KR102201770B1 - Capillary electrophoresis-electrospray ionization-mass spectrometry using single droplet micro-extraction and large-volume stacking method - Google Patents

Abstract

In the present invention, the single droplet micro-extraction method (SDME) and the large-volume stacking method (LVSEP) using an electro-osmotic flow pump are interlocked with CE-ESI-MS (capillary electrophoresis-electrospray ionization-mass spectrometry) to extract organic droplets. It relates to a method of continuously stacking, separating and analyzing samples.
By the application of the method of the present invention, the concentration coefficient of the extracted sample can be significantly improved, the sample can be analyzed with high sensitivity, and a low-concentration soil contaminant sample can be conveniently and usefully analyzed without going through a complicated process.

Description

Capillary electrophoresis-electrospray ionization-mass spectrometry using single droplet micro-extraction and large-volume stacking method

In the present invention, the single droplet micro-extraction method (SDME) and the large-volume stacking method (LVSEP) using an electroosmotic flow pump are interlocked with CE-ESI-MS (capillary electrophoresis-electrospray ionization-mass spectrometry) to extract organic droplets. It relates to a method of continuously stacking, separating and analyzing samples.

The capillary electrophoresis method is a highly charged type of separation method and is very excellent, but many efforts have been made to increase the sensitivity of the very low concentration, and among them, there is a method of stacking samples in a capillary tube. Large-volume stacking using the electroosmotic flow pump (LVSEP) involves injecting a large amount of anion sample dissolved in a solution with low electrical conductivity into a capillary tube and applying a reverse voltage to the matrix of the sample. It is removed toward the sample injection part by electroosmotic flow (EOF), so that the sample is concentrated while having a high degree of separation, so a high concentration effect can be expected. LVSEP is a relatively easy method, and a high concentration effect can be expected. Recently, a method of using it in connection with MS has been reported (Korean Patent Registration No. 10-1144974). In the reported method, a buffer vial containing a run buffer was placed in the capillary outlet and added while it was concentrated, but the degree of stacking efficiency was proportional to the conductivity ratio between the matrix of the sample and the run buffer. , There was a hassle requiring pretreatment of the sample.

Meanwhile, single drop microextraction (SDME) is a method in which a single drop of microliter volume is made at the tip of a microsyringe needle and the droplet is used as an extractant. to be. Based on this basic method of SDME, it is possible to increase the concentration effect by significantly reducing the volume of the acceptor and making the difference in volume with the donor larger. In addition, recently, a method of performing sample pretreatment and separation analysis at once by grafting SDME method to CE and MS methods has been reported (Korean Patent Registration No. 10-1160389).

However, it was observed that the previously reported individual LVSEP and SDME were not basically satisfactory for high-sensitivity analysis of low-concentration pollutant samples contained in soil with a high concentration factor.In addition, for the above analysis, a complex pretreatment was required. In addition, it was necessary to go through a complicated process of separately concentrating the sample material of low concentration. Therefore, there is a need for a method capable of analyzing a low concentration pollutant sample contained in the soil with a high concentration coefficient without going through a complicated process.

In the present invention, in accordance with the above requirements, both the single droplet micro-extraction method (SDME) and the large-volume stacking method (LVSEP) using an electroosmotic flow pump are CE-ESI-MS (capillary electrophoresis-electrospray ionization-mass spectrometer) and It is intended to provide a method of continuously stacking, separating, and analyzing samples extracted as organic droplets by interlocking.

In addition, in the present invention, by linking both SDME and LVSEP to CE-ESI-MS, it is intended to provide a method that can significantly improve the concentration coefficient of the extracted sample and analyze the sample with high sensitivity.

In addition, in the present invention, by linking both SDME and LVSEP to CE-ESI-MS, a method of remarkably improving the concentration coefficient of the extracted low-concentration soil contaminant sample and analyzing the sample with high sensitivity without going through a complicated process. I want to provide.

