Systems and methods for analyzing analytes extracted from a sample using adsorbent materials - Patents.com

Abstract

To provide systems and methods for analyzing an analyte extracted from a sample using an adsorbent material.SOLUTION: A method of extracting an analyte from a sample is provided, the method comprising: introducing a sample into a capillary; having the sample within the capillary interact with an adsorbent material within the capillary such that at least one analyte is extracted from the sample and to be bound to the adsorbent material; introducing an extraction solvent into the capillary; and having the extraction solvent interact with the adsorbent material in order to elute the at least one analyte from the adsorbent material.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 345,161, filed June 3, 2016, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention relates generally to systems and methods for analyzing analytes extracted from a sample using an adsorbent material. [Background technology]

[0003] background Mass spectrometry (MS) is a powerful tool for the analysis of complex mixtures, providing specific molecular information based on the molecular weight and chemical structure of target compounds. Traditionally, complex biomedical samples are usually separated using chromatographic techniques, such as high-performance liquid chromatography, gas chromatography, and electrophoresis, prior to MS analysis to minimize matrix effects and preconcentrate analytes. With the development of ambient ionization methods, direct MS analysis can now be performed without a separation procedure for rapid and straightforward analysis of complex biomedical samples. Meanwhile, simpler protocols with reduced sample consumption and time are still desired for qualitative and quantitative analysis. Summary of the Invention [Means for solving the problem]

[0004] overview The present invention provides a new approach to liquid-solid-liquid extraction, as well as systems and methods that allow sample preparation and pretreatment coupled with an ionization process. In particular, the present invention allows liquid-solid-liquid extraction to be performed in very small diameter capillaries (e.g., capillaries with an internal diameter as small as 500 μm). The liquid in the capillary may be moved back and forth, allowing for control of the extraction process, i.e., the extraction can be turned on and off. This movement causes an interaction between the sample and the solid phase material, leading to the extraction of one or more target analytes from the sample onto the solid phase material. An extraction solvent is then used to elute the target analytes from the solid phase material. An added benefit of using narrow capillaries is the ability to handle small sample volumes for quantitative analysis.

[0005] Furthermore, the extraction capillary can serve as the ionization probe. In this way, the present invention provides systems and methods that allow target analytes in a sample to be extracted and analyzed by mass spectrometry without separate sample preparation and pretreatment protocols. Rather, the systems and methods of the present invention are configured so that sample preparation and preconcentration occur within the ionization probe. The purified analytes can then be ionized (although not necessarily) directly from the ionization probe where the sample preparation and pretreatment occurred and injected into the mass spectrometer.

[0006] Aspects of the present invention are achieved using a solid-phase extraction material. A solvent and a sample are placed within the ionization probe of the present invention. One or more target analytes from the sample are extracted onto the solid-phase extraction material, while non-target components of the sample (e.g., salts in urine) remain in the sample. The target analytes are then eluted from the solid-phase extraction material using an extraction solvent. Electrodes are then operably connected to the extraction solvent within the body of the ionization probe, and the target analytes are ionized and injected into a mass spectrometer. In this way, sample preparation and pretreatment are combined with the ionization process, and when a voltage is applied to the solvent, the target analytes do not have to compete with non-target components of the sample for charge. Thus, the systems and methods of the present invention effectively suppress the effects of the matrix, allowing for better ionization of target analytes from samples, particularly biological samples such as blood, saliva, urine, or spinal fluid. The systems and methods of the present invention also have the added benefit of pre-concentrating target analytes from a sample in an extraction solvent, thereby avoiding expensive chromatographic equipment and time-consuming separation protocols, and enabling point-of-care sample analysis systems and methods.

[0007] In certain embodiments, the present invention provides a method for extracting an analyte from a sample, comprising: introducing a sample into a capillary; allowing the sample in the capillary to interact with an adsorbent material in the capillary such that at least one analyte is extracted from the sample and bound to the adsorbent material; introducing an extraction solvent into the capillary; and allowing the extraction solvent to interact with the adsorbent material to elute at least one analyte from the adsorbent material. In certain embodiments, the capillary is kept separate (i.e., separated or disconnected) from the solvent flow. Instead, the sample and extraction solvent are equally divided in the capillary to perform the method of the present invention. Depending on the technique used, the extraction solvent may be immiscible or miscible with the sample.

[0008] There are many different ways to have an adsorbent material in a capillary. In one way, the adsorbent material coats a portion of the wall of the capillary. In this way, the capillary is first moved so that the sample interacts with the adsorbent material, and then the extraction solvent is moved so that the sample interacts with the adsorbent material after the sample has interacted with the adsorbent material. In certain embodiments, both the sample and the extraction solvent are present in the capillary and are separated from each other by an air gap. In other embodiments, the sample and the solvent contact each other without mixing with each other, i.e., the sample and the solvent are immiscible with each other.

[0009] In another approach, a probe includes an adsorbent material and is configured to fit within a capillary. The probe is then inserted into the sample in the capillary in such a way that the adsorbent material penetrates the sample. The probe is then moved from the sample into the extraction solvent already present in the capillary in such a way that the adsorbent material penetrates the extraction solvent. Alternatively, the sample is removed from the capillary and the extraction solvent is introduced into the capillary in such a way that the adsorbent material interacts with the extraction solvent.

[0010] The methods of the present invention may further involve analyzing the extracted analytes. In certain embodiments, analyzing involves applying a voltage to an extraction solvent containing at least one analyte in a capillary, causing the analyte to be expelled from the capillary, thereby generating analyte ions, and analyzing the ions (online method). In other embodiments, analyzing involves removing the solvent containing at least one analyte from the capillary and performing an assay to analyze the analyte (offline method).

[0011] The method of the present invention may also involve reacting with a target analyte in a sample. In certain embodiments, the adsorbent material comprises one or more molecules that react with at least one target analyte, and a reaction is carried out between the one or more molecules and the at least one target analyte to produce a reaction product, which can then be analyzed by any of the above-mentioned methods (e.g., online or offline). In other embodiments, the extraction solvent comprises one or more molecules that react with at least one target analyte, and a reaction is carried out between the one or more molecules and the at least one target analyte to produce a reaction product, which can then be analyzed by any of the above-mentioned methods (e.g., online or offline).

