KR101160389B1 - The coupling system of single drop microextraction with capillary electrophoresis-mass spectrometry - Google Patents

Abstract

The present invention relates to a SDME-CE-MS binding system that can be analyzed at high sensitivity while performing the pretreatment and separation analysis of the sample by combining the SDME method with CE and MS methods. The present invention for this purpose, the outlet vials containing the acceptor solution at the end of the capillary toward the MS (mass spectrometer) orifice (orifice), injecting and filling the acceptor solution in the capillary; Injecting a small amount of octanol into the capillary, and immersing the tip of the capillary in a donor solution (sample solution); Drawing octanol injected into the capillary toward the capillary inlet to form a drop at the end of the capillary; Extracting a sample from the donor solution into the droplets; Injecting the extracted sample into the capillary by applying pressure to the acceptor solution in the concentrated droplet of the sample, dipping the capillary of the inlet into the run buffer and applying a separation voltage; Analyzing the extracted sample in conjunction with the CE-MS; provides an SDME-CE-MS binding system comprising a.

SDME, CE, ESI, MS

Description

The coupling system of single drop microextraction with capillary electrophoresis-mass spectrometry

The present invention relates to a SDME-CE-MS binding system that can be analyzed at high sensitivity while performing the pretreatment and separation analysis of the sample by combining the SDME method with CE and MS methods.

In sample analysis of biological matrices, most of the conventional methods undergo two or more experiments by performing a separate analysis in the next step after pretreatment of the sample. At this time, it takes a lot of time to go through several stages of the experiment, contamination during the sample transfer or re-dilution to analyze the pre-treated sample.

On the other hand, when detecting biological samples and environmental samples by mass spectrometry, sensitivity is lost due to the matrix effect, and thus pretreatment of the samples is necessary. Sample preparation is the most commonly used method of liquid liquid extraction (LLE) and solid phase extraction (SPE), which requires a long time, requires a lot of samples and solvents, and SPE is expensive. It has the disadvantage of being expensive. Solid phase microextraction (SPME) is a recent advance in technology that allows for rapid pretreatment and solvent-free use, while sample carry-over and fiber ) Has a weak durability.

In order to overcome the drawbacks of this sample preparation method, a simple and inexpensive liquid phase microextraction (LPME) was introduced, and compared to this method, the volume of the acceptor is significantly reduced by reducing the volume of the acceptor. A single drop microextractio (SDME) method was introduced by our laboratory that could increase the concentration effect by making it larger.

After the pretreatment of the sample, it is used in connection with chromatography and mass spectrometer. The connection with GC-MS is reported the most, and the method with ICP-MS and recently, matrix assisted laser desorption ionization (MALDI) is introduced. It became.

On the other hand, although SDME is a very simple and efficient pretreatment method, when used in connection with MS, the syringe is injected into the analytical device using a syringe after extraction, and thus, when the syringe is reused, the syringe is washed cleanly. In case of GC-MS, derivatization process is required to reduce polarity of sample, and in case of MALDI-MS, separate crystallization process is required after extraction. For best crystallization it is necessary to heat the target plate to evaporate the extracted organic solvent.

In the present invention, a very simple sample preparation method that can be performed at a time by directly connecting capillary electrophoresis and an electrospray ionization mass spectrometer is introduced. The method of extracting single drop from capillary injection was introduced. In order to connect this method directly to MS, the following two items were developed. Firstly, during the extraction process, the outlet vial was temporarily used to fill the amount of acceptor used to make the drops, and secondly, a single drop using vacuum. Formed. After extracting the sample into 3-phase SDME at the capillary inlet by the above method, it is directly injected into the CE and separated, and then detected by MS. have.

The present invention for this purpose, the outlet vials containing the acceptor solution at the end of the capillary toward the MS (mass spectrometer) orifice (orifice), injecting and filling the acceptor solution in the capillary; Injecting a small amount of octanol into the capillary, and immersing the tip of the capillary in a donor solution (sample solution); Drawing octanol injected into the capillary toward the capillary inlet to form a drop at the end of the capillary; Extracting a sample from the donor solution into the droplets; Injecting the extracted sample into a capillary by applying pressure to an acceptor solution in a concentrated droplet of the sample, dipping the capillary of the inlet into a run buffer and applying a separation voltage; Analyzing the extracted sample in conjunction with the CE-MS; provides an SDME-CE-MS binding system comprising a.