Hereinafter, the present invention will be described in more detail, and facts that are obvious to those skilled in the art will be briefly mentioned or omitted in order not to obscure the subject matter of the invention. For example, a general ESI-MS mass spectrometer is well known to those of ordinary skill in the art, and detailed structural descriptions thereof are omitted.

For the above object, the present invention

a) filling the capillary tube by injecting a run buffer;

b) placing an outlet storage container containing a run buffer in the capillary outlet on the orifice side of the mass spectrometer (MS) so that the capillary outlet is immersed;

c) injecting pentanol, an organic acceptor, into the capillary tube, and then dipping the inlet portion of the capillary tube into a donor solution (sample solution);

d) pulling pentanol injected into the capillary tube toward the inlet portion of the capillary tube to form an organic droplet at the end of the inlet portion of the capillary tube;

e) extracting a sample from the donor solution into the droplet;

f) injecting the acceptor solution in the droplet in which the sample is concentrated into the capillary tube, and immersing the inlet portion of the capillary tube in the run buffer;

g) stacking the extracted sample by applying a reverse voltage to the inlet portion of the capillary tube, observing the current change in capillary electrophoresis, and removing the outlet storage container when the current change occurs rapidly; And

h) flowing a sheath liquid from a mass spectrometer and applying an electrospray ionization voltage (ESI voltage) to move the stacked concentrated sample to the mass spectrometer;

A single droplet micro-extraction method (SDME) and a large-volume stacking method (LVSEP) using an electroosmotic flow pump, characterized in that it comprises a CE-ESI-MS (capillary electrophoresis-electrospray ionization-mass spectrometer) effectively interlocked. By doing so, it provides a method of continuously stacking, separating, and analyzing samples extracted into organic droplets.

In the method of the present invention, in order to stably maintain the organic droplet formed in step d) at the end of the inlet portion of the capillary tube, the inlet portion of the capillary tube may be coated with a Teflon sleeve, which is a hydrophobic support (Fig. 1). ). At this time, the organic droplet must have a size sufficient to facilitate the subsequent LVSEP application, and in order to stably maintain the size sufficient for the smooth LVSEP application, the inlet portion of the capillary tube is a Teflon sleeve, which is a hydrophobic support. It is preferable that it is coated with a sleeve).

In step b) of the method of the present invention, an outlet storage container containing a buffer solution may be detachably provided between the capillary outlet of the capillary electrophoresis device and the sampler orifice cone of the mass spectrometer. At this time, it is very important to configure the capillary outlet to be immersed in the buffer solution of the outlet storage container to contact it.

In step c) of the method of the present invention, pentanol is used as an organic acceptor and extraction solvent in order to smoothly implement the methodological feature of the linkage between SDME and LVSEP. Meanwhile, in the case of interlocking SDME and LVSEP by injecting octanol, a high-quality alcohol as an organic acceptor and extraction solvent into a capillary tube, octanol is used as a sample matrix when LVSEP is applied due to the high viscosity (9.85 cP, 20°C) of octanol. It is not smoothly removed from the capillary tube, and there is a problem in that the inside of the capillary tube is blocked. In step c) of the method of the present invention, pentanol is preferably injected in a small amount into the capillary tube, and more specifically, it is preferable to inject 5 to 20% of the total volume of the capillary tube (Fig. 2(a)).