[0012] In certain embodiments, the adsorbent material includes an internal standard. The internal standard may be eluted from the adsorbent material by an extraction solvent. The method may further involve analyzing the internal standard and one or more target analytes using any of the above techniques (e.g., online or offline techniques). In such embodiments, the analyzing may be performed in a quantitative manner. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method for extracting an analyte from a sample, comprising: introducing a sample into the capillary; allowing the sample in the capillary to interact with the adsorbent material in the capillary such that at least one analyte is extracted from the sample and bound to the adsorbent material; introducing an extraction solvent into the capillary; interacting the extraction solvent with the adsorbent material to elute the at least one analyte from the adsorbent material; A method comprising: (Item 2) 2. The method of claim 1, wherein the adsorbent material coats a portion of the wall of the capillary. (Item 3) 3. The method of claim 2, wherein the capillary is moved a first time to cause the sample to interact with the adsorbent material, and then a second time to cause the extraction solvent to interact with the adsorbent material after the sample has interacted with the adsorbent material. (Item 4) 4. The method of claim 3, wherein the sample and the extraction solvent are both present in the capillary and separated from each other by an air gap. (Item 5) Item 10. The method of claim 1, wherein a probe comprises the adsorbent material, the probe configured to fit within the capillary. (Item 6) 6. The method of claim 5, wherein the probe is inserted into the sample in the capillary in such a way that the adsorbent material penetrates the sample. (Item 7) Item 8. The method of item 6, wherein the probe is moved from the sample into the extraction solvent already present in the capillary in such a manner that the adsorbent material penetrates the extraction solvent. 7. The method of claim 6, wherein the sample is removed from the capillary and the extraction solvent is introduced into the capillary in a manner that causes the adsorbent material to interact with the extraction solvent. (Item 9) 2. The method of claim 1, further comprising analyzing the extracted analyte. (Item 10) The analyzing step applying a voltage to the extraction solvent containing the at least one analyte in the capillary, such that the analyte is expelled from the capillary, thereby generating ions of the analyte; analyzing the ions; Item 10. The method according to item 9, comprising: (Item 11) The analyzing step removing the solvent containing the at least one analyte from the capillary; conducting an assay to analyze said analyte; Item 10. The method according to item 9, comprising: (Item 12) 2. The method of claim 1, wherein the solvent is immiscible with the sample. (Item 13) Item 10. The method of claim 1, wherein the solvent is miscible with the sample. (Item 14) 2. The method of claim 1, wherein the adsorbent material comprises one or more molecules that react with the at least one target analyte. (Item 15) 15. The method of claim 14, further comprising the step of reacting the one or more molecules with the at least one target analyte to produce a reaction product. (Item 16) Item 16. The method of item 15, further comprising analyzing the reaction product. (Item 17) The analyzing step applying a voltage to the extraction solvent containing the reaction product in the capillary, such that the reaction product is expelled from the capillary, thereby producing ions of the analyte; analyzing the ions; Item 17. The method according to item 16, comprising: (Item 18) The analyzing step removing the extraction solvent containing the reaction product from the capillary; conducting an assay to analyze the reaction product; Item 7. The method according to item 6, comprising: (Item 19) 2. The method of claim 1, wherein the extraction solvent comprises one or more molecules that react with the at least one target analyte. (Item 20) 20. The method of claim 19, further comprising the step of reacting the one or more molecules with the at least one target analyte to produce a reaction product. (Item 21) 21. The method of claim 20, further comprising analyzing the reaction product. (Item 22) The analyzing step applying a voltage to the extraction solvent containing the reaction product in the capillary, such that the reaction product is expelled from the capillary, thereby producing ions of the analyte; analyzing the ions; 22. The method according to item 21, comprising: (Item 23) The analyzing step removing the extraction solvent containing the reaction product from the capillary; conducting an assay to analyze the reaction product; 22. The method according to item 21, comprising: (Item 24) 2. The method of claim 1, wherein the adsorbent material comprises an internal standard. (Item 25) 25. The method of claim 24, wherein the internal standard is eluted from the adsorbent material by the extraction solvent. (Item 26) 26. The method of claim 25, further comprising analyzing the internal standard and the one or more target analytes. (Item 27) 27. The method of claim 26, wherein the analyzing step is performed in a quantitative manner. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows a schematic diagram of coated capillary-based microextraction.

[0014] [Figure 2] Panel A of Figure 2 shows a photograph of a capillary coated with poly(EGDMA-co-AA). Panel B of Figure 2 shows a scanning electron micrograph of the poly(EGDMA-AA) layer.

[0015] [Figure 3] Figure 3 shows the mass spectrum of fatty acids in a healthy human blood sample using polymer-coated capillary extraction.

[0016] [Figure 4] Figure 4 shows the effect of extraction cycles and sample volume on the extraction efficiency of fatty acids in bovine blood samples. Linoleic acid-d11 was used as the internal standard, and 5 μL of ethyl acetate was used as the extraction solvent.

[0017] [Figure 5] FIG. 5 is a schematic diagram of coated polymer-based microextraction with an air plug between the biological sample and the elution solvent.

[0018] [Figure 6] FIG. 6 is a schematic diagram of a coated metal wire for in-capillary extraction.

[0019] [Figure 7] Panels A-B of Figure 7 show spectra of rat spinal cord using polymer-coated capillary extraction: Panel A shows a full scan in negative mode, and Panel B shows a full scan in positive mode.

[0020] [Figure 8] Panel A of Figure 8 is a schematic diagram of the introduction of an internal standard into coated capillary-based microextraction, and Panel B of Figure 8 is a graph showing the ratio of the intensity of fatty acid 18:2 extracted from bovine blood to the intensity of two internal standards introduced by the polymer coating.

[0021] [Figure 9A] Figure 9A shows the MS spectrum of the PB reaction of a mixture of fatty acids 18:1 (9Z) (5.0 μM) and (11Z) (5.0 μM) isomer mixture in the negative ion mode. [Figure 9B] Figure 9B shows the PB-MS / MS spectrum of m / z 339 formed in Figure 9A. Diagnostic ions of two fatty acid 18:1 isomers are shown. [Figure 9C]FIG. 9C is a graph showing the linear relationship established between the diagnostic ion ratio (I11Z / I9Z) and the molar ratio (c11Z / c9Z) of two fatty acid 18:1C=C positional isomers. [Figure 9D] FIG. 9D shows the PB-MS / MS spectrum of fatty acid 18:1 isomers from a human blood sample using polymer-coated capillary extraction.

[0022] [Figure 10] Panel A of Figure 10 shows the composition of fatty acid 18:1 isomers in homogenized solutions of bovine, human, and rat blood and spinal cord. Panel B of Figure 10 shows a comparison of the relative percentage of the ΔC=C positional isomer from fatty acid C18:1 between breast tissues from normal and cancerous mice.

