sample injection time (30,60,120,180,240 s); the analyte concentration was 1mg/L, and the other conditions were the same as in FIG. 6.

FIG. 8A is a labeled electrophoretogram 60s (a) and 5s (b) for analyzing Laminaria japonica under optimal conditions.

FIG. 8B is an electrophoretogram of standard solutions superimposed and typically injected by a two-step process; two-step stacking: a sample solution containing 1mg/L analyte cocktail and 20mM PAPS was injected at-10 kV for 180 s. Methanol (60%) was injected at 50mbar for 5s, typical injection: micelle free target analyte was injected at 50mbar for 3s with a mixed arsenic concentration of 10.0 mg/L.

Detailed Description

As previously mentioned, in view of the prior artThe inventor of the present invention has made a long-term study and a great deal of practice to provide the technical solution of the present invention, which is mainly based on at least: 1) the present invention utilizes the zwitterionic surfactant 3- (N, N-dimethylpalmitoylamino) propanesulfonate (PAPS) in the co-electroosmotic flow (co-EOF) for four arsenic species [ As (V), As (III), sodium monomethylarsonate (MMA) and sodium Dimethylarsinate (DMA) by electrokinetic injection and micelle-to-solvent stacking (MSS)]An efficient and sensitive two-step stacking Capillary Zone Electrophoresis (CZE) was performed. (2) The capillary walls were coated with Hypromellose (HDMB) to reverse the EOF to meet the condition of the same EOF direction between the anion and the capillary. (3) In the invention, a zwitterionic surfactant 3- (N, N-dimethyl Palmityl Amino) Propane Sulfonate (PAPS) is used as a micelle, mixed with a target analyte and used as a sample solution, and is electrically injected by negative voltage, and then the sample solution is separately injected to form an MSS boundary so as to realize sample accumulation. When a negative separation voltage is applied, all compounds migrate towards the anode under strong reverse EOF. The presence of the organic solvent reduces the retention factor (κ). When the kappa is reduced to the effective electrophoretic mobility of the analyte [ mu ]_ep(a′)]Effective electrophoretic mobility of anionic analytes [ mu ] when inverted and transferred directly to the anode_ep ^*(a′)]This can be obtained from equation (1):

in the formula, mu_ep(a') is the electrophoretic mobility of the analyte,. mu._ep(mc) is the electrophoretic mobility of the micelle. When micelle collapse causes μ_ep ^*When κ of (a') is 0,. mu._ep(a') is directed to the cathode where the analyte accumulates. Thus, equation (1) can be simplified as:

finally, all surfactants passed through the MSSB and stacked analytes were separated by CZE under continuous electric field. Since the four As species have different migration rates in electrophoresis, they are sequentially detected and analyzed by an ultraviolet detector.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the respective embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

FIG. 1 is an enrichment mechanism diagram of the present invention, a method for simultaneously stacking, enriching and separating a plurality of arsenic forms in a natural drug on line based on micelle-solvent stacking, comprising the following steps:

step (1), preparation of target analyte sample:

cleaning natural medicine, drying, pulverizing, sieving, and mixing with HNO₃Mixing, ultrasonic treating at 40-45 deg.C for 0.5-1 hr, shaking at room temperature for 1.5-2 hr, centrifuging at high speed for 10-15min, and collecting supernatant. Regulating the supernatant to be neutral by NaOH to obtain a target analyte sample;

preferably, the natural drug refers to kelp;

preferably, the natural drug is mixed with HNO₃The mass to volume ratio of (2) is 200 mg: 5 mL;

preferably, HNO₃Is 2% (w/v);

preferably, the rotation speed of the centrifugation is 4000-5000 r/min;

step (2), enriching the target analyte sample:

2-1, activating capillary column

Preferably, the concentration of the borax buffer solution is 25-100mM, and preferably 100 mM.

Preferably, the micelle concentration is 10-40mM, more preferably 20 mM.

Preferably, the injection time of the micelle is 180 s.

Preferably, the negative injection pressure of the micelle is-10 kV.

The technical solutions of the present invention are further explained below with reference to some preferred embodiments, but the experimental conditions and the setting parameters should not be construed as limitations of the basic technical solutions of the present invention. And the scope of the present invention is not limited to the following examples.

The CE conditions were:

detection wavelength: 192 nm. Column temperature: at 20 deg.c.

A capillary column: an inner diameter of 50 μm, an outer diameter of 375 μm, a length of 60cm and an effective length of 51.5 cm. Before the first use, the new capillary column was rinsed with 1.0M NaOH solution for 20 min, 0.1M NaOH solution for 15min, pure water for 10min, 1% HDMB coating for 30min, running buffer for 10 min. To achieve good reproducibility, the space between the two needles was rinsed with 0.1M NaOH solution for 2 minutes, purified water for 2 minutes, and running buffer for 5 minutes.

