WO2011031005A2

WO2011031005A2 - Single drop microextraction method combined with capillary electrophoresis-mass spectrometry

Info

Publication number: WO2011031005A2
Application number: PCT/KR2010/004751
Authority: WO
Inventors: Doosoo Chung; Kihwan Choi; Jihye Kim; Zeid A Alothman; Saud I Alresayes; Ahmed Yacine Badjah Hadj Ahmed
Original assignee: SNU R&DB Foundation; Intellectual Property And Tech Licensing Program SA
Current assignee: SNU R&DB Foundation; Intellectual Property And Tech Licensing Program SA
Priority date: 2009-09-08
Filing date: 2010-07-20
Publication date: 2011-03-17
Anticipated expiration: 2012-03-08
Also published as: KR20110026658A; KR101160389B1; WO2011031005A3

Abstract

The present invention relates to a single-drop microextraction (SDME) method combined with capillary electrophoresis (CE)-mass spectrometry (MS) in which the SDME method is combined with CE and MS such that the pretreatment, separation and analysis of a sample can be carried out continuously while the sample can be analyzed with high sensitivity.

Description

SINGLE DROP MICROEXTRACTION METHOD COMBINED WITH CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY

Conventional methods for analyzing biological matrix samples mostly comprise an experimental process consisting of at least two steps, including pretreating the samples and then carrying out the separation and analysis of the samples. In this experimental process consisting of multiple steps, a great deal of time is consumed, contamination may occur during the transfer of the sample, and an operation of diluting the pretreated sample may be carried out before analysis.

Meanwhile, when biological samples and environmental samples are detected with a mass spectrometer, sensitivity is lost due to a matrix effect, and for this reason, the pretreatment of the sample is necessarily required. Methods which are most generally used for the pretreatment of samples include liquid-liquid extraction (LLE) and solid-phase extraction (SPE). The LLE has disadvantages in that it is very time consuming and requires large amounts of a sample and a solvent, and the SPE has the disadvantage of being expensive. Solid-phase microextraction (SPME), a recently developed technique, has advantages in that it can perform pretreatment within a short time and can be carried out without any solvent, whereas it has problems of sample carry-over and weak fiber durability.

To overcome the disadvantages of these sample pretreatment methods, a simple and inexpensive liquid-phase microextraction (LPME) technique was recently introduced. Also, a single-drop microextraction (SDME) technique was introduced by the present inventors. In the SDME technique, a concentration effect can be improved by greatly reducing the volume of an acceptor so as to increase the volume difference between the acceptor and a donor compared to the LPME technique. After the pretreatment of samples, techniques combined with chromatography and mass spectrometry are used, and among them, techniques combined with GC-MS were most frequently reported. In addition, techniques combined with ICP-MS and with matrix-assisted laser desorption ionization (MALDI) were recently introduced.

Meanwhile, the SDMEis a very simple and efficient pretreatment technique, but when it is used in combination with MS, injection into an analytical instrument is carried out using a syringe after extraction. Thus, there is a problem in that the syringe used for injection must be washed clean for reuse. Moreover, in the case of GC-MS, a derivation process is required to reduce the polarity of a sample. In addition, in the case of MALDI-MS, a separate crystallization process is required after extraction, and in the crystallization process, it is required to heat a target plate so as to volatilize an extracted organic solvent for the best crystallization conditions.

An object of the present invention is to introduce a very simple sample pretreatment method which is combined directly with capillary electrophoresis and electrospray ionization mass spectrometry so as to be able to carry out all the pretreatment, separation and analysis of a sample. A method of extracting a single drop formed at the capillary inlet is introduced in the present invention, and in order to combine this extraction method directly with MS, the following two points were developed in the present invention. First, in an extraction process, the volume of solution used to form a drop was replenished with an acceptor by temporarily using an outlet vial. Second, a single drop was formed using vacuum. According to this method, a sample is extracted by 3-phase SDME, and then immediately the extracted sample is separated by capillary electrophoresis (CE) and analyzed by mass spectrometry (MS). Thus, this method has an advantage in that the sample is separated and analyzed immediately after sample pretreatment without a separate process.

To achieve the above object, the present invention provides a single-drop microextraction (SDME) method combined with capillary electrophoresis (CE)-mass spectrometry (MS), the method comprising the steps of: placing an acceptor solution-containing outlet vial at a capillary tip directed toward the mass spectrometer (MS) orifice, and injecting an acceptor solution into the capillary; injecting a small amount of octanol into the capillary, and then immersing the capillary inlet tip in adonor solution (sample solution); drawing the octanol injected into the capillary toward the capillary inlet to form a drop at the capillary tip; extracting the sample from the donor solution into the drop; applying pressure to the acceptor solution in the drop containing the extracted sample to inject the extracted sample into the capillary, immersing the capillary inlet in run buffer, and applying separation voltage to the capillary; and analyzing the extracted sample by CE-MS.