[0023] [Figure 11] FIG. 11 is a photograph showing the various components and their arrangement in a miniature mass spectrometer. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description The present invention generally relates to systems and methods for analyzing analytes extracted from samples using adsorbent materials. Aspects of the present invention describe a capillary sample pretreatment method that integrates capillary modification, sequential in-capillary microextraction, internal standard introduction, spray generation, and optional online chemical reactions in a pulled glass capillary, and is useful for rapid qualitative and quantitative analysis of complex mixtures. In certain embodiments, the inner surface of the capillary is modified with a layer of adsorbent material. Analyte extraction is achieved by molecular exchange between three phases: sample solution, organic solvent for elution, and coated adsorbent. As a plug of sample solution moves through the adsorbent region, the analyte is first adsorbed onto the coated adsorbent. The adsorbed analyte is then eluted into the plug of organic solvent. This process is repeated multiple times to enrich the analyte in the organic solvent (Figure 1). The capillary can be used online or offline and coupled to a nano-ESI-MS. During each extraction cycle, the amount of analyte that can be extracted into the organic solvent (n i ) is determined by equation (1) found below, where C is the analyte concentration in the sample solution before extraction, and V S and V C are the volumes of the sample and coating, respectively, K is the partition coefficient of the analyte between the coating and the sample matrix, and a i is the elution rate of the organic phase that elutes the analyte from the coating).

number

[0025] Other aspects of extraction and analysis of extracted samples are described, for example, in Ouyang et al. (U.S. Patent Application Publication No. 2016 / 0201051), the contents of which are incorporated herein by reference in their entirety.

[0026] The performance of the coated capillary-based microextraction-nanoESI system was first investigated by directly profiling free fatty acids in blood samples. A thin layer of poly(ethylene glycol dimethacrylate-co-acrylamide) (poly(EGDMA-co-AA)) was synthesized on the inner surface of a glass capillary. The thin polymer layer was polyporous, with numerous large, meso, and micropores that facilitated contact between the biological sample and the polymer surface and increased extraction efficiency (Figure 2, panels A-B). Ethyl acetate was used as the organic solvent due to its excellent elution ability for fatty acids from the poly(poly(EGDMA-co-AA)) layer. Extraction was completed within 1-2 min, and the ethyl acetate extract was then injected for nanoESI-MS analysis. Using a small amount of raw human blood sample (approximately 5 μL), several peaks corresponding to free fatty acids could be detected (Figure 3). The reported data identified at least 24 fatty acids. Rat blood and bovine blood were analyzed.

[0027] Although only a small amount of analyte can be adsorbed onto the coating during each extraction cycle, more cycles can be applied to increase extraction efficiency very easily and simply. The effect of extraction cycles was studied using bovine blood spiked with linoleic acid-d11 as an internal standard (Figure 4A). Approximately 50 cycles were required to achieve extraction equilibrium, but the total extraction time could be as short as 1 minute. Typically, the ratio of organic phase to blood sample is 1:1 (5 μL and 5 μL). However, the volume of the blood sample can be increased to several times the volume of the organic phase. The extracted fatty acids steadily increased with increasing blood volume (Figure 4B).

[0028] The blood and organic solvent plugs were connected to each other to simplify the extraction procedure. However, the exchange of analytes between the blood and organic phases may limit the final extraction efficiency of this method because analytes eluted in the organic phase may diffuse back into the blood sample. An air plug of approximately 5 mm can remain between the biological sample and the extraction solvent (Figure 5). In such an embodiment, the biological sample and the extraction solvent do not come into contact with each other during the extraction process, thereby limiting the exchange of analytes between the biological sample and the extraction solvent and resulting in increased extraction efficiency.

[0029] Adsorbents can also be coated onto thin metal wires, such as steel wires. The biological sample and dissolution medium are then injected into the capillary one at a time. Adsorption occurs when the coated steel wire is inserted into the biological sample solution, and desorption occurs immediately when the coated steel wire is inserted into the dissolution medium (Figure 6). Take the example of blood lipid extraction: 5 μL of ethyl acetate and 5 μL of blood are injected into a glass capillary. A polymer-coated steel wire is first inserted into the blood, allowing some lipids to adsorb onto the coating. As the coated wire is continued to move into the ethyl acetate, the lipids adsorbed onto the coating are desorbed into the ethyl acetate. By repeatedly moving the coated steel wire between the blood and ethyl acetate, the lipids are enriched in the ethyl acetate.

[0030] The coated capillary can be applied to liquid samples, including blood samples and tissue homogenization solutions. We investigated the extraction of rat spinal cord using the polymer-coated capillary. First, healthy rat spinal cord was homogenized in PBS solution; the concentration of ATP in rat spinal cord could be as low as 0.05 mg / mL. Next, 5 μL of ethyl acetate and the homogenized rat spinal cord solution were pushed into the poly(EGDMA-co-AA) capillary, and in-capillary extraction was performed. Fatty acids in the homogenized rat spinal cord solution were fully extracted and could be detected by MS (Figure 7, panels A-B). Several other types of lipids, such as phosphocholine (PC) and phosphoethanolamine (PE), were also extracted and detected in the rat spinal cord samples. Without being limited to any particular theory or mechanism of action, it is believed that small tissue fragments can be adsorbed by the polyporous polymer coating, and then the PC and PE in these tissues are eluted and enriched with ethyl acetate.

[0031] The inner coating of the capillary can be used as a substrate for the introduction of internal standards for extraction or analysis. Two isotope-labeled fatty acids, linoleic acid-d11 and docosahexaenoic acid-d5, were used as internal standards for the extraction and analysis of fatty acids in blood samples. First, linoleic acid-d11 and docosahexaenoic acid-d5 were dissolved in ethyl acetate; then, 1 μL of the solution was carefully injected onto the polymer layer. The polyporous polymer layer was able to adsorb the solution, and after drying, the internal standard was retained on the polymer, achieving immobilization of the internal standard. In a typical extraction, when ethyl acetate and a blood sample were introduced into the capillary, the internal standard eluted from the polymer layer and dissolved into one or both of the liquid plugs, achieving internal standard introduction. The reproducibility of this method was good, with a relative standard deviation of less than 12.8% for five replicate extractions (Panels A-B of Figure 8).