And, it is preferable that the acceptor solution compensates for the empty space of the column on the capillary outlet side during the formation of the drop, and the outlet vial on the MS inlet side (outlet side from the capillary position) after applying the separation voltage. For the mass spectrometer, the inlet side is preferably removed, and the MS is preferably a tandem (MS / MS) analysis.

The method of the present invention can perform sample preparation and separation analysis of biological or environmental substances at once. In addition, it has a nanomolar analysis limit through the extraction for a short time, it is also useful for structural analysis using tandem (MS / MS).

In other words, by overcoming the limitations that SDME could not directly combine with MS, it is very simple to perform extraction and separation analysis in one step, without the problem of sample carryover, and to carry the extracted sample directly. It does not transfer, avoiding cross contamination and keeping in mind the minimum sample volume needed for further analysis after extraction, and by mass spectrometry MS / MS structure analysis It is helpful to identify.

SDME creates a single drop of microliter volume at the tip of a microsyringe needle and extracts the droplet using an extractant. In the present invention, a double-drop or single-drop SDME-CE method is used in combination with MS as a method of blowing with pressure from the opposite side of the capillary inlet to the inlet. In the present invention, by directly combining the SDME with CE-ESI-MS / MS, the detection sensitivity can be greatly improved by extracting for a short time, and extraction and separation analysis can be performed at once.

1 shows schematically the method of the present invention. Two layers of droplets are formed at the end of the separation capillary inlet tip, which is made of an acidic aqueous solution, and the envelope surrounding the droplets uses octanol as the organic layer. To make a very thin film. These two layers of droplets are made in a basic aqueous solution. The weak base drug sample is neutralized in a basic aqueous solution, which is a donor phase, and extracted into the organic layer. The drug sample is extracted again with an acidic aqueous solution. In order to bind the SDME to the CE-MS, it is necessary to fill the solution by the pressure to make the drop and the volume of the solution which has been drawn out of the column to make the drop. That is, after filling the capillary with the acceptor solution (acceptor solution), a small amount of organic phase (octanol) is injected into the capillary, and then pressure is blown to the capillary inlet to make an extraction drop. Since a typical CE-MS interface does not use an outlet vial, a drop was made using a vacuum on the capillary inlet side instead of the reverse pressure required to make the drop. At this time, an empty space is formed in the column on the outlet side by the volume of the droplet exiting the column (the capillary column on the opposite side of the capillary inlet) to form the drop. Therefore, the empty space of the column on the outlet side of the capillary tube should be filled to maintain electrical contact. Sheath liquid was first used to suck the sheath liquid into the column by the volume that escaped during droplet formation. A volume equivalent to about 5% of the entire column was used with a sheath solution containing an aqueous acceptor solution (sometimes acting as a run buffer) and an organic solvent of another component. At this time, the current value of CE was unstable, and the ESI injection was also unstable, which was not suitable for quantitative analysis. Thus, in the present invention, while forming a drop, an outlet vial containing an acceptor solution (run buffer) is placed at the end of a capillary (outlet-side capillary) toward the orifice of the mass spectrometer (MS), and after the drop is formed, the outlet vial SDME was carried out in a CE / MS system as a method of removing the same.

This method is very simple and reproducible because the entire process is automated except that the outlet vial is removed and removed before the experiment is run. In addition, since the organic droplets fall off automatically during the process of changing the capillary injector into a run buffer vial after extraction, there is no problem of sample carryover, and there is an advantage in that it can be made and used all the time without any cost burden.

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 may be changed to other embodiments equivalent to substitutions and equivalents without departing from the technical spirit of the present invention. Will be apparent to those of ordinary skill in the art. For example, it is to be understood that the octanol used to form the drop is illustrative.

<Sample preparation>

Phenethlyamine was purchased from Aldrich (Milwaukee, WI, USA). Methoxyphenamine, mephentermine, methaphentamine, 1-octanol, octadecyl trimethoxysilane (ODTS), ammonium formate, sodium hydroxide, and ammonium acetate are Sigma ( St. Louis, Mo, USA). HPLC grade methanol and isopropyl alcohol were purchased from Mallinckrodt Baker (Paris, Kentucky, USA) and glacial acetic acid from MERCK (Darmstadt, Germany). Deionized water was prepared using the Milli-Q system (Millipore, Bedford, Mass., USA). The run buffer is 20 mM ammonium formate adjusted to pH 2.5 with concentrated formic acid. The run buffer solution was sonicated to degas before use.