Separation voltage: -20 kV.

And (3) data recording: HP chemical workstation (Agilent).

Example 1 examination of the Effect of micelle type on detection

Cleaning and drying kelp, pulverizing, sieving with 100 mesh sieve, weighing 200mg sample, placing into 15mL centrifuge tube, adding 5mL 2% HNO₃Ultrasonic treating for 1h (40 deg.C), shaking for 2h at room temperature, and centrifuging at 4000r/min for 15 min. The extract was neutralized with NaOH prior to analysis.

Fused silica capillaries were coated with 1% Hypromellose (HDMB) for 20 minutes to satisfy the condition of the same EOF direction between the anion and the capillary. The activated capillary column was washed with 100mM borax buffer at pH 9.2 for 5 minutes before injection. In a capillary column, 10mM micelles and 10mg/L target analytes are mixed and then used as sample solution to be injected into a stack for 60s under-10 kV, then 100% MeOH5s is injected under-10 kV at 50mbar, and the scanned analytes are brought to the MSS boundary by the micelles to realize sample accumulation and separation; wherein the micelle adopts 3- (N, N-dimethyl palmityl amino) propane sulfonate PAPS. Capillary electrophoresis conditions: the detection wavelength is 192nm, the separation negative pressure is-20 kV, and the temperature is 20 ℃.

Cationic surfactants are key factors in the formation of MSS, and significantly affect the efficiency of enrichment and separation. This example employed three surfactant solutions, PAPS, CTAB, or CTAC, as micelles to evaluate the effect of micelles on the stacking efficiency of four target analytes.

As shown in fig. 2, CTAB (b) use can achieve longer migration time and higher sensitivity than use of CTAC (c), whose baseline is less uniform than CTAB. Meanwhile, when CTAB and CTAC act as micelles, the As (V) peak is not significant. Clearly, the detection sensitivity is highest for the four analytes and the resolution is highest for satisfactory detection when paps (a) is applied. This is probably due to the fact that PAPS is a quaternary ammonium amphoteric surfactant containing quaternary ammonium cations and sulfonic acid anions. Because CMC (0.79X 10-4M) is lowest, zwitterionic surfactants can reverse EOF, reducing adsorption of analytes and coating walls. It has good surface properties and has a significant synergistic effect with the analyte. Thus, PAPS was chosen as the micelle for subsequent work.

Example 2 examination of the Effect of PAPS concentration on the assay

In addition to example 1, PAPS was selected as micelles, and the PAPS concentration was changed to 0,5,10,20,30, and 40mM, while the other conditions were not changed.

The concentration of micelles is a key parameter that affects the formation of micelles to the solvent packing boundary (MSSB). Therefore, different concentrations of PAPS in the sample solution were analyzed, and the results are shown in FIG. 3. The peak areas of As (V), MMA and DMA gradually increased when the surfactant concentration was varied from 0 to 20mM, while the peak area of As (III) sharply decreased when the micelle concentration was 5 to 10 mM. While the PAPS concentration was further increased from 20mM to 40mM, the peak heights were slightly decreased for all four analytes. It was demonstrated that an increase in PAPS concentration negatively affected micelle formation during MSS, since low concentrations of organic solvents did not meet the conditions to reverse μ ep (a') for high κ analytes. If its concentration is too high, the affinity between the zwitterionic surfactant and the analyte is too strong to reverse μ ep (a') at MSSB, resulting in MSS failure. As a result, 20mM PAPS was selected in the sample matrix.

Example 3 examination of the influence of methanol content on the test results

On the basis of example 1, PAPS was selected as micelles, and the MeOH concentration was changed to 40, 60, 80, 100% (v/v), with the other conditions being unchanged.

Methanol is typically an organic solvent in MSS, which affects the interaction between the analyte and the micelles, reversing the effective electrophoretic mobility to achieve enrichment. This example combines two separate injections of MeOH, optimizing the percentage of MeOH in the range of 40-100% (v/v) for optimal resolution and enrichment of analytes under other conditions.

The results are shown in FIG. 4. In addition to As (III), the peak areas of the other three compounds were each smaller than the peak area when 60% was used at a methanol concentration of 40% (v/v). However, the peak intensities of the three compounds [ As (V), MMA, DMA ] decreased when the MeOH content was 60-100% (v/v), and the migration time of the analyte increased with the percentage MeOH. The reason for this may be that the analyte has a weaker affinity for the micelle, resulting in a lower kappa value, which occurs in the presence of an organic solvent. As the percentage of methanol increases, the kappa value decreases due to the decreased affinity of the analyte for the micelles, thereby extending the length of the scan zone. However, the detection sensitivity of As (III) is highest when 80% (v/v) MeOH is used. Therefore, 60% (v/v) MeOH was chosen for further study.

