Analyzing extracted sample using immiscible extraction solvent

Abstract

To provide a system and method for analyzing a sample extracted using an immiscible extraction solvent.SOLUTION: In an embodiment, a present invention provides a system for analyzing a specimen in a sample. The system includes an ionization probe and a mass spectrometer. The ionization probe optionally includes a hollow body including a distal tip. The hollow body is configured such that no substrate is present in the body and electrodes are not disposed on a surface of the body. An electrode is at least partially disposed within the hollow body.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (Related Applications) This application claims the benefit of and priority to U.S. Provisional Patent Application No. 61 / 942,949, filed February 21, 2014, and U.S. Provisional Patent Application No. 62 / 013,007, filed June 17, 2014, the contents of each of which are incorporated herein by reference in their entirety.

[0002] (Government support) This invention was made with government support under No. GM106016 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] FIELD OF THE INVENTION The present invention relates generally to systems and methods for analyzing samples extracted using immiscible extraction solvents. [Background technology]

[0004] (background) Rapid analysis of complex biological samples (e.g., complex mixtures such as blood, saliva, or urine) is of great interest in clinical, forensic, and many other applications. A problem in analyzing such samples using mass spectrometry is that non-target components of the biological sample (e.g., salts) compete with targets in the sample for charge during the ionization process. Such competition leads to the non-target components of the sample suppressing the ionization of targets in the sample, known as the matrix effect. To minimize the suppression effect on analyte ionization and pre-concentrate the analytes, complex biological samples are routinely extracted and then separated using chromatography prior to mass spectrometry measurement. However, such processes can only be performed in a laboratory setting, using expensive chromatography equipment and time-consuming sample preparation protocols. Summary of the Invention [Means for solving the problem]

[0005] (Abstract) The present invention provides a new approach for liquid-liquid extraction and also provides systems and methods that allow sample preparation and pretreatment to be combined with the ionization process. In particular, the present invention allows liquid-liquid extraction to be performed in very small diameter capillaries (e.g., capillaries with an inner diameter as small as 500 μm), which provide a very small interface between the two liquids. This is typically considered highly unfavorable for conventional liquid-liquid extraction. The liquid within the capillary is moved back and forth, allowing control of the extraction process; i.e., extraction can be turned on and off. This motion induces circulation inside each plug of immiscible fluid, facilitating extraction. An additional advantage of using thin capillaries is that small sample volumes can be handled for quantitative analysis.

[0006] In addition, the extraction capillary can also serve as the ionization probe. Thus, 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 pre-treatment protocols. Rather, the systems and methods of the present invention are configured so that sample preparation and pre-concentration occur within the ionization probe. The purified analytes can then be directly ionized (although not required) and injected into the mass spectrometer from the ionization probe, where sample preparation and pre-treatment occur.

[0007] Aspects of the present invention are accomplished using a solvent that is immiscible with the sample. The solvent and sample are placed within an ionization probe of the present invention. One or more target analytes from the sample are extracted into the solvent, while non-target components of the sample (e.g., salts in urine) remain in the sample. Electrodes are then operably coupled to the 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; 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 matrix effects, enabling 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 advantage of pre-concentrating target analytes from the sample into the extraction solvent, thereby avoiding expensive chromatography equipment and time-consuming separation protocols and enabling point-of-care sample analysis systems and methods.

[0008] Aspects of the present invention provide methods for analyzing analytes in a sample. These methods may involve introducing a solvent into a hollow body, optionally having a distal tip. The sample is also introduced into the hollow body. The solvent does not mix with the sample. The method then involves extracting at least one analyte from the sample into the solvent. A voltage is applied to the solvent containing the extracted analyte within the hollow body such that the analyte is expelled from the distal tip of the body, thereby generating analyte ions. The ions are then analyzed. 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 some embodiments, the solvent is introduced first, followed by the sample. In other embodiments, the sample is introduced first, followed by the solvent. In some embodiments, the sample and solvent are immiscible. In some embodiments, more than one solvent is used. For example, a second solvent can be introduced 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 the first solvent, which in such embodiments are typically mutually miscible.

[0009] In some embodiments, the sample and solvent are slowly mixed prior to application of the voltage. This can be done manually by slowly tilting the capillary tube or through the use of a moving mechanism. In some embodiments, the two or more phases do not intermix. An exemplary moving mechanism is a pump that applies alternating air pressure within the hollow body, pushing and pulling the sample within the body, thereby causing slow movement. In some embodiments, a high voltage is applied to the solvent, expelling it from the hollow body and desolvating and ionizing the sample. In other embodiments, a nebulizing gas is also applied to the extracted sample, either in pulses or as a continuous flow.

[0010] The methods of the present invention can be used with any type of sample. In some embodiments, the sample is a biological fluid such as blood, urine, saliva, or spinal fluid. The sample typically includes a target of interest. In the case of a biological sample, the target may be a therapeutic drug, a drug of abuse, or another molecule such as a steroid. The target may be a component derived from the sample or artificially introduced into the sample. In some embodiments, an internal standard is also introduced.

[0011] In some 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. Introducing a drug such as hydroxylamine into the sample adds a charged group to the steroid, making it more susceptible to spray ionization.

[0012] The extraction solvent selected may depend on the target to be extracted. It is also important to consider the effectiveness of the solvent for ionization. An ideal solvent is good for both extraction and ionization. An exemplary such solvent is ethyl acetate, but one skilled in the art may recognize that other solvents are also effective for both extraction and ionization of targets in a sample. In embodiments where multiple analytes are extracted from a sample into a solvent, the solvent may also be capable of differentially extracting the analytes.

[0013] Numerous methods exist for analyzing ions. In certain embodiments, analysis involves introducing ions into a mass analyzer of a mass spectrometer or a miniature mass spectrometer. An exemplary miniature mass spectrometer is described, for example, in Gao et al. (Anal. Chem. 2008, 80, 7198-7205), the contents of which are incorporated herein by reference in their entirety. Compared to pumping systems used for laboratory-scale instruments involving thousands of watts of power, miniature mass spectrometers require pumping of 5 L / min (0.3 m) for the system described in Gao et al. 3 Generally, they have smaller pumping systems, such as an 18 W pumping system with only a 1000 W (1 / hr) diaphragm pump and an 11 L / sec turbo pump. Other exemplary miniature mass spectrometers are described, for example, in Gao et al. (Anal. Chem., 2006, 80:7198-7205, 2008), Hou et al. (Anal. Chem., 83:1857-1861, 2011), and Sokol et al. (Int. J. Mass Spectrom., 2011, 306, 187-195), the contents of each of which are incorporated herein by reference in their entirety.

[0014] Another aspect of the present invention provides a system for analyzing analytes in a sample. The system includes an ionization probe and a mass spectrometer. As previously described, the mass spectrometer may be a benchtop mass spectrometer or a miniature mass spectrometer. The ionization probe includes a hollow body (e.g., a glass capillary tube extended to a tip) including a distal tip. The hollow body is configured such that no substrate is present within the body and no electrodes are disposed on the surface of the body. Rather, the electrodes are at least partially disposed within the hollow body. In certain embodiments, the electrodes are spaced from the surface of the hollow body, i.e., do not touch the surface of the hollow body. In certain embodiments, the electrodes are disposed coaxially within the hollow body. In certain embodiments, the electrodes extend to a distal portion of the hollow body. An exemplary electrode is a metal wire.

[0015] In some embodiments, the probe operates without air pressure assistance. In other embodiments, the system includes an nebulizing gas source. The nebulizing gas source may be configured to provide pulses of gas or a continuous flow of gas.