[0032] Another application of coated capillary-based microextraction is the rapid identification and quantification of C=C positional isomers of lipids in raw biological samples. Extraction was coupled with an online photochemical Paterno-Buchi (PB) reaction, in which acetone was used as the PB reagent for 250 nm UV irradiation. The PB reaction product ion has a mass shift of +58 Da due to acetone addition to intact unsaturated lipids. CID of the product causes cleavage at the original C=C position, giving a C=C diagnostic ion, which was used to determine the C=C position (Figure 9A-C). The determination of C=C isomers of FA18:1 in raw blood and tissue samples was investigated. Ethyl acetate and 5 μL of blood were introduced into a polyporous poly(EGDMA-co-AA)-coated capillary for extraction. After extraction, the ethyl acetate was evaporated by sparging with nitrogen, which took exactly 30 seconds. The residual fraction of the extract was separated using acetonitrile / water (1:1, v / v) and loaded into a pulled capillary tip for photochemical reaction and nanoESI-MS analysis. The ratio of C=C isomers of FA18:1, i.e., Δ9 to Δ11 isomers, in raw blood or tissue samples could be obtained in a total time of 2–3 min (Figure 9D). This is much faster than that obtained by conventional extraction methods, which usually take longer than 1 h. Four types of samples were investigated, including human blood, bovine blood, rat blood, and homogenized solutions of rat spinal cord. This method was attempted to determine the difference in the ratio of C=C isomers of FA18:1 between healthy and diseased biological samples (Figure 10, panels A–B). The Δ11 isomer was significantly increased in mouse cancer breast tissue ( *** P<0.0005) was observed (23.4±0.9% vs. 4.1±0.4%, cancer vs. normal).

[0033] Other materials may also be modified within the capillary. For example, by modification with a hydrophilic polymer layer, the in-capillary microextraction method may be used to extract polar lipids, metabolites, or drugs in raw blood or tissue samples. By modification with specific components such as titanium dioxide or materials containing boric acid groups, the in-capillary microextraction method may be used to extract phosphate compounds or polysaccharides.

[0034] The coating in the capillary can also be a platform for biological / chemical reactions. For example, an enzyme can be immobilized on the coating to trigger an enzymatic reaction when a solution of a biological sample or any other type of sample is introduced into the capillary. The product can be rapidly extracted with a plug of extraction solvent for further reaction or analysis. Some reactive compounds can be modified on the coating to trigger a chemical reaction within the coating. The product can be rapidly extracted with a plug of extraction solvent for further reaction or analysis. Some reactions may be interrupted by a complex biological matrix; the coating can be an intermediate zone for solving problems. For example, by placing a plug of air between the sample and the plug of extraction solvent, the analytes in the sample adsorb onto the coating as the sample solution flows through the coating. As the extraction solvent flows through the coating, the analytes for further reaction are obtained without contacting the sample solution.

[0035] There are numerous methods for analyzing ions. In certain embodiments, analyzing involves introducing ions into a mass analyzer of a mass spectrometer or a miniature mass spectrometer. Any type of mass spectrometer known in the art can be used with the probes of the present invention. For example, the mass spectrometer can be a standard benchtop mass spectrometer. In other embodiments, the mass spectrometer is a miniature mass spectrometer. An exemplary miniature mass spectrometer is described, for example, in Gao et al. (Anal. Chem., 2006, 78, 5994-6002). ), the contents of which are incorporated herein by reference in their entirety. Compared to pumping systems used in laboratory-scale instruments with thousands of watts of power, miniature mass spectrometers typically require pumping systems of 5 L / min (0.3 m) for the system described in Gao et al. 3 and smaller pumping systems, such as an 18W pumping system with only a 1L / hr diaphragm pump and an 11 L / sec turbo pump. Other exemplary miniature mass spectrometers are described, for example, by Gao et al. (Anal. Chem., 2008, Vol. 80, pp. 7198-7205), Hou et al. (Anal. Chem., 2011, Vol. 83, pp. 1857-1861), and Sokol et al. (Int. J. Mass Spectrom., 2011, Vol. 306, pp. 187-195), the contents of each of which are incorporated herein by reference in their entirety. Miniature mass spectrometers are described, for example, by Xu et al. (JALA, 2010, Vol. 15, pp. 433-439), Ouyang et al. (Anal. Chem., 2009, Vol. 81, pp. 2421-2425), Ouyang et al. (Ann. Rev. Anal. Chem., 2009, Vol. 2, pp. 187-214), Sanders et al. (Euro. J. Mass Spectrom., 2009, Vol. 16, pp. 11-20), Gao et al. (Anal. Chem., 2006, 78 (Chem. Comm., 2006, pp. 1709-1711), and Fico et al. (Anal. Chem., 2007, Vol. 79, pp. 8076-8082), the contents of each of which are incorporated herein by reference in their entirety.

[0036] In certain embodiments, the inlet of the mass spectrometer is located remotely from the ionization probe, and an ion transfer member is used to transfer ions over longer distances. Exemplary ion transfer members are described, for example, in Ouyang et al. (U.S. Patent No. 8,410,431), the contents of which are incorporated herein by reference in their entirety.

[0037] In certain embodiments, the ionization probes of the present invention operate without pneumatic assistance. That is, when using the probes of the present invention, pneumatic assistance is not required to transport the analyte; rather, a voltage is simply applied to a substrate held in front of the mass spectrometer. However, in certain embodiments, a nebulizing gas may be used with the systems of the present invention to assist desolvation. The nebulizing gas may be pulsed or provided as a continuous flow. In other embodiments, a gas-generating device is operably coupled to the probe so that it can inject gas into the hollow body to force the sample and solvent into the distal end of the probe. The gas is typically an inert gas such as nitrogen or argon, but could also be air.

[0038] In certain embodiments, the ionization probe is kept separate (i.e., isolated or disconnected) from a solvent flow, such as a continuous flow of solvent. Instead, separate volumes of solvent and sample are introduced into the hollow body of the probe. The probe is then connected to a voltage source to generate ions of the sample, which are subsequently mass analyzed. The sample is transported through the hollow body without the need for a separate solvent flow. As previously mentioned, no pneumatic assistance is required to transport the analytes; rather, a voltage is simply applied to the solvent in the probe containing the extracted analytes, which is held in front of the mass spectrometer.