Preferably, an empty space occurring in a column located at the capillary outlet side during formation of the drop is replenished with the acceptor solution. Preferably, the outlet vial located at the MS inlet side (the outlet side with respect to the capillary, but the inlet side with respect to the MS) is removed after applying separation voltage. Preferably, the MS is tandem MS/MS analysis.

According to the method of the present invention, the pretreatment, separation and analysis of a biological or environmental sample can be performed continuously. Also, the method of the present invention has a detection limit of nanomolar level even when the extraction is carried out for a short time, and the method of the present invention is also useful in structural analysis utilizing tandem (MS/MS).

Namely, by overcoming the limitation of SDME that could not be coupled directly with MS, the method of the present invention is very simple because the extraction, separation and analysis of a sample are carried out continuously. Also, it has no problem of sample carryover, and avoids cross-contamination because the extracted sample is not manually transferred. In addition, it eliminates the need to consider the minimum sample volume required for additional analysis after extraction and assists in identifying unknown substances, extracted from samples, by MS/MS structural analysis.

FIG. 1 is a conceptual schematic diagram illustrating the present invention.

FIG. 2 shows CE-MS scans for four drugs.

FIG. 3 shows SDME-CE-MSscans of the present invention for four drugs.

FIG. 4(a) shows the CE/MS analysis resultsobtained without carrying out SDME after adding four samples (phenethylamine, methamphetamine, mephenermine and methoxyphenamine) to urine, and FIG. 4(b)shows the CE/MSanalysis results obtained after carrying out SDME.

SDME is a method in which a single drop of microliter volume is formed at the tip of a microsyringe needle and extracted with an extractant. In the present invention, the SDME method of drawing octanol toward the capillary inlet to form a drop at the capillary inlet tip is combination with CE-MS. In the present invention, by combining SDME directly with CE-ESI-MS/MS, detection sensitivity can be greatly improved even when the extraction is carried out for a short time, and the extraction, separation and analysis of a sample can be performed continuously.

FIG. 1 schematically shows the method of the present invention. A two-layer drop is formed at the separation capillary inlet tip, in which the inner drop is formed of an acidic aqueous solution, and the outer drop surrounding the inner drop is formed as a very thin film using octanol as an organic layer. The two-layer drop is formed in a basic aqueous solution. Weakly basic drug samples become neutral in a basic aqueous solution as the donor phase and are extracted into the organic layer. Then, the drug samples are extracted into the acidic aqueous solutionthat is the inner drop. In order to use the SDMEcombined with CE-MS, pressure for forming a drop is needed and it is required to replenish a solution in a volume corresponding to the volume of solution discharged from the column in order to form the drop. After filling the capillary with the acceptor solution and injecting a small amount of an organic phase (octanol) into the capillary, pressure for drawing the organic phase toward the capillary inlet to form a drop is required. At a general CE-MS interface, no outlet vial is used. For this reason, in the present invention, vacuum in place of backward pressure is applied to the capillary inlet to form a drop. At this time, in the column located at the outlet side of the capillary, an empty space corresponding to the volume of solution which comes out of the column (the capillary column opposite to the capillary inlet) to form a drop. For this reason, the empty space of thecolumn located at the capillary outlet should be filled with solution, such that electrical contact can be maintained. In an experiment conducted in the present invention, sheath liquid was introduced into the column in a volume corresponding tothe volume of solution coming out of the column during drop formation. In the case in which 5% of total volume of the column was comprised of an aqueous acceptor solution (also serving as run buffer) and a sheath liquid containing an organic solvent, when separation voltage was applied, the current value of CE was unstable and ESI spray was also unstable, suggesting that these conditions were not suitable for quantitative analysis. For this reason, in the present invention, SDME was carried out in the CE/MS system by placing an acceptor solution (run buffer)-containing outlet vial directed toward the mass spectrometer (MS) orifice during drop formation, and then removing the outlet vial after drop formation. All the processes of the method of the presentinvention, except for placing the outlet vial before the experiment and removing the outlet vial by applying separation voltage, are automatically carried out in a very simple and reproducible manner. Also, the organic drop is automatically detached in the process replacing the capillary inlet with a run buffer vial, thus eliminating a problem of sample carryover, and can be freshly prepared in a cost-effective manner as required.

Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention. Also, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention. For example, octanol used to form a drop should be understood to be illustrative only.

<Sample preparation>

Phenethlyamine was purchased from Aldrich (Milwaukee, WI, USA). Methoxyphenamine, mephentermine, methaphentamine, 1-octanol, octadecyl trimethoxysilane (ODTS), ammonium formate, sodium hydroxide, and ammonium acetatewere purchased from Sigma (St. Louis, Mo, USA). HPLCgrade 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 a Milli-Q system (Millipore, Bedford, MA, USA). A run buffer was 20 mM ammonium formateadjusted to pH 2.5 with concentrated formic acid. The run buffer solution was degassed by sonication before use.

<Capillary electrophoresis (CE)>

CE was performed using an MDQ CE system with 32 Karat software version 5.0 from Beckman (Fullerton, CA, USA). Capillaries (Postnova, Landsberg am Lech, Germany) were uncoated fused-silica capillaries having a total length of 100 cm, an ID of 50 mm ID and an OD of 280 mm.New capillaries were flushed at 80 psi with 1 M NaOHfor 10 min, water for 10 min and run buffer for 10 min. The temperature of the capillary cartridge was set at 25 ℃.

<Mass spectrometry (MS)>

MS was performed with a triple quadrupole mass spectrometer (Quattro LC, Waters-Micromass, Manchester, UK)and controlled with Masslynx 3.3 software. The system is equipped with a Z-spray nanoflow electrospray source. A cone voltage of 20 V was used in the positive ionization mode. Nitrogen with a flow rate of 30 L/h was used as nebulizer gas. The ionization source block temperature was set at 80 ℃. For MS/MSmeasurement, the argon pressure in the collision cell was kept at 2.0 x 10^-3 mbar. MS spectral date for CE-MS/MS and SDME CE-MS/MS were obtained in the multiple reaction monitoring (MRM) mode.

<CE-MS/MS>

First, a capillary was washed at 80 psi with 0.1 M NaOH, water and run buffer for 3 minutes each. A mixture of 1 mM of drugs in run buffer was injected for 10 sec at 1 psi. Electrophoresis was carried out by applying a pressure of 1 psi and a voltage of +20 kV across the capillary. The ESI voltage was set at +3.3 kV to show a net voltage of 16.7 kV for electrophoresis. Under such conditions, the capillary current was 13.3 mA. A sheath liquid of 1% acetic acid in a mixed solvent of water/isopropyl alcohol/methanol (20:30:50 v/v) was transferred at a rate of 0.8 ㎕/min by a syringe pump.

<SDME CE-MS/MS>

In order to make it easier to form a single drop at the capillary tip, a capillary having an outer diameter (OD) of 280 mm which was larger than 180 mm for existing capillaries was used. To use the capillary having an OD of 280 mm, the existing stuff (M955423BD1, Waters-Micromass, Manchester, UK) of the stainless steel capillary in the Z-sprayer (Waters-Micromass, Manchester, UK) was replaced by a stainless steel tube (HTX-25X-06-10, SmallParts, miramar, FL, USA)having an OD of 515 mm, an ID of 363 mm ID and a wall thickness of 76 mm, the 1 cm portion at both ends of the stainless steel tube was ground with a grinder (a nail decorator, Ningbo Junway Plastic Electric Manufacturer, Ningbo, China) such that the stainless steel tube was fitted into a peak tube.

Before extraction, the capillary inlet was coated in order to facilitate the adhesion of an organic drop to the surface of the capillary inlet. The coating treatment was carried out using hydrophobic ODTS at the first experimental stage once each day. In the process of coating the capillary inlet, the capillary surface was washed in ethanol for about 1-2 minutes, and then the capillary inlet tip was a surface coating solution (5% ODTS and 0.1% acetic acid in ethanol) for about 6 seconds. Then, the coated portion was dried in as an empty vial for 5 minutes.