Example 4 examination of the Effect of Borax concentration on the test results

On the basis of example 1, PAPS was selected as micelles, and the concentration of borax buffer was changed to 25,50,75,100mM, and the other conditions were unchanged.

In the BGS, borax buffer was used as the electrolyte, and this example explores that the concentration of the electrolyte in the BGS has a significant effect on both migration time and peak intensity.

The electropherogram of FIG. 5A shows that from 25 to 100mM, the apparent mobility (. mu.app) decreases, inversely proportional to the migration time, with the results shown in FIG. 5B. It is noteworthy that the increase in solution current is due to the increase in concentration. It was demonstrated that EOF was affected by the concentration of borax buffer, which decreased with increasing concentration of borax buffer in BGS. In addition, the increased current generates more joule heating, which results in an increase in baseline noise (16-65 μ A). In general, 100mM borax buffer is preferred in the present invention.

Example 5 examination of the Effect of buffer pH on assay

On the basis of example 1, PAPS is selected as micelle, and the pH of borax buffer is changed to 8.8,9.0,9.2 and 9.4, and other conditions are not changed.

The migration of arsenic compounds in CE is also influenced by their pKa values, so it is necessary to study the effect of BGS pH on arsenic species analysis. Arsenic species in the present invention are polygenic acids whose apparent charge is related to their own pKa and the pH of the BGS. BGS pH was 8.8-9.4, as pre-experimental studies show that two-step stacking at alkaline pH is more effective in analyzing BGS. In addition, the alkaline conditions ensure that all As migrates to the anode in the form of anions.

The results showed that the apparent mobility reached a maximum at a pH of 9.0 (fig. 6). When the pH was raised from 9.0 to 9.4, the apparent mobility began to decrease and the migration time was extended. In the co-current mode, As the surface of fused silica is modified by HDMB, the As species migrate in the same direction As EOF. Therefore, the separation speed of the analyte is faster. Meanwhile, when the pH of BGS is 9.2, the degree of separation between the four analytes is good, and the maximum peak is obtained at pH 9.2. To obtain higher sensitivity, when the apparent mobility is lower than 9.0, pH 9.2 is preferably selected as BGS.

Example 6 examination of the Effect of sample injection time on test results

On the basis of example 1, PAPS was selected as micelles, and the injection time of the sample solution was changed to 30,60,120,180, and 240s, and the other conditions were not changed.

Injection time affects the detection sensitivity of analytes in MSS, satisfactory separation efficiency and high stacking performance by directly changing the injection volume. The injection time of 60% methanol was kept at 5s (50mbar) and the injection time of the sample solution was varied from 30 to 240 s.

As can be seen from fig. 7, it can be seen from the histogram that when the injection time is 30 to 180s, by enlarging the injection volume, the peak area of the target analyte is increased, and satisfactory stacking efficiency is obtained. However, with increasing injection time, the peak shape becomes broader, the baseline is disturbed, and the analysis time is relatively longer. At 240s injection, the peak area was not further enlarged compared to 180s, and a minimum resolution of four analyte peaks (1.17) occurred, resulting in catastrophic separation of the analytes. Based on the above results, 180s is preferred as the optimum injection time to obtain satisfactory deposition efficiency and good resolution.

Repeatability survey

Under the optimal experimental conditions described above, the linearity, limits of detection (LODs) and quantification (LOQs) (n ═ 3) of the intravascular and intercapillary reproducibility of the four arsenic species were calculated. The analytical figures for performance are summarized in table 1. Preparing standard sample solutions containing arsenic at concentration levels of 0.05, 0.10, 0.25, 0.50, 1.00mg/L and 20mM PAPS5 to obtainTo the calibration curve. Determination coefficient (R) calculated from the peak areas of As (V), MMA, DMA and As (III)²) 0.9992, 0.9990 and 0.9928 respectively. The migration time and the% reproducibility of the peak area RSD were both below 2.7%. The concentration was 1mg/L of the standard solution, which was used to calculate the% RSD in capillary reproducibility. As (V), MMA, DMA and As (III) have capillary migration time RSD% of 1.0, 0.8, 0.7 and 0.5, respectively. The% intravascular RSD of the target analyte peak area is in the range of 0.3 to 0.7. Standard solutions (10mg/L) were used to obtain% RSD reproducibility between capillaries. The percentage of intercapillary RSD for As (V), MMA, DMA and As (III) migration times was 1.1, 1.6, 2.3 and 2.7, respectively. The peak area of four arsenic species has an inter-capillary RSD% range of 0.5-0.9%. The migration time and the reproducibility RSD% of the peak area were both satisfactory. Peak areas LODs (S/N ═ 3) for As (v), MMA, DMA and As (iii) were 0.382, 0.633, 0.911 and 0.659ng/mL, respectively. LOQ is in the range of 1.27-3.04 ng/mL, and the signal-to-noise ratio is calculated to be 10. As (V), As (III), MMA and DMA were enriched by factors 1230, 840, 3820 and 1450, respectively.