[0039] The present invention is not necessarily limited to capillaries, and other hollow bodies can be used with the methods of the present invention. The hollow body can have a distal end for ejecting a spray of solvent that is loaded into the probe. An exemplary hollow body is a nano-ESI probe capillary having a distal end. An exemplary nano-ESI probe is described, for example, in Karas et al. (Fresenius (J. Anal. Chem. 2000, 366, 669-76) and El-Faramawy et al. (J. Am. Soc. Mass. Spectrom. 2005, 16, 1702-1707), the contents of each of which are incorporated herein by reference in their entirety. NanoESI needles are commercially available from Proxeon Biosystems (Odense, Denmark) and New Objective Inc. (Woburn, MA). In other embodiments, the system may include a sample cartridge containing one or more spray tips and one or more electrodes.

[0040] An exemplary hollow body is a 0.86 mm inner diameter borosilicate glass capillary with an elongated tip. The tip typically has a diameter of about 2 μm to about 50 μm. Plastic and rubber tubing can also be used for the hollow body. For example, the hollow body can be constructed from PEEK tubing (polyetheretherketone polymer tubing), TEFLON® tubing (polytetrafluoroethylene (PTFE) polymer tubing), or TYGON tubing (flexible tubing made from a variety of substrates).

[0041] Exemplary hollow bodies are 0.5 mm or 0.25 mm inner diameter fused silica capillaries, with or without an elongated tip.

[0042] The methods of the present invention can be used with any type of sample, such as organic or non-organic, biological or non-biological. In certain embodiments, the sample is derived from biological tissue or is a biological fluid, such as blood, urine, saliva, or spinal fluid. The sample may contain an analyte of interest to be analyzed. The analyte may be inherent to the sample or may be introduced into the sample. Exemplary analytes include therapeutic drugs, drugs of abuse, and biomarkers. Using the systems and methods of the present invention, effective suppression of matrix effects is achieved, for example, for therapeutic drugs, drugs of abuse, and biomarkers. In certain embodiments, the systems and methods of the present invention can be used for the direct analysis of biological fluid or liquid samples.

[0043] The solvent may be any solvent, even an ideal solvent, that works for both sample extraction and ionization. Typically, the solvent selected depends on the sample being analyzed and / or the analytes of interest believed to be present in the sample. A factor to consider is the polarity of the solvent. If the solvent is immiscible with the sample, ideally, the solvent has a different polarity from the sample and / or the analytes of interest believed to be present in the sample. For example, aqueous samples typically have high polarity, and therefore a good choice of solvent would be a low-polarity organic solvent (e.g., methanol or ethyl acetate, or a mixture containing these solvents, e.g., a water / methanol mixture or a water / ethyl acetate mixture). Oil-based samples typically have low polarity, and therefore a good choice of solvent would be a high-polarity solvent, such as a water / methanol mixture. Those skilled in the art can determine the appropriate solvent to use based on the sample being analyzed.

[0044] Another consideration for a solvent is that in addition to being good for extracting analytes from a sample, it can also be used to ionize the sample. That is, a solvent can be compatible with both extraction and ionization of extracted analytes. Methanol and ethyl acetate work well for analyte extraction and analyte ionization, while chloroform works well for analyte extraction but not for analyte ionization. Typically, any solvent compatible with electrospray ionization can likely be used with the systems and methods of the present invention, as long as the solvent is also immiscible with the sample and capable of extracting analytes from the sample. Those skilled in the art with experience in mass spectrometry will know of specific solvents that are compatible with electrospray ionization.

[0045] Those skilled in the art will recognize that the order in which the sample and solvent are introduced into the hollow body does not matter. In certain embodiments, the solvent is introduced first and the sample is introduced second. In other embodiments, the sample is introduced first and the solvent is introduced second. In certain embodiments, the sample and solvent are immiscible. In certain embodiments, more than one solvent is used. For example, a second solvent can be introduced that is located between the first solvent and the sample (a three-phase embodiment). The second solvent can act as a solvent bridge and is immiscible with the sample and first solvent, which in such embodiments are typically miscible with each other.

[0046] In certain embodiments, the sample and solvent are gently moved. This can be done manually by gently tilting the capillary or through the use of a moving mechanism. An exemplary moving mechanism is a pump that uses alternating air pressure within the hollow body to push and pull the sample within the body, thereby causing gentle movement. Other mechanisms can be standard sample agitators or mixers known to those skilled in the art. In certain embodiments, magnetic beads are added to the sample and extraction solvent, and an alternating magnetic field is applied to induce movement of the magnetic beads within the sample and solvent, thereby promoting turbulence inside the hollow body to transport analytes that interact with the adsorbent material within the hollow body.

[0047] In certain embodiments, the target to be analyzed is, for example, a target that is not efficiently ionized by spray ionization.In such embodiments, it is beneficial to derivatize the target molecule by introducing a drug that can add a charged group to the target, thereby making it more susceptible to ionization.For example, steroids are difficult to ionize by spray ionization.By introducing a drug such as hydroxylamine into the sample, a charged group is added to the steroid, making it more susceptible to spray ionization.

[0048] In certain embodiments, the hollow body is configured so that no electrodes are disposed on the surface of the body. Instead, the electrodes are disposed at least partially within the hollow body. In exemplary embodiments, the electrodes can be metal wires extending into the hollow body. Typically, any metal used in electrodes can be used for the metal electrodes. The metal wire is connected to a voltage power source, such as a high-voltage power source. The metal wire can extend into the hollow body to any length. In certain embodiments, the metal wire can extend to the distal end of the hollow body. Alternatively, the metal wire can be significantly shorter and may not extend far into the body. The length of the metal wire is determined by the amount of solvent added to the hollow body, as the wire must extend far enough into the hollow body to interact with the solvent added to the hollow body.

[0049] The metal wire may be coaxially disposed within the hollow body, but this is not required. Typically, the metal wire does not contact the walls of the hollow body. The metal wire electrode and its connection can be removably or permanently attached to the hollow body. In certain embodiments, the metal wire electrode and its connection are removably attached to the hollow body. This allows the proximal end of the hollow body to act as a port for the introduction of fluid into the body. In such embodiments, the metal wire electrode and its connection are removed from the hollow body, leaving an opening through which fluid is introduced into the body. Once introduced, the metal wire electrode and its connection are attached to the hollow body, sealing the hollow body.

[0050] In other embodiments, the attachment is a permanent attachment and one or more separate fluid ports along the body are used to introduce fluid into the hollow body. Even if the attachment of the metal wire electrode and its connection to the hollow body is a removable attachment, the hollow body still includes one or more separate ports along the body to allow fluid to be introduced into the hollow body.