1.8 ml of a solution of a sample in a donor phase (0.1 M NaOH) was placed in a 2-ml buffer vial, and an acceptor solution was placed in an outlet vial and attached to the sprayer at the MS detector. At this time, the flow of sheath liquid and nebulizer gas was turned off. The extraction process at the stage before CE was carried out in the following order: 1) an acceptor solution (20 mM ammonium formate pH 2.5 also serving as run buffer) was injected into a capillary at 80 psi for 1 min; 2) a small amount of octanol was injected into the capillary at 5 psi for 8 sec; 3) After the capillary inlet tip has been immersed in a donor solution, a two-layer dropwas formed by drawing the octanol injected into the capillary toward the capillary inlet under vacuum at 1.2 psi for 63 sec; 4) the drop was extracted; and 5) after completion of the extraction, the acceptor solution containing the extracted sample was injected into the capillary by applying a pressure of 1 psi for 10 sec, the capillary inlet was immersed in run buffer (it is the same solution as the acceptor solution, but performs a role different from the role of the acceptor solution), and voltage was applied to the capillary. Then, the outlet vial located at the MS inlet side (the outlet side with respect to the capillary, but the inlet side with respect to the MS) was removed, sheath liquid was delivered, the flow of nebulizer gas and collision gas was turned on, and the extracted sample was analyzed by CE-MS/MS. The removal of the outlet vial was performed when the CE current reached 15.4 mA by the application of separation voltage after injecting the extracted sample into the capillary. Then, when a voltage of 3.3 kV was applied for ESI spray, the CE current reached 13.3 mA. Then, sheath liquid was delivered, the flow of nebulizer gas and collision gas turned on, and the extracted sample was analyzed in the MRM mode.

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

To compare the method of the present invention with the prior art method, three kinds of drugs (mephentermine, mthoxyphenamine and phenthylamine) in addition to methaphentamine in human urine were analyzed. First, a blank test was carried out without spiking a drug substance into a urine sample. Herein, a donor was an urine containing only 10 vol% 1 M NaOH. The analysis of the urine sample was carried out in the same manner as described above, except that MS detection was carried out in MS scan mode in a scan range from 20 to 500 m/z. The blank urine was analyzed by SDME and CE/MS and, as a result, it was observed through TLC that an unknown substance of m/z 114 was present in the urine sample. Also, it was observed that the unknown substance was detected at the same migration time even after spiking a drug material sample into a donor. The unknown substance of m/z 114 was fragmented into m/z 44 and m/z 86 (neutral loss, C=O, m/z 28) in the form of [M+H⁺] through MS/MS and found to be a creatinine of C₄H₈N₃O⁺.

Then, to detect the above-described four drug substances, SDME CE/MS/MSwas carried out. Each of the four drug substances was spiked into urine at a concentration of 50 nM, and 1 M NaOHwas added to the urine in order to adjust the donor to basic pH. SDMEwas carried out for 3 min using 20 mM ammonium formate (pH 2.5) as an acceptor phase. In the method of the present invention, a limit of detection (LOD) of nM level is possible, and the analysis results are shown in FIGS. 2 and 3. According to the method of the present invention, sensitivity was improved by 40-150 times compared to the CZE MS/MS when the extraction process was carried out for 3 min. In addition, according to the method of the present invention, unknown components extracted from urine by SDME could be identified using tandem mass spectrometry analysis.

FIG. 4(a) shows the CE/MS analysis results obtained without carrying out SDME after adding four samples (phenethylamine, methamphetamine, mephenermine and methoxyphenamine) to urine, and FIG. 4(b) shows the CE/MS analysis results obtained after carrying out SDME.As can be seen in FIGS. 4(a) and 4(b), when SDME is not carried out, the signals of the drug substances are interfered with by the components contained in urine, whereas, when SDME is carried out, the components interfering with the analysis of the sample are removed, thus making it easy to detect the drug substances.

In short, the present invention has great advantages in that the pretreatment, separation and analysis of biological or environmental samples are achieved in a convenient and fast way because they are continuously carried out and in that the qualitative and quantitative analyses of the samples can be performed with high sensitivity.

Claims

A single-drop microextraction (SDME) method combined with capillary electrophoresis (CE)-mass spectrometry (MS), the method comprising the steps of:

placing an acceptor solution-containing outlet vial at a capillary tip directed toward the mass spectrometer (MS) orifice, and injecting an acceptor solution into the capillary;

injecting a small amount of octanol into the capillary, and then immersing the capillary inlet tip in a donor solution (sample solution);

drawing the octanol injected into the capillary toward the capillary inlet to form a drop at the capillary tip;

extracting the sample from the donor solution into the drop;

applying pressure to the acceptor solutionin the drop containing the extracted sample to inject the extracted sample into the capillary, immersing the capillary inlet in run buffer, and applying separation voltage to the capillary; and

analyzing the extracted sample by CE-MS.
The method of Claim 1, wherein an empty space occurring in a column located at the capillary outlet side during formation of the drop is replenished with the acceptor solution.
The method of Claim 1, wherein the outlet vial located at the MS inlet side is removed after applying separation voltage.
The method of Claim 1, wherein the MS is tandem MS/MS analysis.