In step c) of the method of the present invention, the sample applied to the donor solution may preferably be a soil contaminant. Examples of the contaminants are (4-chloro-2-methylphenoxy)acetic acid ((4-Chloro-2-methylphenoxy)acetic acid; MCPA: p K _a 3.14), 2,4,5-trifluorobenzoic acid (2,4,5-trifluorobenzoic acid: p K _a 2.53), 2,3,4,5-tetrafluorobenzoic acid (2,3,4,5-tetrafluorobenzoic acid: p K _a 2.87), 2-methyl- 4,6-dinitrophenol (2-Methyl-4,6-dinitrophenol; 4,6-DNOC: p K _a 4.70), 2,4-dinitrophenol (2,4-dinitrophenol; 2,4-DNP: p K _a 4.07) or mixtures thereof. The exemplary pollutants are harmful substances that may be included in the soil, and also correspond to useful analytes that can be applied to the method of the present invention. When applying the exemplary contaminants to the donor solution, it is preferred that the donor solution has a pH of 0.5 to 2. The exemplary contaminant is mainly present in a neutral state when the donor solution has a pH range of 0.5 to 2 as long as it exhibits _a p K _a range of 2.53 to 4.70, and thereafter in step e) The exemplary contaminants in the neutral state can be smoothly extracted by pentanol, which is an organic acceptor, and further, low electrical conductivity of the acceptor solution is secured, so that the application of LVSEP is smooth. On the other hand, ionic forms such as salts or basic compounds in the form of some of the exemplary contaminants in the donor solution are difficult to be smoothly extracted by pentanol, an organic acceptor. In addition, as a sample applied to the donor solution, soil contaminants may be included in the donor solution at a low concentration of preferably 70 nM or less, and more preferably at a low concentration of 1 to 70 nM.

In step d) of the method of the present invention, in order to form an organic droplet at the end of the inlet portion of the capillary tube (Fig. 2(b)), a pressure in the reverse direction required to make the droplet may be applied, or a vacuum at the inlet portion of the capillary tube You can also use to make a drop. At this time, there is an empty space in the column on the outlet side as much as the volume of the droplet exiting the column (capillary column on the other side of the capillary inlet) to make a droplet. Therefore, it is necessary to fill the empty space of the column on the outlet side of the capillary tube with a run buffer to maintain electrical contact. In addition, it is necessary to lower the concentration of salt in the formed organic droplets to a level that allows smooth application of the LVSEP afterwards.

In step e) of the method of the present invention, a sample is extracted from the donor solution (sample solution) into pentanol drops for at least 5 minutes (Fig. 2(b)). At this time, in order to maintain the stability of the droplet in the extraction process, it is preferable to apply a predetermined pressure to counteract the surface force of the droplet. More specifically, the pressure condition is for 5 to 20 minutes. It may be 0.5 to 1.0 psi. In addition, by appropriately adjusting the pH of the donor solution (sample solution), it is necessary to ensure that the sample is well extracted into the pentanol drop in a neutralized state.

In step f) of the method of the present invention, the acceptor solution in the droplet in which the sample is concentrated is injected into the capillary tube, and the inlet portion of the capillary tube is immersed in the run buffer. More specifically, the sample extracted by applying hydraulically to the acceptor solution in the droplet in which the sample is concentrated is injected into the capillary tube (Fig. 2(c)), and the hydraulic condition is preferably 2 to 6 psi for 0.5 to 1 minute. Can be At this time, the acceptor solution including the extracted sample is preferably injected in an amount of 5 to 15% of the total volume of the capillary tube. In particular, when the acceptor solution is injected in an amount greater than 15% of the total volume of the capillary tube, the final concentration coefficient of the sample may be lowered, and it may take a lot of time for sample stacking according to the subsequent application of LVSEP. Efficiency can be hindered.

For the extracted sample injected into the capillary tube through step f) (SDME application) of the method of the present invention, LVSEP is applied from step g) (Fig. 2(d)). In the method of the present invention, pentanol acts as an organic acceptor for SDME application, and also acts as a sample matrix for LVSEP application. When applying LVSEP, if a reverse voltage is applied to the inlet of the capillary tube, a large electric field is applied to the extracted sample, and at the same time, the EOF is in the direction toward the inlet of the capillary tube, so the sample matrix escapes from the capillary tube. Accordingly, the extracted sample is stacked in the concentration region between the sample matrix region and the run buffer exiting the capillary tube (Fig. 2(d)).