[0016] In another aspect, the present invention provides methods for extracting an analyte from a sample. These methods involve introducing a solvent into a capillary. A sample containing the analyte is also introduced into the capillary. The solvent is immiscible with the sample. The sample and solvent are moved within the capillary, inducing circulation within the sample and solvent, thereby extracting the analyte from the sample into the solvent. In some embodiments, the solvent is introduced first, and the sample is then introduced. In other embodiments, the sample is introduced first, and the solvent is then introduced. In some embodiments, the sample and solvent are immiscible. In some embodiments, more than one solvent is used. For example, a second solvent can be introduced between the first solvent and the sample (a three-phase embodiment using a bridging solvent). The second solvent can act as a solvent bridge and, in such embodiments, is typically immiscible with the sample and first solvent, which are mutually miscible.

[0017] In certain embodiments, the method may additionally involve analyzing the extracted analyte. The analysis may involve applying a voltage to a solvent containing the extracted analyte within the capillary such that the analyte is expelled from the capillary, thereby generating ions of the analyte, and analyzing the ions. In other embodiments, the analyzing may involve removing the solvent containing the extracted analyte from the capillary and performing an assay to analyze the analyte.

[0018] The method of the present invention allows a reaction to be monitored. To monitor a reaction, the method may additionally involve stopping movement of the sample and solvent within the capillary and analyzing an amount of analyte extracted into the solvent. Based on the results of the analyzing step, the method of the present invention may additionally involve resuming movement of the sample and solvent within the capillary based on the results of the analyzing step.

[0019] Another aspect of the invention provides a method for extracting an analyte from a sample, which involves introducing multiple solvents into a capillary tube, where each two adjacent solvents are immiscible with each other, displacing the solvents within the capillary tube and inducing circulation within each solvent, thereby transferring chemical compounds between the solvents. The present invention provides, for example, the following. (Item 1) 1. A system for analyzing an analyte in a sample, the system comprising: A system comprising: an ionization probe comprising a hollow body, the hollow body having no substrate within the body and no electrodes disposed on a surface of the body, an electrode disposed at least partially within the hollow body, and a mass spectrometer. (Item 2) Item 10. The system of item 1, wherein the probe operates without air pressure assistance. (Item 3) Item 10. The system of item 1, further comprising a source of atomizing gas. (Item 4) Item 10. The system of item 1, wherein the atomizing gas source is configured to provide pulses of gas. (Item 5) Item 10. The system of item 1, wherein the atomizing gas source is configured to provide a continuous flow of gas. (Item 6) Item 10. The system of item 1, wherein the electrodes are coaxially arranged within the hollow body. (Item 7) 7. The system of claim 6, wherein the electrode extends to a distal portion of the hollow body. (Item 8) 8. The system of claim 7, wherein the electrode is a metal wire. (Item 9) Item 10. The system of item 1, wherein the hollow body comprises glass. (Item 10) Item 10. The system of item 1, wherein the mass analyzer is a miniature mass analyzer. (Item 11) 1. A method for analyzing an analyte in a sample, comprising: introducing a solvent into a hollow body having a distal tip; introducing a sample into the hollow body, wherein the solvent is immiscible with the sample; extracting at least one analyte from the sample into the solvent; applying a voltage to a solvent containing the extracted analyte within the hollow body such that the analyte is expelled from the distal tip of the body, thereby generating ions of the analyte; analyzing the ions; A method comprising: (Item 12) Item 12. The method of item 11, further comprising the step of introducing an agent that imparts a charged functional group to the analyte. (Item 13) 12. The method of claim 11, wherein the solvent is compatible with both the extraction and the ionization of the extracted analytes. (Item 14) 12. The method of claim 11, wherein the analyzing step comprises introducing the ions into a benchtop mass spectrometer or a miniature mass spectrometer. (Item 15) Item 12. The method of item 11, wherein the solvent is introduced into the hollow body prior to the introduction of the sample. (Item 16) 12. The method of claim 11, further comprising the step of mixing the sample and the solvent prior to the step of applying the voltage. (Item 17) 12. The method of claim 11, wherein multiple analytes are extracted into the solvent. (Item 18) 18. The method of claim 17, wherein the analytes are differentially extracted into the solvent. (Item 19) The step of applying a voltage includes: inserting a metal wire through the sample into the solvent; applying the voltage to the solvent via the metal wire. (Item 20) 12. The method of claim 11, wherein a nebulizing gas is also applied to the extracted sample. (Item 21) 1. A method for extracting an analyte from a sample, comprising: introducing a solvent into the capillary; introducing a sample containing an analyte into the capillary tube, wherein the solvent does not mix with the sample; moving the sample and the solvent within the capillary tube, inducing circulation within the sample and the solvent, thereby extracting the analyte from the sample into the solvent; A method comprising: (Item 22) 22. The method of claim 21, further comprising analyzing the extracted specimen. (Item 23) The analyzing step includes: applying a voltage to a solvent containing the extracted analyte within the capillary such that the analyte is expelled from the capillary, thereby producing ions of the analyte; analyzing the ions; Item 23. The method according to Item 22, comprising: (Item 24) The analyzing step includes: removing the solvent containing the extracted analyte from the capillary tube; performing an assay to analyze the sample; Item 23. The method according to Item 22, comprising: (Item 25) 22. The method of claim 21, wherein the solvent is first introduced into the capillary tube. (Item 26) 22. The method of claim 21, wherein the sample is first introduced into the capillary tube. (Item 27) stopping movement of the sample and the solvent within the capillary; removing a portion of the solvent; 22. The method of claim 21, further comprising monitoring the extraction by analyzing the amount of analyte extracted into the solvent. (Item 28) 28. The method of claim 27, further comprising the step of restarting movement of the sample and the solvent within the capillary tube based on the results of the analyzing. (Item 29) 22. The method of claim 21, wherein the solvent is immiscible with the sample. (Item 30) 22. The method of claim 21, wherein the solvent is miscible with the sample, and the method further comprises introducing a bridging solvent into the capillary tube that is immiscible with the solvent and the sample in such a manner that the bridging solvent is between the solvent and the sample. [Brief explanation of the drawings]

[0020] [Figure 1A] FIG. 1A illustrates an exemplary system of the present invention.

[0021] [Figure 1B] Figure 1B illustrates a method for using the system of the present invention. In this illustration, two immiscible phases, a liquid sample and an organic solvent, are injected side by side into a capillary with an extended tip. The liquid phase is moved back and forth within the capillary by tilting the capillary or applying gas pressure to facilitate microextraction. The liquid phase is then pushed by applying gas pressure as the extraction phase reaches the extended tip of the capillary. A wire electrode is inserted into the extraction solvent and a DC voltage is applied for nano-ESI.

[0022] [Figure 2] Figure 2, panels A and C, show calibration curves for the quantification of different compounds in synthetic urine samples. Figure 2, panel A, shows methamphetamine. Figure 2, panel B, shows nicotine. Figure 2, panel C, shows benzoylecgonine. 10 μL of synthetic urine containing drug and internal standard was used as the sample for measurement. 5 μL of ethyl acetate (EA) was used as the extraction phase for extraction, purification, and spraying. Internal standards: methamphetamine-d8 (0.8 ng / mL), nicotine-d3 (2 ng / mL), benzoylecgonine-d3 (1 ng / mL). Single reaction monitoring (SRM) transitions used were: methamphetamine m / z 150 → 91, methamphetamine-d8 m / z 158 → 93; nicotine m / z 163 → 130, nicotine-d3 m / z 166 → 130; benzoylecgonine m / z 290 → 168, benzoylecgonine-d3 m / z 293 → 171. Partition coefficients: LogP methamphetamine = 2.07; LogP nicotine = 1.17, LogP benzoylecgonine = -0.59.