[0051] In an exemplary embodiment, the introduction of a high voltage to the liquid within the hollow body causes the liquid to be ejected from the distal end of the hollow body in the form of a spray. The inlet of the mass spectrometer is operably positioned to receive the liquid ejected from the probe. This distance is typically less than 10 mm, but any distance that allows a signal from the sample to be generated within the mass spectrometer is appropriate. This distance can be determined by one skilled in the art simply by adjusting the spacing between the probe and the inlet of the mass spectrometer and monitoring the readings generated by the mass spectrometer.

[0052] In other embodiments, the outer wall of the elongated tip can be coated with metal, and a high voltage can be applied through the metal coating for spray ionization.

[0053] In certain embodiments, more than one analyte (e.g., multiple analytes) are extracted from a sample onto an adsorbent material. The multiple analytes are then extracted from the adsorbent material using one or more extraction solvents. That is, typically, based on the polarity of the analytes and the polarity of the solvent, multiple analytes can be simultaneously extracted using a single solvent, or the analytes are differentially eluted from the adsorbent material using one or more solvents.

[0054] Although the methods of the present invention have been discussed using two fluids (sometimes two immiscible fluids), the systems and methods of the present invention are not limited to the use of two fluids. Any number of fluids, such as three fluids, four fluids, five fluids, etc., can be used with the systems and methods of the present invention. In certain embodiments, a three-fluid system is used. In certain exemplary embodiments, two miscible fluids are separated by an immiscible fluid. In this exemplary embodiment, the polarity of the sample-solvent bridge-extraction / spray solvent can be high-low-high or low-high-low.

[0055] In certain embodiments, the systems and methods of the present invention can also be used to prepare samples that are subsequently analyzed. The extraction solvent can be stored as a liquid sample or deposited onto a paper substrate or MALDI plate to prepare dried sample spots. An internal standard can be incorporated into the dried sample spots during the extraction process. The target analyte can be chemically modified during the extraction process.

[0056] In other embodiments, the hollow body does not require a distal end because the extraction capillary is not used as an ionization probe. In such embodiments, extraction simply occurs in the capillary, as described above. After extraction is complete, the solvent containing the extracted analytes is removed from the capillary and then analyzed using any method known in the art. For example, the solvent containing the extracted analytes may be loaded into a separate ionization probe and then analyzed by mass spectrometry. In other embodiments, the analytes are analyzed in a different manner, such as by any spectroscopy technique or other assay known in the art.

[0057] Further aspects of the invention are further described below.

[0058] Ion generation Any method for generating ions known in the art may be used.Exemplary mass spectrometry techniques that utilize an ionization source at atmospheric pressure for mass analysis include electrospray ionization (ESI; Fenn et al., Science, 1989, vol. 246, pp. 64-71 and Yamashita et al., J. Phys. Chem., 1984, vol. 88, pp. 4451-4459); atmospheric pressure ionization (APCI; Carroll et al., Anal. Chem., 1975, vol. 47, pp. 2369-2373). ), and atmospheric pressure matrix-assisted laser desorption ionization (AP-MALDI; Laiko et al., Anal. Chem., 2000, 72, 652-657, and Tanaka et al., Rapid Commun. Mass Spectrom., 1988, Vol. 2, pp. 151-153). the contents of each of which are incorporated herein by reference in their entirety.

[0059] An exemplary mass spectrometry technique utilizing direct ambient ionization / sampling methods is desorption electrospray ionization (DESI; Takats et al., Science, 2004, 30 6, pp. 471-473 and U.S. Pat. No. 7,335,897), Direct Analysis in Real Time (DART; Cody et al., Anal. Chem., 2005, Vol. 77, pp. 2297-2302), Atmospheric Pressure Dielectric Barrier Discharge Ionization (DBDI; Kogelschatz, Plasma Chem. and Plasma P., 2003, Vol. 23, pp. 1-46 and PCT International Publication WO2009 / 1027 66), ion generation using wet porous materials (Paper Spray, U.S. Pat. No. 8,859,956), and electrospray-assisted laser desorption / ionization (ELDI; Shiea et al., Rapid Comm. Mass Spectrom., 2005, Vol. 19, pp. 3701-3704). The contents of each of these references are incorporated herein by reference in their entirety.

[0060] Generation of ions can be achieved by placing a sample on a porous material and generating ions of the sample from the porous material or other type of surface, as shown in U.S. Patent No. 8,859,956 to Ouyang et al., the contents of which are incorporated herein by reference in their entirety. Alternatively, the assay can be performed and ions generated from a non-porous material. See, for example, Cooks et al., U.S. Patent Application No. 14 / 209,304, the contents of which are incorporated herein by reference. See, e.g., Cooks et al., U.S. Patent Application Publication No. 20140264004, the contents of which are incorporated herein in their entirety. In certain embodiments, a solid needle probe or surface to which a high voltage can be applied is used to generate ions of a sample (see, e.g., Cooks et al., U.S. Patent Application Publication No. 20140264004, the contents of which are incorporated herein in their entirety). See, e.g., pp. 111-114, 2003, which is incorporated herein by reference in its entirety.

[0061] In certain embodiments, sample ions are generated using nanospray ESI. Exemplary nanospray tips and methods for preparing such tips are described, for example, in Wilm et al. (Anal. Chem. 2004, Vol. 76, pp. 1165-1174), the contents of which are incorporated herein by reference in their entirety. NanoESI is described, for example, in Karas et al. (Fresenius J. Anal. Chem. 2000, Vol. 366, pp. 669-676), the contents of which are incorporated herein by reference in their entirety.