TABLE 1 Linear regression data, LOD and LOQ

Recovery rate experiment

The utility of the developed technology was evaluated by analyzing the added kelp as a standard solution at 1 mg/L. As (III) and As (V) were not detected but contained trace amounts of MMA and DMA in the kelp sample according to the sample detection results. The results of the analysis of the spiked samples are shown in Table 2. The recovery rate of the kelp is 89.3-120.6%. The purpose of three parallel experiments was to obtain reproducibility of the method. The migration time and% RSD of peak area varied from 5.3 to 6.2 and 0.9-4.1, respectively.

TABLE 2 analysis results of spiked samples

Claims

1. A micelle-solvent accumulation based method for simultaneously accumulating, enriching and separating a plurality of arsenic forms in natural medicines on line is characterized by comprising the following steps:

step (1), preparation of target analyte sample:

cleaning natural medicine, drying, pulverizing, sieving, and mixing with HNO₃Mixing, ultrasonic treating at 40-45 deg.C for 0.5-1 hr, shaking at room temperature for 1.5-2 hr, centrifuging at high speed for 10-15min, and collecting supernatant; regulating the supernatant to be neutral by NaOH to obtain a target analyte sample;

step (2), enriching a target analyte sample:

2-1, activating capillary column

Adopting a fused quartz capillary tube with a HMB coating for 20-25 minutes to meet the condition of the same EOF direction between anions and the capillary tube;

2-2, washing the activated capillary column with borax buffer solution for 5-10 minutes before sample injection;

2. The method for simultaneous on-line accumulation, enrichment and separation of multiple forms of arsenic in natural drugs based on micelle-solvent accumulation as claimed in claim 1, wherein the natural drug in step (1) is kelp.

3. The method for simultaneous on-line stacking enrichment and separation of multiple arsenic morphologies in natural drug based on micelle-solvent stacking as claimed in claim 1, wherein the natural drug and HNO in step (1)₃The mass-to-volume ratio of (A) is 200 mg: 5 mL; wherein HNO₃The mass volume concentration of (2%).

4. The method for simultaneously stacking, enriching and separating multiple arsenic forms in a natural drug on line based on micelle-solvent stacking as claimed in claim 1, wherein the borax buffer solution in the step (2-2) has a concentration of 25-100mM and a pH of 8.8-9.4.

5. The method for simultaneously stacking, enriching and separating a plurality of arsenic morphologies in a natural drug on line based on micelle-solvent stacking as claimed in claim 4, wherein the borax buffer solution in the step (2-2) has a concentration of 100mM and a pH of 9.2.

6. The method for simultaneous on-line accumulation, enrichment and separation of multiple forms of arsenic in natural drugs based on micelle-solvent accumulation as claimed in claim 1, characterized in that the capillary electrophoresis conditions in step (2-3) are as follows: the detection wavelength is 192nm, the separation negative pressure is-20 kV, and the temperature is 20 ℃.

7. The method for simultaneous on-line stacking enrichment and separation of multiple arsenic morphologies in natural drugs according to claim 1, wherein the micelle concentration in step (2-3) is 10-40 mM.

8. The method for simultaneously accumulating, enriching and separating a plurality of arsenic morphologies in a natural drug on line based on micelle-solvent accumulation as claimed in claim 7, wherein the micelle concentration in the step (2-3) is 20mM, the injection time is 180s, and the injection negative pressure is-10 kV.

9. The method for simultaneous on-line accumulation, enrichment and separation of multiple forms of arsenic in natural drugs based on micelle-solvent accumulation as in claim 1, wherein the methanol aqueous solution in the step (2-3) has a volume content of 40-100%, and the injection parameters are injection power of-10 kV, injection pressure of 50mbar, and injection time of 5 s.

10. The method for simultaneous on-line stacking enrichment and separation of multiple arsenic forms in natural drugs based on micelle-solvent stacking As claimed in claim 1, wherein the enrichment factors of multiple arsenic forms As (v), As (iii), sodium monomethylarsonate MMA, sodium dimethylarsinate DMA are 1230, 840, 3820 and 1450 respectively.