[0023] [Figure 3A] Figure 3A shows reactive slug flow microextraction nanoESI, in which a sample phase, a derivatization reagent phase, and an extraction phase are injected adjacently. The extraction phase is immiscible with either the reagent or sample phase. The sample and reagent phases can be miscible. Analytes in the sample phase are derivatized and extracted into the extraction phase during the SFME operation. The liquid phase is then pushed so that the extraction phase reaches the extended tip of the capillary. A wire electrode is inserted into the extraction phase, and a DC spray voltage is applied for nanoESI.

[0024] [Figure 3B] FIG. 3B shows binding of a target in a sample with a charged reagent to form a charged complex, which makes the uncharged target more susceptible to spray ionization.

[0025] [Figure 4]Figure 4, panels A and B, show MS / MS spectra obtained using slug-flow microextraction nanoESI. Bovine blood samples containing 40 ng / mL nicotine (Figure 4, panel A) and 40 ng / mL methamphetamine (Figure 4, panel B), respectively, were diluted 10-fold with water and then analyzed using SFME nanoESI. 10 μL of diluted sample was used, followed by 5 μL of ethyl acetate.

[0026] [Figure 5] Figure 5, panels A-D, show MS / MS spectra obtained using reactive slug-flow microextraction nano-ESI with hydroxylamine as the reagent. In synthetic urine, 10 μL of 8 ng / mL epitestosterone (Figure 5, panel A), 5 ng / mL 5α-androstan-3β,17β-diol-16-one (Figure 5, panel B), 5 ng / mL 6-dehydrocholestenone (Figure 5, panel C), and 5 ng / mL stigmastadienone (Figure 5, panel D) were added. 5 μL of an aqueous solution containing 0.1% acetic acid and 10% hydroxylamine was added as the reagent phase. 5 μL of ethyl acetate was used as the extraction phase.

[0027] [Figure 6] Figure 6 shows an exemplary three-phase fluid system. Sample analysis using immiscible three-phase SFME: The sample phase is highly polar, and a relatively highly polar solvent, such as HO and acetonitrile, is used as the extraction solvent. A relatively less polar solvent, immiscible with the sample and extraction solvent, is plugged between them, keeping them separated. Hydrophobic tubing, rather than glass tubing, is used in this case to ensure isolation. Pushing and pulling forces are applied for extraction, inducing slug flow movement. After extraction, the extract can be analyzed either directly or indirectly by nanoESI or stored for further processing.

[0028] [Figure 7]Figure 7 shows microextraction for analyzing chemicals in low polarity samples. A relatively highly polar solvent can be used for extraction and spray ionization.

[0029] [Figure 8] Figure 8 shows the analysis of vegetable oil using the system and method of the present invention. A mixture of water and methanol was used as the extraction solvent. Figure 8, panel A, is an MS spectrum showing diacylglycerol and triacylglycerol species observed in the MS spectrum in positive mode. Figure 8, panel B, is an MS spectrum showing different fatty acids observed in the MS spectrum acquired in negative mode.

[0030] [Figure 9] Figure 9, panels A and B, show the analysis of 100 ng / mL phenylalanine (165 Da, logP = -1.38) in urine using three-phase SFME (Figure 6). MS / MS spectra of the molecular ion were collected. Figure 9, panel A, shows the MS spectrum of a 5 μL sample diluted 10-fold using methanol as the reducing matrix and directly sprayed by nanoESI. Figure 9, panel B, shows the MS spectrum of a 5 μL sample processed by three-phase SFME using hexane / HO:MeOH (1:1) as the crosslinking / extraction solvent and then analyzed by nanoESI.

[0031] [Figure 10] FIG. 10 shows the analysis of 50 ng / mL amitriptyline in bovine whole blood using fused silica capillaries.

[0032] [Figure 11]Figure 11, panel A, shows in-capillary sample extraction using slug flow microextraction. Figure 11, panel B, shows subsequent MS analysis using nanoESI. MS / MS spectra. Figure 11, panel C, shows the analysis of 10 ng mL methamphetamine in 5 μL urine. Figure 11, panel D, shows the analysis of 50 ng mL benzoylecgonine in 5 μL urine. Figure 11, panel E, shows the effect of the number of SFME cycles on analyte extraction. The intensities of the MS / MS product ions were monitored for methamphetamine (m / z 150 → 91), nicotine (m / z 163 → 130), and benzoylecgonine (m / z 290 → 168), respectively, at 50 ng / mL in the urine sample. 2 kV was used for nanoESI.

[0033] [Figure 12] Figure 12, panels AB, show that movement of the liquid plug inside the capillary can be generated in two ways. Figure 12, panel A, shows gently tilting the capillary up and down. Figure 12, panel B, shows applying pushing and pulling forces by air pressure through the pipette. The pipette volume was set to 10 μL for this purpose.

[0034] [Figure 13] Figure 13, panels A and B, show spectra recorded for direct MS / MS analysis. Figure 13, panel A, shows 1 ng mL verapamil in 5 μL of undiluted human pooled blood. Figure 13, panel B, shows endogenous creatinine contained in human whole blood.

[0035] [Figure 14] FIG. 14A shows the derivation for calculating the concentration at equilibrium.

[0036] [Figure 14B] Figure 14B shows the derivation for calculating the internal standard (IS) incorporation concentration at equilibrium after SFME.

[0037] [Figure 15]Figure 15 shows the quantitative analysis of whole blood spiked with methamphetamine (1-100 ng mL). Blood samples were diluted 10-fold to reduce viscosity. Methamphetamine-d8 (2 ng mL) in the extraction solvent ethyl acetate.

[0038] [Figure 16] Figure 16, panel A, shows reactive SFME-nanoESI with a reagent plug injected between the biofluid sample and the extraction solvent. The MS / MS spectrum in Figure 16, panel B, shows direct SFME-nanoESI analysis of 200 ng mL epitestosterone in synthetic urine. 5 μL water containing 50 mM hydroxylamine was used for the reagent liquid plug. Figure 16, panel C, shows reactive SFME-nanoESI analysis of 200 ng mL epitestosterone in synthetic urine. 5 μL water containing 50 mM hydroxylamine was used for the reagent liquid plug.

[0039] [Figure 17] Figure 17, panel A, shows the reaction scheme for the enzymatic conversion of acetylthiocholine (ATCh) to thiocholine (TCh) catalyzed by cholinesterase (ChE). Figure 17, panel B, shows the progress curve of ATCh digestion determined by SFME-nanoESI. Incubation was performed for 30 minutes and catalyzed by blood cholinesterase (ChE) at room temperature. Iodinated acetylthiocholine standards were spiked into human whole blood to a final concentration of 1.8 mg / mL before incubation. The intensity ratio of product ions from thiocholine (m / z 102→61) and the enzyme substrate (m / z 162→103) was monitored using MRM. Figure 17, panel C, shows the evaluation of blood ChE with different levels of enzyme inhibition. ChE activity in blood samples was determined by SFME-nanoESI after 5 minutes of incubation.