When applying a reverse voltage in step g) of the method of the present invention, the electroosmotic flow (EOF) is made to be less than the electrophoretic speed of the sample. In the initial application of LVSEP, where a large amount of extracted sample is injected, the EOF is greater than the electrophoretic velocity (EPV) of the sample, but when separation occurs due to the sample matrix escaping, the EOF must be smaller. To this end, the run buffer of the present invention may preferably be in the form of using a non-aqueous solvent capable of significantly lowering EOF as a buffer, and more preferably, the non-aqueous solvent may be methanol. Alternatively, for the purpose of lowering EOF, the run buffer of the present invention may be in a form in which an electroosmotic flow modifier (EOF modifier) is added to a neutral or low pH buffer. Therefore, due to the greatly reduced EOF, the electrophoretic speed of the concentrated extracted sample is separated from each other while overcoming the EOF and proceeding toward the detector. In other words, LVSEP is a method in which a large amount of extracted sample dissolved in a solution with low electrical conductivity is injected into a capillary tube and then reverse voltage is applied, the matrix of the sample is removed toward the sample injection part by EOF and the sample is concentrated while having a high degree of separation. High concentration effect can be expected.

In step g) of the method of the present invention, while the sample extracted by applying a reverse voltage is stacked, pentanol, which is a matrix of the sample, is removed toward the inlet portion of the capillary tube by electroosmotic flow. At this time, even while pentanol is removed toward the inlet portion of the capillary tube by the electroosmotic flow, the run buffer fills the empty space of the column on the capillary outlet side.

In step g) of the method of the present invention, the outlet storage container is removed when the sample is stacked and the current change in capillary electrophoresis is observed. If the current of capillary electrophoresis is observed during LVSEP process, the current changes rapidly when the sample matrix is removed and the run buffer is filled. At this time, the run buffer storage container placed in the capillary outlet is removed. When the current rapidly changes, the degree of rapid increase in the current value is preferably 3.0 μA or more, and more preferably 3.0 to 8.0 μA.

In step h) of the method of the present invention (FIG. 2(e)), the run buffer storage container placed in the capillary outlet has already been removed, and when an electrospray ionization voltage is applied, CE and Since the magnitude of the total voltage across the MS decreases, the current value changes slightly after a few minutes. As a result, the concentrated sample stacked in the previous step is separated by electrophoresis while moving toward the capillary outlet, and can be detected with a mass spectrometer.

In the present invention, by applying a method of linking both SDME and LVSEP to CE-ESI-MS, the concentration coefficient of the extracted sample is significantly improved compared to the case of individual SDME or LVSEP linkage, and the sample can be analyzed with high sensitivity.

In the present invention, by applying a method of linking both SDME and LVSEP to CE-ESI-MS, the concentration coefficient of the extracted low-concentration soil pollutant sample can be significantly improved and the sample can be analyzed with high sensitivity without going through a complicated process.

1 is a schematic diagram of a device feature applied to the method of the present invention.
2 is a conceptual diagram for schematically explaining the method of the present invention.
In Figure 3, (a) shows the mass electropherogram according to the application of the conventional CE-ESI-MS for a sample of 5 μM, (b) is SDME-LVSEP-CE- of the present invention for a sample of 10 nM. It shows the mass electropherogram according to the application of ESI-MS. Sample peak: (1) MCPA; (2) 2,4,5-trifluorobenzoic acid; (3) 2,3,4,5-tetrafluorobenzoic acid; (4) 4,6-DNOC; (5) 2,4-DNP
4 shows a mass electropherogram according to applying SDME-LVSEP-CE-ESI-MS of the present invention to an actual soil sample including a pollutant sample. Immediately before applying SDME-LVSEP-CE-ESI-MS, the spiked concentration of each analyte contained in the sample is 20 μg/L. Sample peak: (1) MCPA; (2) 2,4,5-trifluorobenzoic acid; (3) 2,3,4,5-tetrafluorobenzoic acid; (4) 4,6-DNOC; (5) 2,4-DNP

Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited to the following examples, and can be substituted and equivalent to other examples within the scope not departing from the technical spirit of the present invention. Will be apparent to those of ordinary skill in the art to which the present invention pertains.