CE; Capillary electrophoresis

CE was performed using Beckman (Fullerton, CA, USA) 's MDQ CE system with 32 Karat 5.0 software. The capillary (Postnova, Landsberg am Lech, Germany ) is a non-coated fused-silica capillaries having a total length of 100 cm, 50 m m ID, 280 m and m OD. The new capillary was flushed the column at 80 psi with 1 M NaOH for 10 minutes, water for 10 minutes, and run buffer for 10 minutes. The capillary cartridge was set at 25 ° C.

<MS; Mass spectrometry>

MS was performed with a triple quadrupole mass spectrometer (Quattro LC, Waters-Micromass, Manchester, UK) and controlled with Masslynx 3.3 software. The device has a Z-spray nanoflow electrospray source. Cone voltage of 20 V was used in positive ionization mode. Nitrogen gas with a flow rate of 30 L / h was used as a nebulizer gas. The ionization source block temperature was set to 80 ° C. For MS / MS measurements, the argon pressure in the collision cell was maintained at 2.0 * 10 ⁻³ mbar. MS spectral data for CE-MS / MS and SDME CE-MS / MS were obtained in multiple reaction monitoring (MRM) mode.

<CE-MS / MS>

First, the capillaries were washed at 80 psi with 0.1 M NaOH, water and run buffer, respectively, for 3 minutes. A mixture of 1 m M of chemicals dissolved in a run buffer is injected by the pressure (10 s at 1 psi). Electrophoresis was performed at a pressure of 1 psi and a voltage of +20 kV across the capillary. The ESI voltage is set at +3.3 kV, representing a net voltage of 16.7 kV for electrophoresis. Under these conditions, the capillary current was 13.3 m A. A sheath liquid of 1% acetic acid in a water / isopropyl alcohol / methanol (20:30:50 v / v) mixed solvent was transferred at a rate of 0.8 μl / min by syringe pump.

< SDME CE-MS / MS>

A single drop (single drop) of the capillary OD (outer diameter) to make it easier for the capillary front end was used for a 280 m m m m OD OD than the conventional 180. Inner diameter in existing steel (M955423BD1, Waters-Micromass, Manchester, UK) of stainless steel capillary inside Z-sprayer (Waters-Micromass, Manchester, UK) to utilize 280 OD capillary Replaced with this large 515 mm OD, 363 mm ID, and 76 mm wall stainless steel tube (HTX-25X-06-10, SmallParts, miramar, FL, USA), 1 cm on both ends of the stainless steel tube. The grinder (nail decorator, Ningbo Junway Plastic Electric Manufacturer, Ningbo, China) was used to fit the outer diameter of the length into the peek tube.

Prior to extraction, the capillary inlet was coated in order for the droplets to adhere well to the surface of the capillary inlet. Coating treatment was carried out only once in the first experimental phase every day, using hydrophobic ODTS. The coating process of the capillary inlet is performed by dipping the capillary surface in ethanol for about 1 to 2 minutes to clean the surface, and then the capillary inlet tip is dissolved in surface coating solution (5% ODTS and 0.1% acetic acid in ethanol). After soaking for a second, the coated part is placed in an empty vial and dried for 5 minutes.

The sample was dissolved in the donor phase (0.1 M NaOH) and placed in 1.8 ml in a 2 ml buffer vial, and the acceptor solution was placed in the outlet vial on the MS detector side. Attach to sprayer. At this time, the sheath liquid and the nebulizer gas are turned off. The extraction process in front of the CE is performed in the following order. 1) Fill acceptor solution (20 mM ammonium formate pH 2.5, also acting as run buffer) in capillary by injecting 80 psi for 1 minute. 2) Inject a small amount of octanol into the capillary for 5 psi for 8 seconds. 3) Dip the capillary inlet tip into the donor solution and make a two-layer drop to extract. To make the drop, use a vacuum at 1.2 psi for 63 seconds at the capillary inlet to the capillary tube. The injected octanol is pulled towards the capillary inlet to make a drop. 4) Extract for 3 minutes. 5) After the extraction is completed, the concentrated acceptor solution is injected into the capillary by applying pressure to 1 psi for 10 seconds, and the capillary of the inlet is placed in the same solution as the run buffer (acceptor solution). Because of its role in soaking). After removing the outlet on the mass spectrometer inlet side (outlet side from the capillary point of view and the inlet portion from the mass spectrometer's point of view), flow the sheath solution, turn on the nebulizer gas and the collision gas, and detect the Run MS / MS. When the outlet vial was removed, the extracted sample was injected into the capillary and the separation voltage was applied to remove the outlet when the CE current reached 15.4 m A. Then, when 3.3 kV was added for the ESI injection, the CE current was 13.3 m A, the sheath solution was flowed, and the nebulizer gas and the collision gas were turned on to detect the MRM mode.