[0040] [Figure 18]Figure 18, panel A, shows SFME-nanoESI-MS spectra recorded for SFME-nanoESI MS analysis of samples (5 μL each) obtained immediately (top) and 60 minutes later (bottom) after mixing acetylthiocholine into diluted blood. Figure 18, panel B, shows an investigation of the effect of extraction solvents on enzyme activity. For testing of specific solvents, a 5 μL blood sample (diluted) was injected into a capillary tube along with an extraction solvent plug immediately after mixing acetylthiocholine into the blood. After 5 minutes of incubation in the capillary, the TCh / ATch ratio was measured using SFME-nanoESI MS. For control analysis, 5 minutes after mixing acetylthiocholine into diluted blood, a sample was taken and immediately analyzed using SFME-nanoESI MS with ethyl acetate as the extraction phase. A spray voltage of 1.5 kV was used for nanoESI.

[0041] [Figure 19] Figure 19 shows a comparison of different organic solvents for the extraction phase used in SFME-nanoESI. For each experiment, bovine whole blood spiked with 5 ng mL methamphetamine was diluted 10-fold with water, and 5 μL of the sample was then used to prepare a sample plug, and 5 μL of one organic solvent was used for the extraction plug. SRM (single reaction monitoring) with the transition m / z 150 → 91 was used to record the intensity of the product ion m / z 91 from protonated methamphetamine m / z 150 while the spray voltage was varied.

[0042] [Figure 20] Figure 20, panels A-D, show MS / MS analysis of low concentrations of drugs or steroids in 5 μL biological samples (undiluted). Figure 20, panel A, shows 0.5 ng mL methamphetamine in bovine whole blood. Figure 20, panel B, shows 0.5 ng mL amitriptyline in bovine whole blood. Figure 20, panel C, shows 0.8 ng mL verapamil in bovine whole blood. Figure 20, panel D, shows 9 ng mL epitestosterone in synthetic urine using reactive SFME.

[0043] [Figure 21] FIG. 21 shows the derivatization and MS / MS fragmentation pathway of epitestosterone and a table providing an overview of target ions for MS / MS analysis of steroids using reactive SFME-nanoESI.

[0044] [Figure 22] FIG. 22 is a table showing the LODs obtained using SLME nanoESI and the cutoff concentrations for detection or monitoring. DETAILED DESCRIPTION OF THE INVENTION

[0045] (Detailed explanation) The present invention provides systems and methods for slug flow microextraction (SFME), optionally followed by ionization of extracted analytes for rapid analysis of samples. The systems and methods of the present invention are useful for the analysis of analytes in any commercial or research field, such as biomedical, pharmaceutical, food safety, and environmental fields.

[0046] In one embodiment, the present invention provides a system for analyzing analytes in a sample. FIG. 1A provides an exemplary embodiment of the system of the present invention. The system includes an ionization probe and a mass spectrometer. The ionization probe includes a hollow body with a distal tip. Many different types of hollow bodies can be envisioned by those skilled in the art, all of which can cooperate with the system of the present invention. The hollow body can have a distal tip for ejecting a spray of solvent loaded into the probe. An exemplary hollow body is a nano-ESI probe capillary with a distal tip. Exemplary nano-ESI probes are described, for example, in Karas et al. (Fresenius J Anal Chem. 366(6-7):669-76, 2000) and El-Faramawy et al. (J Am Soc Mass Spectrom, 16:1702-1707, 2005), 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 that includes one or more spray tips and one or more electrodes.

[0047] An exemplary hollow body is a 0.86 mm inner diameter borosilicate glass capillary tube with an elongated tip. The tip may typically have 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 include PEEK tubing (polyetheretherketone polymer tubing) or TEFLON® tubing (polytetrafluoroethylene (PTFE) polymer tubing), or TYGON tubing (flexible tubing made from a variety of base materials).

[0048] An exemplary hollow body is a 0.5 mm or 0.25 mm internal diameter fused silica capillary tube, with or without an extended tip.

[0049] As shown in FIG. 1A, the hollow body is loaded with at least two immiscible fluids, such as a solvent and a sample that is immiscible with the solvent, and extraction occurs within the hollow body of the probe. These aspects of the invention are discussed in more detail below. In certain embodiments, to perform extraction within the probe body, the body should be devoid of any other material. For example, there is no substrate (e.g., a porous substrate such as a paper substrate), filter, beads, gel, or other substance disposed within the body. Rather, the body remains completely empty, devoid of other material, to receive the immiscible fluids that would be involved in the extraction.

[0050] In certain embodiments, magnetic beads are added to the sample and solvent plugs, and an alternating magnetic field is applied to induce movement of the magnetic beads inside the liquid plugs, thereby promoting turbulent flow inside each plug for transport of analytes to and from the liquid-liquid interface.

[0051] In some embodiments, the inner surface of the body is coated to adjust the hydrophobicity of the inner surface of the body. The hydrophobic region may be coated on the surface using known techniques, such as photolithography, printing, or plasma treatment. Martinez et al. (Angew. Chem. Int. Ed. 2007, 46, 1318-1320); Martinez et al. (Proc. Natl. Acad. Sci. USA 2008, 105, 19606-19611); Abe et al. (Anal. Chem. 2008, 80, 6928-6934); Bruzewicz et al. (Anal. Chem. 2008, 80, 3387-3392); Martinez et al. (Lab Chip 2008,8,2146-2150); and Li See, e.g., S. et al. (Anal. Chem. 2008, 80, 9131-9134), the contents of each of which are incorporated herein by reference in their entirety. In certain embodiments, the body is prepared to have a uniform hydrophobicity. In other embodiments, the body can be prepared to have multiple distinct regions, each with a different hydrophobicity that may be based on the type of liquid that fills that region of the body. For example, the region of the body that receives an oil-based sample can be treated to be more hydrophobic than the region of the body that receives water and methanol-based solvents.

[0052] In some embodiments, the hollow body is configured so that the electrode is not disposed on the surface of the body. Instead, the electrode is disposed at least partially within the hollow body. As shown in FIG. 1A, the electrode can be a metal wire extending into the hollow body. Any metal typically used for electrodes can be used for the metal electrode. The metal wire is connected to a voltage source, such as a high-voltage source. The length of the metal wire shown in FIG. 1A is merely exemplary. The metal wire can extend any length into the hollow body. The metal wire can extend to the distal end of the hollow body, as shown in FIG. 1A. Alternatively, the metal wire can not extend as far into the body and can be much shorter than shown in FIG. 1A. The amount of solvent added to the hollow body can determine the length of the metal wire, as the wire should extend sufficiently into the body to interact with the solvent added to the body.

[0053] As shown in FIG. 1A, the metal wire may be coaxially disposed within the hollow body, but this is not required. Typically, the metal wire does not touch the walls of the hollow body, as shown in FIG. 1A. The metal wire electrode and its connector can be removably or permanently attached to the hollow body. As shown in FIG. 1A, the metal wire electrode and its connector 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 an embodiment, the metal wire electrode and its connector are removed from the hollow body, leaving an opening through which fluid can be introduced into the body. Once introduced, the metal wire electrode and its connector are attached to the hollow body, sealing the hollow body.

[0054] 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 coupling 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.

[0055] As shown in FIG. 1A, the introduction of a high voltage to the liquid within the hollow body causes the liquid to be ejected from the distal tip of the hollow body in the form of a spray. The inlet of the mass spectrometer is operatively positioned to receive the liquid ejected from the probe. This distance is typically less than 10 mm; however, any distance that allows a signal from the sample to be generated in the mass spectrometer is suitable. This distance can be determined by one skilled in the art by simply adjusting the spacing between the probe and the inlet of the mass spectrometer and monitoring the readings generated by the mass spectrometer.

[0056] In other embodiments, the outer wall of the extended tip can be coated with a metal, and a high voltage can be applied through the metal coating for spray ionization.