<Sample preparation>

2,4,5-trifluorobenzoic acid, 2,3,4,5-tetrafluorobenzoic acid, and 2,4-dinitrophenol were purchased from Aldrich (Milwaukee, WI, USA). (4-chloro-2-methylphenoxy)acetic acid (MCPA) was purchased from TCI (Tokyo, Japan). 2-methyl-4,6-dinitrophenol (4,6-DNOC), ammonium formate, sodium hydroxide, ammonium hydroxide solution, ammonium acetate, hydrochloric acid (hydrochloric acid), HPLC-grade methanol, HPLC-grade pentanol (1-pentanol), and HPLC-grade isopropyl alcohol (HPLC-grade) isopropyl alcohol) was purchased from Sigma (St. Louis, Mo, USA). Deionized water was prepared using a Milli-Q system (Millipore, Bedford, MA, USA).

<CE-ESI-MS>

The basic shape of the capillary is an uncoated fused silica capillary (Postnova, Landsberg am Lech, Germany) with a total length of 100 cm, 50 μm ID, and 280 μm OD, and the capillary is 0.1 M NaOH, water and run buffer at 80 psi. Washed for 10 minutes each. The run buffer is 25 mM ammonium formate adjusted to pH 8.0 with methanol. The capillary cartridge was set at 25°C.

CE was performed using the MDQ CE system of Beckman (Fullerton, CA, USA) with 32 Karat software, and electrophoresis was performed with a reverse voltage of -22 kV. MS was performed with a triple quadrupole mass spectrometer (Quattro LC, Waters-Micromass, Manchester, UK), and was controlled with Masslynx 3.3 software. In MS, a sheath liquid of 2 mM ammonium acetate in isopropyl alcohol/water (80:20 v/v) mixed solvent was flowed at a rate of 1.0 μL/min by a syringe pump. In MS, 20 V (cone voltage of 20 V) was used in negative ionization mode. In MS, the ionization source block temperature was set to 80°C, and nitrogen gas with a flow rate of 20 L/h was used as the nebulizer gas. The ESI voltage was set to -2.5 kV.

Analytical sample is a mixture consisting of MCPA, 2,4,5-trifluorobenzoic acid, 2,3,4,5-tetrafluorobenzoic acid, 4,6-DNOC and 2,4-DNP, 5 μM in run buffer It was injected into the capillary tube under 1 psi pressure for 10 seconds in a dissolved state at a concentration of.

MS spectral data for CE-ESI-MS of the analysis sample were recorded under the MRM (multiple reaction monitoring) mode in the range of m/z 20 to 500 ([Table 1]), and also observed a mass electropherogram (Fig. 3(a )).

<SDME-LVSEP-CE-ESI-MS: Example 1>

The capillary used is a fused silica capillary (Postnova, Landsberg am Lech, Germany) having a total length of 100 cm, 50 μm ID, and 280 μm OD, and the run buffer used is 25 mM ammonium formate ( ammonium formate).

Prior to the full-scale application of SDME, the capillary was washed with 1 M NaOH and water for 10 minutes, respectively, and the inlet portion of the capillary was coated with a hydrophobic support, Teflon sleeve (500 μm OD, Zeus, SC, USA) (FIG. 1 ).

In the full-scale application of SDME, run buffer was injected into a capillary tube coated with a Teflon sleeve to fill the inlet. In addition, an outlet storage container containing a run buffer was placed in the capillary outlet on the orifice side of the mass spectrometer (MS) so that the capillary outlet was immersed.

Next, pentanol, an organic acceptor, is injected into the capillary at 10% (180 nL) of the total volume of the capillary tube (Fig. 2(a)), and then the inlet of the capillary is filled with a donor solution (0.1 M HCl acidic aqueous solution: pH). 1.0). At this time, the donor solution (sample solution) contains an analysis sample, and the analysis sample is a soil contaminant such as MCPA, 2,4,5-trifluorobenzoic acid, 2,3,4,5-tetrafluorobenzoic acid, 4, It was a mixture consisting of 6-DNOC and 2,4-DNP (concentration in donor solution: 10 nM). It was confirmed that at pH 1.0, which is the pH condition of the donor solution, the contaminants mainly exist in a neutral state.