<CZE-MS / MS VS SDME CE-MS / MS>

In order to compare the present invention and the conventional method, three kinds of drugs (mephentermine, Methoxyphenamine, Phenethlyamine) in addition to metaphentamine were analyzed in human urine.

First, a blank test was conducted without spiking a drug substance into a urine sample. First, the donor did not add anything else to the urine containing 1 M NaOH in a 10% volume. The rest of the method is the same as the previous method, except that only MS detection was scanned in the range of 20 ~ 500 m / z in MS scan mode. Blank urine (SDME) was performed by CE / MS. As a result, an unknown substance of m / z 114 was observed through TIC. It was also observed that this substance was detected with the same migration time even after spiking a drug sample into the donor. Unknown of m / z 114 was fragmented into m / z 44, m / z 86 (neutral loss, C = O, m / z 28) in the form of [M + H ⁺ ] through MS / MS, and C ₄ It was found to be creatinine of H ₈ N ₃ O ⁺ .

Next, it was applied to SDME CE / MS / MS to detect four drug substances. Four drugs were spiked to 50 nM each in urine, and 1 M NaOH was mixed in urine at 10% by volume to make the donor basic pH. SDME was extracted using 20 mM ammonium formate pH 2.5 as an acceptor phase for 3 minutes. The method of the present invention enables a limit of detection (LOD) in the range of ˜nM and the results are shown in FIGS. 2 and 3. The method of the present invention improved the sensitivity of 40-150 times with an extraction time of 3 minutes compared to the CZE MS / MS method. In addition, using the tandem of the mass spectrometer it was possible to identify the unknown components extracted by the SDME in the urine.

In Figure 4 (a) is the result of CE / MS analysis without SDME after adding four samples phenethylamine, methamphetamine, mephenermine, methoxyphenamine in the urine, (b) is the result of CE / MS analysis after performing SDME to be. Without SDME, the components in the urine obstruct the signal from the sample, while SDME facilitates sample detection by removing components that interfere with the analysis.

In short, the present invention has a great advantage in that the sample preparation and separation analysis of biological or environmental substances can be performed at one time for convenient, fast and high-quality qualitative and quantitative analysis.

1 is a conceptual diagram for explaining the present invention,

2 is a CE-MS scan of the four drugs,

3 is an SDME-CE-MS scan of the present invention for four drugs.

In Figure 4 (a) is the result of CE / MS analysis without SDME after adding four samples phenethylamine, methamphetamine, mephenermine, methoxyphenamine in the urine, (b) is the result of CE / MS analysis after performing SDME to be.

Claims (4)

Placing an outlet vial containing the acceptor solution at the end of the capillary head toward the MS (mass spectrometer) orifice and injecting and filling the acceptor solution into the capillary; Injecting a small amount of octanol into the capillary, and immersing the tip of the capillary in a donor solution (sample solution); Drawing octanol injected into the capillary toward the capillary inlet to form a drop at the end of the capillary; Extracting a sample from the donor solution into the droplets; Injecting the extracted sample into the capillary by applying pressure to the acceptor solution in the concentrated droplet of the sample, dipping the capillary of the inlet into the run buffer and applying a separation voltage; Analyzing the extracted sample in conjunction with the CE-MS; SDME-CE-MS binding system comprising a. The method of claim 1, SDME-CE-MS binding system, characterized in that the acceptor solution supplements the empty space of the column toward the capillary outlet during the formation of the droplet. The method of claim 1, SDME-CE-MS coupling system, characterized in that to remove the outlet vial on the MS inlet side after applying the separation voltage. 4. The method according to any one of claims 1 to 3, The MS is tandem (MS / MS) analysis, characterized in that the SDME-CE-MS binding system.

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