[0057] Any type of mass spectrometer known in the art can be used in conjunction 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. Exemplary miniature mass spectrometers are described, for example, in Gao et al. (Z. Anal. Chem, 2006, 78, 5994-6002), the contents of which are incorporated herein by reference in their entirety. Compared to pumping systems used for laboratory-scale instruments involving thousands of watts of power, miniature mass spectrometers generally require pumping of 5 L / min (0.3 m) for the system described in Gao et al. 3 and smaller pumping systems, such as an 18 W pumping system with only a 1 L / s turbo pump and a 1 L / s diaphragm pump. Other exemplary miniature mass spectrometers are described, for example, in Gao et al. (Anal. Chem., 80:7198-7205, 2008), Hou et al. (Anal. Chem., 83:1857-1861, 2011), and Sokol et al. (Int. J. Mass Spectrom., 2011, 306, 187-195), the contents of each of which are incorporated herein by reference in their entirety. Miniaturized mass spectrometers have also been described, for example, by Xu et al. (JALA, 2010, 15, 433-439); Ouyang et al. (Anal. Chem., 2009, 81, 2421-2425); Ouyang et al. (Ann. Rev. Anal. Chem., 2009, 2, 187-214); Sanders et al. (Euro. J. Mass Spectrom., 2009, 16, 11-20); Gao et al. (Anal. Chem., 2006, 78(17), 5994-6002); Mulligan et al. (Chem. Com., 2006, 1709-1711); and Fico et al. al. (Anal. Chem., 2007, 79, 8076-8082), the contents of each of which are incorporated herein by reference in their entirety.

[0058] In some embodiments, the mass spectrometer inlet 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.

[0059] In certain embodiments, the ionization probes of the present invention operate without pneumatic assistance. That is, with 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 in conjunction with the systems of the present invention to assist in desolvation. The nebulizing gas may be either pulsed or provided as a continuous flow. In other embodiments, a gas-generating device is operably coupled to the probe so as to inject gas into the hollow body and push the sample and solvent to the distal tip of the probe. The gas is typically an inert gas such as nitrogen or argon, but could also be air.

[0060] In certain embodiments, the ionization probe is kept discrete (i.e., separate or decoupled) from a solvent flow, such as a continuous flow of solvent. Instead, discrete amounts 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 within the probe containing the extracted analytes, which is held in front of the mass spectrometer.

[0061] FIG. 1B illustrates an exemplary method of use for a system of the present invention. In certain embodiments, such a method involves introducing a solvent into a hollow body including a distal tip. A sample is also introduced into the hollow body. The solvent is immiscible with the sample and extracts at least one analyte from the sample into the solvent. A voltage is applied to the solvent containing the extracted analyte within the hollow body such that the analyte is ejected from the distal tip of the body, thereby generating analyte ions. These ejected ions are then analyzed.

[0062] FIG. 1B shows two immiscible phases, a liquid sample and an organic solvent, injected adjacently into a capillary tube with an extended tip. Given the different polarities of the different phases, one or more analytes migrate from the sample into the solvent (analyte extraction from the sample into the solvent). This extraction process can be facilitated by moving the liquid phase back and forth within the capillary, such as by tilting the capillary or applying gas pressure, to facilitate microextraction. The liquid phase can then be pushed as the extraction phase reaches the extended tip of the capillary by applying gas pressure (from a gas-generating device operably coupled to the probe). A wire electrode is inserted into the extraction solvent and applies a DC voltage for nano-ESI. The voltage causes the solvent to exit the distal tip of the hollow body as a spray that reaches the inlet of the mass spectrometer.

[0063] The methods of the present invention can be used with any type of sample, organic or inorganic, biological or non-biological, etc. 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 derived from the sample or may be introduced into the sample. Exemplary analytes include therapeutic drugs, drugs of abuse, and other biomarkers. Examples herein demonstrate that effective suppression of matrix effects has been achieved for therapeutic drugs, drugs of abuse, and other 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.

[0064] The solvent can be any solvent as long as it is immiscible with the sample and functions for both sample extraction and ionization. Typically, the solvent selected will depend on the sample to be analyzed and / or the analytes of interest believed to be in the sample (FIG. 19). A factor to consider is the polarity of the solvent. In a two-phase extraction system, ideally, the solvent has a different polarity than the sample and / or the analytes of interest believed to be in the sample. For example, aqueous samples typically have high polarity, so a good choice of solvent would be an organic solvent with low polarity (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 samples typically have low polarity, so a good choice of solvent would be a solvent with higher polarity, such as a water / methanol mixture. One skilled in the art will be able to determine the appropriate solvent to use based on the sample to be analyzed.

[0065] Another consideration for a solvent is that it be good for extracting analytes from a sample and also be able to be used to ionize the sample. That is, the solvent can be compatible with both extraction and ionization of the extracted analytes. As illustrated in the examples, methanol and ethyl acetate work well for extracting analytes and ionizing the analytes, while chloroform works well for extraction but not for ionizing the analytes. Typically, solvents compatible with electrospray ionization can potentially 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 be familiar with specific solvents that are compatible with electrospray ionization.

[0066] The methods of the present invention can also involve real-time chemical reactions that can be used to improve the overall analytical efficiency of target analytes. To perform such derivatization, a solution containing an agent that imparts a charged functional group to the analyte is introduced into the hollow body. This solution is typically introduced between the solvent and the sample. The agent in the solution interacts with the analyte in the sample, imparting a charged functional group to the sample and allowing for ionization of the analyte.

[0067] In certain embodiments, more than one analyte (e.g., multiple analytes) are extracted from a sample into a solvent. Multiple analytes can be extracted simultaneously. Alternatively, analytes are typically differentially extracted into a solvent based on the polarity of the analyte and the polarity of the solvent.

[0068] Although the methods of the present invention have been discussed using two immiscible fluids, the systems and methods of the present invention are not limited to the use of two fluids. Any number of fluids can be used with the systems and methods of the present invention, including three, four, five, and so on. In certain embodiments, a three-fluid system is used. In such embodiments, two miscible fluids are separated by an immiscible fluid. An exemplary three-fluid system is shown in FIG. 6. The polarity of the sample-solvent bridge-extraction / spray solvent can be high-low-high or low-high-low. A capillary surface with appropriate hydrophobicity can be selected to stabilize the solvent bridge, which separates the sample phase and extraction solvent phase of similar polarity (meaning they are miscible). As an example, a urine sample plug and a methanol / water plug for extraction can be separated by ethyl acetate or hexane, and a Teflon capillary with a hydrophobic surface can be used.

[0069] In certain embodiments, the systems and methods of the present invention can also be used to prepare samples that will be subsequently analyzed. The extraction solvent can be stored as a liquid sample or deposited on a paper substrate or MALDI plate to prepare dried sample spots. Internal standards can be incorporated into the dried sample spots during the SFME process. Target analytes can be chemically modified during the SFME process.

[0070] In other embodiments, the hollow body does not require a distal tip because the extraction capillary is not used as an ionization probe. In such embodiments, extraction simply occurs within 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, as shown in FIG. 6, and then analyzed by mass spectrometry. In other embodiments, the analytes are analyzed in a different manner, such as any spectroscopy technique or other assay known in the art.

[0071] (Incorporated by reference) References and citations to other documents are made throughout this disclosure, including patents, patent applications, patent publications, journals, books, articles, web content, etc. All such documents are incorporated herein by reference in their entirety for all purposes.