Next, by applying reverse pressure at 2 psi for 20 minutes, pentanol injected into the capillary was pulled toward the inlet portion of the capillary to form organic droplets at the end of the inlet portion of the capillary. The space was filled with the run buffer of the outlet storage container. An analysis sample was extracted from the donor solution (sample solution) for 10 minutes with pentanol organic droplets formed at the end of the inlet portion of the capillary tube (Fig. 2(b)), and at this time, the surface force of the droplet surface at 0.8 psi for 10 minutes force), thereby maintaining the stability of the drop in the extraction process.

Next, the extracted analytical sample was injected into the capillary by applying hydraulically (hydrodynamically) at 5 psi for 0.9 minutes to the acceptor solution in the pentanol drop in which the analysis sample was concentrated (Fig. 2(c)), at this time, the acceptor solution Was injected into the capillary tube at 10% of the total volume of the capillary tube. Subsequently, the inlet portion of the capillary is immersed in the run buffer, and specifically, the storage container containing the inlet portion of the capillary tube is changed from the donor solution storage container to the run buffer storage container. SDME application up to this point proceeded for 10 minutes.

Next, the application of LVSEP was started by applying a reverse voltage of -22 kV to the inlet portion of the capillary and stacking the extracted analysis sample (Fig. 2(d)), and the pentanol of the acceptor solution injected into the capillary tube was LVSEP. It also served as a sample matrix for application. As the EOF is directed toward the inlet part of the capillary tube by the application of LVSEP, pentanol, the sample matrix, escapes from the capillary tube, and at this time, the empty space of the column on the capillary outlet side was filled by supplementing the run buffer of the outlet storage container. . The extracted analysis sample is stacked in the concentration area between the sample matrix area exiting the capillary tube and the run buffer (Fig. 2(d)), and the current of capillary electrophoresis is from about 0 μA to 5.5 ± 0.3 μA in 13 ± 2 minutes. When it increased rapidly, the outlet storage container was manually removed, and sample stacking was completed by confirming that the current of capillary electrophoresis changed rapidly as described above. Sample stacking was completed after 15 minutes from the time point when the acceptor solution was injected into the capillary tube.

Next, interworking with CE-ESI-MS was established. CE was performed using the MDQ CE system of Beckman (Fullerton, CA, USA) with 32 Karat software, and the sample separation by electrophoresis was performed by applying a voltage of -22 kV under 0.7 psi pressure. MS was performed with a triple quadrupole mass spectrometer (Quattro LC, Waters-Micromass, Manchester, UK), and was controlled with Masslynx 3.3 software. In MS, a sheath liquid of 2 mM ammonium acetate in isopropyl alcohol/water (80:20 v/v) mixed solvent was flowed at a rate of 1.0 μL/min by a syringe pump. In MS, 20 V (cone voltage of 20 V) was used in negative ionization mode. In MS, the ionization source block temperature was set to 80°C, and nitrogen gas with a flow rate of 20 L/h was used as the nebulizer gas. The ESI voltage was set to -2.5 kV. With the application of ESI voltage, stacked concentrated samples consisted of MCPA, 2,4,5-trifluorobenzoic acid, 2,3,4,5-tetrafluorobenzoic acid, 4,6-DNOC and 2,4-DNP. Each individual substance in the mixture is dissociated into an anion from which hydrogen ions are released. Therefore, the mixture sample composed of the individual substances dissociated into anions is separated by electrophoresis while moving toward the capillary outlet, so that it can be detected with a mass spectrometer (Fig. 2(e)), and accordingly, the mass electropherogram Was observed (Fig. 3(b)).

Meanwhile, in the mass electropherogram (FIG. 3(b)) for the SDME-LVSEP-CE-ESI-MS of the present invention according to FIG. 3(b), the migration time of the peak for each analyte is CE- It is observed that it is about 15 minutes longer than in the case of ESI-MS (Fig. 3(a)). In the case of the present invention, it is considered that the time difference is caused by the time required for sample stacking according to the application of LVSEP of 15 minutes.