[0062] Ion Analysis In one particular embodiment, the ions are analyzed by directing them into a mass spectrometer (benchtop or miniature mass spectrometer). Figure 11 is a photograph showing the various components and their arrangement in a miniature mass spectrometer. The control system of the Mini 12 (Linfan Li, Tsung-Chi Chen, Yue Ren, Paul I. Hendricks, R. Graham Cooks, and and Zheng Ouyang “Miniature Ambient Mass Analysis System” Anal. Chem. 20 14, vol. 86, pp. 2909-2916 DOI:10.1021 / ac403766c; and Paul I. Hendricks, Jon K. Dalgleish, Jacob T. Shelley, Matthew A. Kirleis, Matthew T. McNicholas, Linfan Li, Tsung-Chi Chen, Chien-Hsun Chen, Jason S. Duncan, Frank Boudreau, Robert J. Noll, John P. Denton, Timothy A. Roach, Zheng Ouyang, and R. Graham Cooks, “Autonomous in-situ analysis and real-time chemical detection using a miniature mass spectrometer: concept, instrumentation development, and performance,” Anal. Chem., 2014, vol. 86, pp. 2900-2908 DOI:10.1021 / ac 403765x (the contents of each of which are incorporated herein by reference in their entirety), and the Mini 10 vacuum system (Liang Gao, Qingyu Song, Garth E. Patterson, R. Graham Cooks, and Zheng Ouyang, "Handheld Rectilinear Ion "Among the miniature mass spectrometers shown in FIG. 11, a dual-LIT configuration may be fabricated using a combination of a dual-LIT and a dual-LIT probe, such as those described in "Among the Miniature Mass Spectrometers," Anal. Chem., 2006, Vol. 78, pp. 5994-6002, DOI: 10.1021 / ac061144k (the contents of which are incorporated herein by reference in their entireties)," which may have a size similar to that of a shoebox (20 cm high x 25 cm wide x 35 cm deep). In certain embodiments, the miniature mass spectrometer uses a dual-LIT configuration as described, for example, in Owen et al. (U.S. Patent Application No. 14 / 345,672) and Ouyang et al. (U.S. Patent Application No. 61 / 865,377), the contents of each of which are incorporated herein by reference in their entireties.

[0063] Mass spectrometers (miniature or benchtop) may be equipped with a discontinuous interface, as described, for example, in Ouyang et al. (U.S. Pat. No. 8,304,718) and Cooks et al. (U.S. Patent Application Publication No. 2013 / 0280819). and US Pat. No. 6,229,999, the contents of each of which are incorporated herein by reference in their entirety.

[0064] sample As discussed above, the systems and methods of the present invention can be used to analyze many different types of samples, including a wide range of heterogeneous samples, such as biological samples, environmental samples (including, for example, industrial and agricultural samples), and food / beverage product samples.

[0065] Exemplary environmental samples include, but are not limited to, groundwater, surface water, saturated soil water, unsaturated soil water; industrial processes such as wastewater, cooling water; other systems with chemicals used in processes, chemical reactants in industrial processes, and leachate from dumps; waste and water injection processes; detection of leaks in or around storage tanks; discharge water from industrial facilities, water treatment plants or facilities; wastewater and leachate from agricultural land, wastewater from urban uses such as surface, subsurface and sewer systems; water from waste treatment technologies; and wastewater from mineral extraction or other processes that extract natural resources such as petroleum production and in situ energy production.

[0066] Further exemplary environmental samples include, but are certainly not limited to, agricultural samples, such as crop samples, including grain and feed products, e.g., soybeans, wheat, and corn. Data regarding product constituents such as moisture, protein, oil, starch, amino acids, extracted starch, density, test weight, digestibility, cell wall content, and any other constituent or characteristic that has market value is often desired.

[0067] Exemplary biological samples include human tissues or bodily fluids, which may be collected in any clinically acceptable manner. Tissues include connective cell masses and / or extracellular matrix materials, such as skin tissue, hair, nails, nasal tissue, CNS tissue, nervous tissue, ocular tissue, liver tissue, kidney tissue, placental tissue, mammary gland tissue, gastrointestinal tissue, skeletal muscle tissue, urogenital tissue, bone marrow, etc., derived from, for example, a human or other mammal, and include connective and liquid materials associated with cells and / or tissues. Bodily fluids are, for example, liquid materials derived from a human or other mammal. Such bodily fluids include, but are not limited to, mucous membranes, blood, plasma, serum, serum derivatives, bile, blood, maternal blood, phlegm, saliva, spit, sweat, amniotic fluid, menstrual fluid, breast fluid, ascites, urine, semen, and cerebrospinal fluid (CSF), such as lumbar or ventricular CSF. Samples may also be fine needle aspirates or tissue biopsies. The sample may be a medium containing cells or biological material. The sample may also be a blood clot, for example a blood clot obtained from whole blood after serum has been removed.

[0068] In one embodiment, the biological sample can be a blood sample from which plasma or serum can be extracted. Blood can be obtained by standard exsanguination procedures and then separated. A typical separation method for preparing a plasma sample includes centrifugation of the blood sample. For example, protease inhibitors and / or anticoagulants can be added to the blood sample immediately after blood extraction. The tube can then be cooled, centrifuged, and then placed on ice. The resulting sample is separated into the following components: a clear solution of plasma in the upper phase; a buffy coat, which is a thin layer of white blood cells mixed with platelets; and erythrocytes (red blood cells). Typically, approximately 2.5 to 3.0 mL of plasma can be obtained from 8.5 mL of whole blood.

[0069] Serum is prepared in a very similar manner. Venous blood is drawn and then mixed with protease inhibitors and coagulants by inversion. The blood is allowed to clot by standing the tube upright at room temperature. The blood is then centrifuged, and the resulting supernatant is designated serum. Serum samples should then be placed on ice.

[0070] Before analyzing a sample, the sample may be purified, for example, using filtration or centrifugation. These techniques can be used, for example, to remove particulates and chemical interferences. Various filtration media for removing particles include filter paper, such as cellulose, and membrane filters, such as regenerated cellulose, cellulose acetate, nylon, PTFE, polypropylene, polyester, polyethersulfone, polycarbonate, and polyvinylpyrrolidone. Various filtration media for removing particulates and matrix interferences include functionalized membranes, such as ion exchange membranes and affinity membranes; SPE cartridges, such as silica-based cartridges and polymer-based cartridges; and SPE (solid phase extraction) disks, such as PTFE-based and fiberglass-based. Some of these filters can be provided in disk format for loose placement in a filter holder / housing, others in disposable tips that can be placed, for example, in standard blood collection tubes, and still others in the form of an array with wells for receiving aliquots of sample. Another type of filter is a spin filter. Spin filters consist of polypropylene centrifuge tubes with a cellulose acetate filter membrane and are used in conjunction with centrifugation to remove particulates from samples, such as serum and plasma samples, that are typically diluted with aqueous buffers.

[0071] Filtration is affected, in part, by the value of porosity, such that higher porosities filter out only larger particulates, while lower porosities filter out both lower and higher porosities. Typical porosity values ​​for sample filtration are 0.20 and 0.45 μm porosities. If the sample contains colloidal material or a large amount of small particles, significant pressure may be required to force the liquid sample through the filter. Therefore, for samples such as soil extracts or wastewater, a pre-filter or depth filter bed (e.g., a "2-in-1" filter) can be used, placed on top of the membrane to prevent clogging by samples containing these types of particulates.