[0072] (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 a complete reading of this document, including the scientific and patent references cited herein. The subject matter herein contains important information, examples, and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents. [Example]

[0073] Example 1: Microextraction Protocol The system and method of the present invention were used to analyze 10 μL urine samples containing benzoylecgonine, nicotine, or methamphetamine, achieving LODs of over 1 ng / mL (Figure 2, panels A-C). Chemical equilibrium was achieved more rapidly with a higher ramp rate (approximately 30 min-1). Significant signal improvements were observed even for analytes with relatively low partition coefficients to the extraction solvent, due to effective suppression of matrix effects. Different solvents were tested for extraction. Nonpolar solvents such as chloroform were found to be efficient for extraction but relatively poor for subsequent ionization. Online injection of methanol could be used to facilitate direct ionization of analytes extracted into these solvents. However, ethyl acetate was found to be effective for both extraction and ionization, such as by nano-ESI. Various methods were also explored for incorporating an internal standard for quantification while keeping the operating procedure simple. Calibration of nicotine with good linearity (R = 0.99) was obtained (Figure 2, panel B).

[0074] Example 2: Real-time derivatization The method of the present invention can also involve real-time chemical reactions, which can be used to improve the overall analytical efficiency of target analytes. This is exemplified by the analysis of steroids in urine. The efficiency was expected to be high for extracting steroids from urine. However, steroids are difficult to ionize by spray ionization. Real-time derivatization was performed for SFME-nanoESI by injecting 3 μL of an aqueous solution with 5% hydroxylamine between the extraction solvent (ethyl acetate) and the urine sample (Figure 3A-B). The reactant solution, which was rapidly mixed with the sample and steroids, was derivatized with charged functional groups while being extracted into the organic phase. The signal in the MS spectrum improved by several orders of magnitude. LODs of 0.2, 0.7, 0.6, and 0.8 ng / mL were obtained for 5α-androstan-3β, 17β-diol-16-one, epitestosterone, 4,6-cholestadien-3-one, and stigmastadienone, respectively, in urine samples of less than 10 mL. (Reaction scheme) [ka] (product ions) Scheme 1. Reaction between hydroxylamine and carbonyl groups on steroids

[0075] Figure 5, panels A-D, show MS / MS spectra obtained using reactive slug-flow microextraction nanoESI combined with hydroxylamine as a reagent. In synthetic urine, 10 μL of 8 ng / mL epitestosterone (Figure 5, panel A), 5 ng / mL 5α-androstan-3β,17β-diol-16-one (Figure 5, panel B), 5 ng / mL 6-dehydrocholestenone (Figure 5, panel C), and 5 ng / mL stigmastadienone (Figure 5, panel D) were added. A 5 μL aqueous solution containing 0.1% acetic acid and 10% hydroxylamine was added as the reagent phase. 5 μL ethyl acetate was used as the extraction phase.

[0076] Analysis of such samples would otherwise need to be performed using conventional laboratory procedures using sample extraction, liquid chromatography, and mass spectrometry with electrospray ionization or atmospheric pressure chemical ionization. The sample volumes required are significantly larger (approximately 1 mL).

[0077] Example 3: Direct analysis of biological fluids with low viscosity Biological samples such as urine were directly analyzed using SFME nanoESI. Figure 2, panels A and C, show the calibration curves for the quantification of methamphetamine (Figure 2, panel A), nicotine (Figure 2, panel B), and benzoylecgonine (Figure 2, panel C) in synthetic urine samples. 10 μL synthetic urine containing drug and internal standard was used as the sample for measurement. 5 μL ethyl acetate (EA) was used as the extraction phase for extraction, purification, and spraying. Internal standards: methamphetamine-d8 (0.8 ng / mL), nicotine-d3 (2 ng / mL), and benzoylecgonine-d3 (1 ng / mL). Single reaction monitoring (SRM) transitions used: methamphetamine m / z 150 → 91; methamphetamine-d8 m / z 158 → 93; nicotine 163 → 130, nicotine-d3 m / z 166 → 130; benzoylecgonine m / z 290 → 168; benzoylecgonine-d3 m / z 293 → 171. Partition coefficient: LogP methamphetamine =2.07;LogP nicotine =1.17, LogP benzoylecgonine =-0.59.

[0078] Matrix effects due to high salt concentrations were minimized. Good LODs were also obtained for benzoylecgonine, a drug of abuse, which has a relatively low partition coefficient with respect to the extract phase. The partition coefficient (LogP) is defined as: LogP=log([solute] octanol / [solute] water ) (This represents the differential solubility of a non-ionized compound in an organic phase, such as octanol, that is immiscible with the aqueous phase at equilibrium.)

[0079] Example 4: Direct analysis of viscous biofluids For viscous biofluid samples, sample dilution was applied to enable operation with the system and method of the present invention. As an example, a blood sample containing a drug was diluted 10-fold before analysis by SFME nanoESI. The data in Figure 4, panels A and B, show that the method of the present invention was able to analyze analytes from blood samples. Figure 4, panels A and B, show MS / MS spectra obtained using slug-flow microextraction nanoESI. Bovine blood samples containing 40 ng / mL nicotine (Figure 4, panel A) and 40 ng / mL methamphetamine (Figure 4, panel B), respectively, were diluted 10-fold with water and then analyzed using SFME nanoESI. 10 μL of diluted sample, 5 μL ethyl acetate, were used.

[0080] Example 5: Summary of analytical performance [Table 1]

[0081] Example 6: Direct analysis of oil samples The preceding examples demonstrate that drug compounds in highly polar aqueous samples, such as blood or urine, were extracted into less polar organic solvents. The systems and methods of the present invention can also be applied to extracting analytes from less polar samples, such as oils, into more polar extraction solvents, such as water / methanol, as shown in FIG. 7 . Results are shown in FIG. 8 , which demonstrates the analysis of vegetable oils using the systems and methods of the present invention. A mixture of water and methanol was used as the extraction solvent. Panel A of FIG. 8 is an MS spectrum showing diacylglycerol and triacylglycerol species observed in the MS spectrum in positive mode. Panel B of FIG. 8 is an MS spectrum showing different fatty acids observed in the MS spectrum acquired in negative mode.

[0082] Example 7: Three-phase method A three-phase method can be performed as illustrated in Figure 6. The polarity of the sample-solvent bridge-extraction / spray solvent can be high-low-high or low-high-low. A capillary surface with appropriate hydrophobicity can be selected to stabilize the solvent bridge (central phase), which separates the sample phase and the extraction solvent phase of similar polarity (meaning they are miscible). As an example, a urine sample plug and a methanol / water plug for extraction can be separated by ethyl acetate or hexane, and a Teflon capillary with a hydrophobic surface can be used. Analysis of phenylalanine from urine is shown in Figure 9, panels AB. Phenylalanine is relatively highly polar. Phenylalanine molecules were extracted from urine with HO:MeOH (1:1) through hexane, which separates them from the salts in the urine. This is a purification process.

[0083] When a two-phase method involving urine and hexane is used, phenylalanine is relatively highly polar, and therefore its solubility in hexane is relatively low, resulting in a low concentration. Also, hexane is less favorable for spray ionization than polar solvents such as HO:MeOH (1:1). A three-phase method involving sample-bridge-spray in a high-low-high polarity order allows highly polar compounds to be concentrated in the highly polar solvent, which is suitable for spray ionization. Subsequent analysis is performed by transferring the extraction solvent to a capillary tube with an extended tip for spray ionization (Figure 6) or using direct spray ionization from the capillary tube, as previously described.

[0084] Real-time chemical derivatization can be applied by adding a reaction reagent into either or both of the crosslinking solvent or the extraction / spray solvent. Real-time internal standard incorporation can be applied by pre-adding an internal standard into either or both of the crosslinking solvent or the extraction / spray solvent.