<Comparison of enrichment factor (EF) of samples according to SDME-LVSEP-CE-ESI-MS, LVSEP-CE-ESI-MS and SDME-CE-ESI-MS>

Example 1 (SDME-LVSEP-CE-ESI-MS) : As described in detail above, according to the application of the SDME-LVSEP-CE-ESI-MS of the present invention, the analysis sample was MCPA, 2,4,5-tree It is a mixture consisting of fluorobenzoic acid, 2,3,4,5-tetrafluorobenzoic acid, 4,6-DNOC and 2,4-DNP. Analytical samples were dissolved in the donor solution at a concentration of 10 nM.

Comparative Example 1 (LVSEP-CE-ESI-MS) : Basically the same method as in Example 1 was applied, except that SDME was not applied and the concentration of the assay sample was different. The analysis sample was the same material as in Example 1. Run buffer is also 25 mM ammonium formate adjusted to pH 8.0 with methanol in the same manner as in Example 1. The analysis sample was dissolved in pentanol at a concentration of 200 nM, and accordingly, a pentanol solution including the analysis sample was injected into the capillary tube at 10% of the total volume of the capillary tube. Next, the samples were stacked by applying a reverse voltage of -22 kV in the same manner as in Example 1, and after completion of the stacking, the same CE-ESI-MS operating conditions as in Example 1 were applied.

Comparative Example 2 (SDME-CE-ESI-MS) : Basically the same method as in Example 1 was applied, except that LVSEP was not applied and the concentration of the assay sample was different. The analysis sample was the same material as in Example 1. Run buffer is also 25 mM ammonium formate adjusted to pH 8.0 with methanol in the same manner as in Example 1. The analysis sample was dissolved in a donor solution (0.1 M HCl acidic aqueous solution) at a concentration of 100 nM, and pentanol was used as in Example 1 as an organic acceptor to extract the analysis sample. Next, in the same manner as in Example 1, an analysis sample was extracted from the donor solution with pentanol organic droplets formed at the end of the inlet portion of the capillary tube for 10 minutes, and the CE-ESI-MS operating conditions after injecting the extracted sample into the capillary tube were It was the same as in Example 1.

In contrast to the application of the conventional CE-ESI-MS, the concentration coefficient of the sample according to the application of each method of Example 1 and Comparative Examples is shown in the following [Table 2] (comparison of concentration coefficients ( n = 4)). Described.

First, according to the description of [Table 2], when the method of Example 1 of the present invention is applied, a concentration factor of several hundred to thousand-fold is confirmed. However, when the method of the comparative examples is applied, a concentration factor of scores-fold is only confirmed. Therefore, as in the comparison of [Table 2], it was confirmed that the concentration coefficient value for each individual substance constituting the analysis sample in Example 1 of the present invention was improved by 10 times or more compared to the case of the comparative examples. . In this case, considering that the concentration of the assay sample in Example 1 of the present invention is 10 nM, which is a low concentration, the improved concentration factor as described above further supports the remarkability of the present invention. That is, when applying the method of the present invention in which both SDME and LVSEP are interlocked, a synergy effect due to double concentration is confirmed. In particular, as long as the analysis sample is a contaminant that may be included in the soil, a low-concentration soil contaminant sample can be analyzed with high sensitivity according to a high concentration factor by the method of the present invention.