[0072] In some cases, centrifugation without a filter can be used to remove particulates, as is often done with urine samples. For example, the sample is centrifuged. The resulting supernatant is then removed and frozen.

[0073] After a sample is obtained and purified, it can be analyzed. With respect to the analysis of a plasma sample, there are many elements present in plasma, such as proteins (e.g., albumin), ions and metals (e.g., iron), vitamins, hormones, and other elements (e.g., bilirubin and uric acid). Any of these elements may be detected. More specifically, the system of the present invention can be used to detect molecules in biological samples that are indicative of a disease state.

[0074] If one or more target molecules in the sample are part of a cell, the aqueous medium may also contain a lysis agent to lyse the cell. A lysis agent is a compound or mixture of compounds that disrupts the integrity of the cell's membrane, thereby releasing the cell's intracellular contents. Examples of lysis agents include, but are not limited to, nonionic detergents, anionic detergents, zwitterionic detergents, low ionic strength aqueous solutions (hypotonic solutions), bactericidal agents, aliphatic aldehydes, and antibodies that induce complement-dependent lysis. Various additional materials may be present in the dilution medium. All materials in the aqueous medium are present in concentrations or amounts sufficient to achieve the desired effect or function.

[0075] In some cases, when one or more target molecules are part of a cell, it may be desirable to fix the cells of the sample. Fixing the cells immobilizes the cells, preserves their structure, and maintains them in a state that closely resembles the in vivo state of the cells and allows the antigen of interest to be recognized by a specific affinity agent. The amount of fixative used is an amount that preserves the cells but does not lead to false results in subsequent assays. The amount of fixative may depend, for example, on one or more of the properties of the fixative and the properties of the cells. In some cases, the amount of fixative is about 0.05% to about 0.15% by weight, or about 0.05% to about 0.10% by weight, or about 0.10% to about 0.15% by weight. Agents for fixing cells include, but are not limited to, crosslinkers, such as aldehyde reagents (e.g., formaldehyde, glutaraldehyde, and paraformaldehyde); alcohols (e.g., C1-C5 alcohols, such as methanol, ethanol, and isopropanol); and ketones (e.g., C3-C5 ketones, such as acetone). The designations C1-C5 or C3-C5 refer to the number of carbon atoms in the alcohol or ketone. One or more washing steps may be performed on the fixed cells using a buffered aqueous medium.

[0076] If necessary, the cell preparation may be permeabilized after fixation. In some cases, the fixative, such as alcohol (e.g., methanol or ethanol) or ketone (e.g., acetone), also causes permeabilization, and no additional permeabilization step is required. Permeabilization allows access to the target molecule of interest through the cell membrane. The amount of permeabilizing agent used is an amount that disrupts the cell membrane and allows access to the target molecule. The amount of permeabilizing agent depends on one or more of the properties of the permeabilizing agent and the properties and amount of cells. In some cases, the amount of permeabilizing agent is about 0.01% to about 10%, or about 0.1% to about 10%. Agents for permeabilizing cells include, but are not limited to, alcohols (e.g., C1-C5 alcohols, such as methanol and ethanol); ketones (e.g., C3-C5 ketones, such as acetone); detergents (e.g., saponin, TRITON X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, t-octylphenoxypolyethoxyethanol, polyethylene glycol tert-octylphenyl ether buffer, etc., available from Sigma-Aldrich), and TWEEN-20 (polysorbate 20, available from Sigma-Aldrich)). One or more washing steps may be performed on the permeabilized cells using a buffered aqueous medium.

[0077] Incorporation by Reference References and citations are made throughout this disclosure to other documents such as patents, patent applications, publications, journals, books, articles, web content, etc. All such documents are hereby incorporated by reference in their entirety for all purposes.

[0078] equivalent Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this document, including the references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

1. 1. A method for extracting an analyte from a sample, comprising: introducing a sample into a capillary containing an adsorbent material coating a portion of the capillary wall; allowing the sample in the capillary to interact with the adsorbent material in the capillary such that at least one analyte is extracted from the sample and bound to the adsorbent material; introducing an extraction solvent into the capillary; allowing the extraction solvent to interact with the adsorbent material to elute the at least one analyte from the adsorbent material; wherein the extraction occurs by repeated back and forth movement of the capillary resulting in interaction of the adsorbent material with the sample and the extraction solvent over multiple occurrences, wherein the sample and the extraction solvent are both present within the capillary.

2. 2. The method of claim 1, wherein the capillary is moved a first time to cause the sample to interact with the adsorbent material and then a second time to cause the extraction solvent to interact with the adsorbent material after the sample has interacted with the adsorbent material.

3. 3. The method of claim 2, wherein the sample and the extraction solvent are separated from each other by an air gap.

4. The method of claim 1 , further comprising analyzing the extracted analytes.

5. The step of analyzing applying a voltage to the extraction solvent containing the at least one analyte in the capillary such that the analyte is expelled from the capillary, thereby generating ions of the analyte; Analyzing the ions. The method of claim 4 , comprising:

6. The step of analyzing removing the solvent containing the at least one analyte from the capillary; performing an assay to analyze said analyte; The method of claim 4 , comprising:

7. The method of claim 1 , wherein the solvent is immiscible with the sample.

8. The method of claim 1 , wherein the solvent is miscible with the sample.

9. The method of claim 1 , wherein the adsorbent material comprises one or more molecules that react with the at least one analyte.

10. 10. The method of claim 9, further comprising performing a reaction between the one or more molecules and the at least one analyte to produce a reaction product.

11. The method of claim 10 further comprising analyzing the reaction products.

12. The step of analyzing applying a voltage to the extraction solvent containing the reaction products in the capillary such that the reaction products are expelled from the capillary, thereby generating ions of the analyte; Analyzing the ions. The method of claim 11 , comprising:

13. 10. The method of claim 1, wherein the extraction solvent comprises one or more molecules that react with the at least one analyte.

14. 14. The method of claim 13, further comprising performing a reaction between the one or more molecules and the at least one analyte to produce a reaction product.

15. The method of claim 14 further comprising analyzing the reaction products.

16. The step of analyzing applying a voltage to the extraction solvent containing the reaction products in the capillary such that the reaction products are expelled from the capillary, thereby generating ions of the analyte; Analyzing the ions.

16. The method of claim 15, comprising:

17. The step of analyzing removing the extraction solvent containing the reaction product from the capillary; conducting an assay to analyze the reaction products; 16. The method of claim 15, comprising:

18. The method of claim 1 , wherein the adsorbent material comprises an internal standard.

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