[0085] Example 8: Microextraction in fused silica tubing (inner diameter 500 μm) SFME sample processing can be performed in smaller diameter fused silica tubing (e.g., tubing with an internal diameter of 500 μm or less), which is typically used as a liquid line in a liquid chromatography system. Extraction can be induced by applying pushing and pulling forces to one side of the tubing. The extract can either be analyzed directly by nanoESI or stored for further processing.

[0086] Figure 10 shows the analysis of 50 ng / mL amitriptyline in bovine whole blood. The MS / MS spectrum of the molecular ion was collected. The blood sample was first diluted 10-fold using HO to reduce viscosity. For extraction, 5 μm of the diluted sample was processed in fused silica tubing (500 μm inner diameter) using the method of the present invention. The extract was then injected into a nanoESI emitter and analyzed by nanoESI.

[0087] Example 9: Direct mass spectrometry analysis of biofluid samples using slug flow microextraction nanoESI Direct mass spectrometry (MS) analysis of biofluids with a simple procedure represents an important step in the transition of MS technology to clinical and point-of-care applications. The current study reports the development of a single-step method using slug flow microextraction and nano-ESI (electrospray ionization) for MS analysis of organic compounds in blood and urine. High sensitivity and quantification accuracy have been achieved for the analysis of therapeutic and illicit drugs in 5 μL samples. Real-time chemical derivatization has been incorporated to analyze anabolic steroids. Enzyme function monitoring has also been demonstrated using cholinesterase in wet blood. The reported research encourages the future development of simple, highly functional disposable cartridges to replace the traditional complex laboratory procedures for MS analysis of biological samples.

[0088] Mass spectrometry (MS) has proven to be a powerful tool for chemical and biological analysis. High specificity, sensitivity, and precision in quantification are traditionally achieved in the laboratory by eliminating matrix effects through sample extraction and chromatographic separation prior to MS analysis. The development of atmospheric pressure ionization, particularly with the recent demonstration using paper spray, has shown promising promise for direct MS analysis using a highly simplified protocol that consumes very small sample volumes while maintaining high quantitative performance. This may be crucial for the transition of MS analysis to non-laboratory applications, particularly point-of-care (POC) diagnostics. The underlying principle for successful development along this line is to minimize sample consumption and achieve high efficiency within an integrated process for analyte extraction and ionization. Slug flow microextraction (SFME) and nano-ESI (electrospray ionization) can be combined to perform one-step analysis of biofluid samples. Excellent sensitivity and high quantitative precision have been obtained using as little as 5 μL of blood and urine samples. More importantly, the SFME-nanoESI method demonstrated how to incorporate a variety of different processes, including liquid-liquid extraction, incorporation of internal standards (IS), chemical derivatization, or even enzymatic reactions, that are necessary for high-performance mass spectrometry analysis using a simple device.

[0089] All experiments were performed using a TSQ Quantum Access Max (Thermo Fisher Scientific, San Jose, CA, USA). Bovine blood was purchased from Innovative Research Inv. (Novi, MI, USA). Human pooled blood for enzyme reaction studies was purchased from Bioreclamation IVT (Baltimore, MD, USA). Synthetic urine was purchased from CST Technologies (Great Neck, NY, USA). Steroids were purchased from Steraloids Inc. (Newport, RI, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0090] A disposable glass capillary tube with an inner diameter of 0.8 mm (Figure 11, panel A) and an extended tip for nanoESI was used to perform the entire sampling-ionization process. Two adjacent liquid plugs were formed by sequentially injecting 5 μL of organic solvent and 5 μL of urine or blood sample into the capillary tube. Liquid-liquid extraction of analytes from biofluids into the organic solvent was expected, but was very inefficient due to the small interfacial contact area. However, the extraction rate could be significantly improved using slug flows induced by the movement of the two liquid plugs, which could be promoted by tilting the capillary tube (Figure 11, panel A and Figure 12, panel A) or by applying pushing and pulling forces through air pressure (Figure 12, panel B). The slug flows formed due to friction with the capillary wall, and the inner flow of each plug (Figure 11, panel A) transported analytes to and away from the liquid-liquid interface, thus significantly improving extraction efficiency. After the extraction process, the organic solvent plug can simply be pushed to the tip of the capillary. A stainless steel wire is then inserted through the biofluid sample to reach the organic solvent plug. A high voltage is applied to generate nanoESI for MS analysis (Figure 11, panel B). The selection of the organic solvent is important; it should be immiscible with the biofluid sample, have good solubility for the target analytes, and be suitable for nanoESI. Several organic solvents were tested (Figure 19), and the weakly polar ethyl acetate was found to provide optimal performance for analyzing a wide range of chemical compounds in urine (Figure 11, panels CD) and blood samples (Figure 13, panels AB).

[0091] The slug-flow extraction process was shown to be highly efficient when tested to extract methamphetamine, nicotine, and benzoylecgonine (a major metabolite of cocaine) from urine samples. Equilibrium was achieved after tilting the capillary tube five times (Figure 11, panel E and Figure 20, panels A-D). A good limit of detection (LOD) of 0.05 ng / mL for verapamil was obtained for whole blood samples using SFME-nanoESI (Table 2). [Table 2]

[0092] When blood samples were diluted to reduce viscosity, fewer extraction cycles were required to reach equilibrium. The distribution of analytes between the sample and extraction phases can be relatively estimated by the partition coefficient (logP, see Figure 14). In the case of methamphetamine, with a logP of 2.1, its concentration in the organic extraction solvent can be 100-fold higher than in the urine sample after SFME, clearly explaining the favorable LOD of 0.03 ng / mL achieved with the urine sample (Table 1). The logP value for benzoylecgonine was -0.6, which means that it has a higher solubility in urine than in organic solvents, and extraction into ethyl acetate was a dilution process. However, an LOD of 0.08 ng / mL was still achieved. This indicates that the limiting factor in the detection of benzoylecgonine in raw urine samples may not be the absolute amount or concentration of benzoylecgonine, but rather interference from matrix effects such as ionization suppression due to the high concentration of salt in the urine sample. Efficient separation of benzoylecgonine from salt was achieved in the SFME process. Even at low concentrations of benzoylecgonine in the extraction phase, the ionization efficiency and overall sensitivity of the analysis were significantly improved.

[0093] In addition to sensitivity, adequate precision in quantification is often essential for clinical and POC applications. A simple means for accurate incorporation of an internal standard is important but can be challenging for the small sample volumes collected by minimally invasive methods. Using SFME-nanoESI, IS compounds can be spiked into the extraction phase (Figure 15) and subsequently mixed with the analyte during the slug-flow extraction process. This method was tested for the quantification of methamphetamine in bovine blood samples with methamphetamine-d8 when the IS was spiked into ethyl acetate at 2 ng / mL. The blood samples were diluted 10-fold and then analyzed using SFME-nanoESI and MRM analysis (transitions m / z 150 to 91 and m / z 158 to 94 for the analyte and IS, respectively) (Figure 15, inset). The measured analyte / IS ratio (A / IS) was plotted as a function of the original analyte concentration in blood, as shown in Figure 15. Good linearity was obtained, which is governed by the partitioning process (see derivation in the Supplementary Information). RSDs of better than 10% were obtained for samples with concentrations higher than 10 ng / mL.