<Applying SDME-LVSEP-CE-ESI-MS of the present invention to an actual soil sample containing a pollutant sample: Example 2>

Analytical samples contained in 0.5 g of soil sample consisted of MCPA, 2,4,5-trifluorobenzoic acid, 2,3,4,5-tetrafluorobenzoic acid, 4,6-DNOC and 2,4-DNP. It is a mixture. The soil sample including the analysis sample was spiked with a standard solution and then completely dried at room temperature overnight. Next, 10 mL of water was added to the soil sample, and the resulting mixture was sonicated for 30 minutes. 1 mL of the upper solution according to the sonication was diluted with 0.2 M HCl and adjusted to 2 mL, and the resulting spiked soil sample (concentration level: 50 nM) was directly added to SDME-LVSEP-CE- ESI-MS was applied (just before SDME-LVSEP-CE-ESI-MS was applied, the spiked concentration of each analyte contained in the sample was 20 μg/L). In the application of SDME-LVSEP-CE-ESI-MS, the sample stacking time according to LVSEP was 20 minutes, and accordingly, the migration time of the peak for each analyte in the mass electropherogram (FIG. 4) was It was observed that it was about 20 minutes longer than in the case of CE-ESI-MS (Fig. 3(a)). In Example 2, except for the donor solution and sample stacking time, basically the same method as in Example 1 was applied, and as confirmed by mass electropherogram (FIG. 4), the analysis sample of low concentration contained in the actual soil sample High sensitivity analysis was also possible.

Claims (11)

a) filling the capillary tube by injecting a run buffer;
b) placing an outlet storage container containing a run buffer in the capillary outlet on the orifice side of the mass spectrometer (MS) so that the capillary outlet is immersed;
c) injecting pentanol, an organic acceptor, into the capillary at 5 to 20% of the total volume of the capillary tube, and then dipping the inlet portion of the capillary tube into a donor solution (sample solution);
d) pulling pentanol injected into the capillary tube toward the inlet portion of the capillary tube to form an organic droplet at the end of the inlet portion of the capillary tube;
e) extracting a sample from the donor solution into the droplet for at least 5 minutes;
f) injecting the acceptor solution in the drop in which the sample is concentrated into the capillary tube at 5 to 15% of the total volume of the capillary tube, and immersing the inlet portion of the capillary tube in the run buffer;
g) stacking the extracted sample by applying a reverse voltage to the inlet portion of the capillary tube, observing the current change in capillary electrophoresis, and removing the outlet storage container when the current change occurs rapidly; And
h) flowing a sheath liquid from a mass spectrometer and applying an electrospray ionization voltage (ESI voltage) to move the stacked concentrated sample to the mass spectrometer;
It characterized in that it comprises a,
In step c), the sample applied to the donor solution is a soil contaminant, the donor solution has a pH of 0.5 to 2, and the soil contaminant is contained in the donor solution at a concentration of 1 to 70 nM,
In the extraction process of step e), a pressure condition of 0.5 to 1.0 psi is applied for 5 to 20 minutes to counteract the surface force of the droplet surface,
In the step f), the extracted sample is injected into the capillary tube by applying a hydraulic pressure of 2 to 6 psi for 0.5 to 1 minute to the acceptor solution in the drop in which the sample is concentrated,
The run buffer using methanol as a buffer contains ammonium formate,
In the step g), when the current rapidly changes, the degree of rapid increase in the current value is 3.0 to 8.0 μA
Stacking of samples extracted as organic droplets by interlocking single droplet micro-extraction (SDME) and large-volume stacking method (LVSEP) using an electroosmotic flow pump with CE-ESI-MS (capillary electrophoresis-electrospray ionization-mass spectrometry) , How to separate and analyze continuously. The method of claim 1,
The method, characterized in that the capillary tube has an outer diameter (OD) of 280 μm, and the inlet portion of the capillary tube is coated with a Teflon sleeve, which is a hydrophobic support.
delete delete delete The method of claim 1,
The method in step g), wherein the electroosmotic flow (EOF) is less than the electrophoretic rate of the sample. delete delete delete The method of claim 1,
In step g), while the sample is stacked by applying a reverse voltage, pentanol, which is a matrix of the sample, is removed toward the inlet portion of the capillary tube by electroosmotic flow. The method of claim 10,
While the pentanol is removed toward the inlet portion of the capillary tube by electroosmotic flow, the run buffer supplements the empty space of the column on the capillary outlet side.

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