[0094] Chemical derivatization is an effective method for modifying the properties of target analytes to improve the efficiency of separation or ionization for MS analysis. For example, steroids in urine or blood samples are expected to be well extracted into the organic phase using SFME. However, the efficiency for subsequent ionization by nanoESI will be low due to the low proton affinity of the steroid molecules. Reaction with hydroxylamine has previously proven effective in improving the ionization efficiency of steroids and was therefore used as an example in this study. An additional liquid plug of 5 μL water containing 50 mM hydroxylamine was added to 5 μL ethyl acetate and 200 ng / mL hydroxylamine. -1A 5 μL urine sample spiked with epitestosterone was injected between the hydroxylamine solution and the urine sample (Figure 16, panel A). Using five SFME cycles, the hydroxylamine solution was well mixed with the urine sample. MS / MS analysis of the reaction product m / z 304 produced a spectrum with significantly improved signal-to-noise ratio (S / N) (Figure 16, panels B-C and Figure 21). Reactive SFME-nanoESI was applied to analyze a series of anabolic steroids in a 5 μL urine sample, including epitestosterone, 6-dehydrocholestenone, 5α-androstan-3β,17β-diol-16-one, and stigmastadienone, and LODs of 0.7, 0.6, 0.2, and 0.8 ng / mL were obtained, respectively (Table 1 and Figure 22).

[0095] By using a liquid-liquid extraction process with SFME, analyses can now be performed directly using wet blood samples. This provides an opportunity to investigate chemical and biological properties that are only present with the original liquid sample. For example, the enzymatic function of proteins is typically suppressed in dried blood spots or after conventional laboratory procedures for sample extraction. SFME-nanoESI was applied to monitor the enzymatic activity of cholinesterase (ChE) in whole blood samples. ChE promotes the enzymatic conversion of acetylthiocholine (ATCh) to thiocholine (TCh) (Figure 17, panel A). The blood sample was diluted 10-fold to slow the reaction rate and promote slug flow for SFME. The substrate, acetylthiocholine iodide, was added to the diluted blood sample at a concentration of 1.8 mg / mL. A 5 μL sample was then immediately withdrawn and injected into a capillary tube along with a 5 μL extraction phase. The capillary tube with the sample and extraction solvent was placed at room temperature (25°C) for incubation. SFME-nanoESI could be performed repeatedly on the same sample, and the ratio of the substrate ATCh to the reaction product TCh could be monitored as a function of time to characterize the enzymatic activity of ChE. A potential problem with this approach would be damage to enzyme function by organic solvents. The impact of the organic extraction phase was investigated for other solvents, such as ethyl acetate and chloroform, using a 5-minute incubation. The decrease in ChE activity due to contact with ethyl acetate was found to be minimal, whereas it was much more severe (>60% decrease) with chloroform. Weakly polar solvents, such as ethyl acetate, may better preserve the enzyme structure.

[0096] Using ethyl acetate as the extraction solvent, SFME-nanoESI was performed repeatedly over 30 minutes, with five cycles of SFME at 1500 V and five seconds (5 s) of nanoESI per analysis. The TCh / ACTh ratio, characteristic of ChE enzymatic activity, is plotted as a function of time in Figure 17, panel B. Enzyme inhibition studies were then performed as a validation of the method. Two ChE inhibitors, donepezil (a therapeutic drug for Alzheimer's disease) and ethion (a neurotoxicant), were spiked separately into blood samples to simulate different degrees of enzyme inhibition. The impaired enzyme activity was then determined using the SFME-nanoESI method with a 5-minute incubation. The measured deficits, compared to blood samples without added inhibitors, are reported in Figure 17, panel C, for blood samples treated with donepezil at 25 ng / mL and 5 μg / mL and ethion at 10 μg / mL. The observed % reduction is consistent with findings reported in previous studies.

[0097] In summary, the combination of slug flow microextraction and nano-ESI has enabled highly sensitive direct analysis of organic compounds in biofluids. Multiple types of sample processing processes, which previously required complex laboratory setups, can now be combined into a one-step analysis with greatly simplified operating procedures. Because biofluid samples are analyzed directly, rather than dried into spots, efficient liquid-liquid extractions can be designed based on their partitioning characteristics. The chemical and biological properties of wet biofluids can also be preserved and thereby characterized. The extraction process can be turned on and off by controlling the movement of the sample and extraction plugs. This enables online monitoring of chemical and biological reactions in biofluid samples as small as 5 μL. With increasing interest in translating MS technology into clinical applications, this development has profound implications for designing disposable sample cartridges with the appropriate functionality for direct analysis. This may ultimately lead to the elimination of traditional laboratory procedures, which require complex setups and specialized knowledge. Its implementation with a miniaturized mass spectrometer will provide a powerful solution for point-of-care diagnostics.

[0098] Example 10: Enzyme activity monitoring by SFME-nanoESI To initiate the enzymatic reaction, acetylthiocholine (final concentration 1.8 mg mL) was added to a human blood sample diluted 10-fold with phosphate-buffered saline (PBS). For the experiment generating the data for Figure 17, panel B, 5 μL of the acetylthiocholine-spiked blood sample was loaded into a capillary tube along with 5 μL of extraction solvent. The enzymatic reaction progress was determined by periodically performing SFME-nanoESIMS analysis of the substrate (m / z 162) and the reaction product thiocholine (m / z 120) (Figure 18, panels A-B). For each SFME-nanoESIMS analysis, a liquid plug was pushed to allow the extraction solvent to reach the tip of the capillary for spraying, and then pulled back after MS analysis. MRM was performed to measure the intensities of TCh (m / z 120 → 61) and ATCh (m / z 162 → 102). The ratio of TCh / ATCh was used to generate the plot in Figure 17, panel B. Three replicates were performed for each time point, and the standard deviations are marked by error bars in Figure 17 panel B.

Claims

1. 1. A method for extracting an analyte from a biological sample, the method comprising: estimating the distribution of the analyte between the biological sample and a solvent by a partition coefficient, thereby determining the solubility of the analyte in said solvent; (i) extracting the analyte from the biological sample into the solvent based on the analyte being more soluble in the solvent than in the biological sample, and (ii) selecting the solvent to be compatible with ionization of the analyte; introducing the solvent into a capillary tube, the capillary tube including an electrode disposed therein and coaxial with a wall of the capillary tube, and no electrode disposed on the wall of the capillary tube; introducing the biological sample containing the analyte into the capillary; moving the biological sample and the solvent back and forth within the capillary tube to induce circulation within the biological sample and the solvent, thereby extracting the analyte from the biological sample; analyzing the extracted analyte, wherein analyzing includes applying a voltage to the solvent containing the extracted analyte in the capillary such that the analyte is expelled from the capillary, thereby producing ions of the analyte, and analyzing the ions in a mass spectrometer; A method comprising:

2. The method of claim 1 , wherein the solvent is first introduced into the capillary tube.

3. The method of claim 1 , wherein the biological sample is first introduced into the capillary tube.

4. stopping movement of the biological sample and the solvent within the capillary; removing a portion of the solvent; analyzing the amount of analyte extracted into the solvent; The method of claim 1 further comprising monitoring the extraction by:

5. The method of claim 4 , further comprising resuming movement of the biological sample and the solvent within the capillary tube based on the results of the analyzing step.

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

7. The method of claim 1 , wherein the method further comprises introducing an agent that imparts a charged functional group to the analyte.

8. The method of claim 1 , wherein analyzing comprises introducing the ions into a benchtop mass spectrometer or a miniature mass spectrometer.

9. The method of claim 1 , wherein the solvent is introduced into the capillary tube prior to the introduction of the biological sample.

10. 10. The method of claim 1, wherein multiple analytes are extracted into the solvent.

11. 11. The method of claim 10, wherein the analytes are differentially extracted in time into the solvent based on the different polarities of the analytes.

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