Patent Document

RELATED APPLICATION 
     This application is related to and claims the benefit of U.S. Provisional Patent Application Ser. No. 61/510,197, filed on Jul. 21, 2011, the disclosure of which is incorporated herein by reference. 
    
    
     TECHNICAL FIELD 
     The present invention is related to apparatii for coupling liquid chromatography (LC) with mass spectrometry (MS) and methods of using the same. 
     BACKGROUND 
     Ambient mass spectrometry is a recent advancement in the field of analytical chemistry and has allowed for the analysis of samples with little-to-no sample preparation. Based on this concept, a variety of ambient ionization methods have been introduced, including desorption electrospray ionization (DESI), direct analysis in real time (DART), desorption atmospheric pressure chemical ionization (DAPCI), electrospray-assisted laser desertion/ionization (ELDI), matrix-assisted laser desorption electrospray ionization (MALDESI), extractive electrospray ionization (EESI), atmospheric solids analysis probe (ASAP), jet desorption ionization (JeDI), desorption sonic spray ionization (DeSSI), desorption atmospheric pressure photoionization (DAPPI), plasma-assisted desorption ionization (PADI), and dielectric barrier discharge ionization (DBDI). 
     DESI is a representative method for ambient mass spectrometry. It has been shown to be useful in providing a rapid and efficient means of desorbing and ionizing a variety of target compounds of interest under ambient conditions. A variety of analytes (for example, pharmaceuticals, metabolites, drugs of abuse, explosives, chemical warfare agents, and biological tissues) have all been studied with these ambient ionization methods. 
     In U.S. Pat. No. 7,915,579, DESI has been shown to analyze liquid samples without sample preparation and may be used for ionizing both small molecules and large biomolecules, such as proteins. Still, it would be useful to use the liquid DESI apparatus in combination with liquid chromatography for the analysis of mixtures; however, the high flow rates associated with some chromatography techniques, such as high performance liquid chromatography (“HPLC”), tend to overwhelm the conventional ion source, such as electrospray ionization (ESI). 
     Furthermore, after analyte separation by chromatography, some analytes are difficult to ionize; therefore, it is often necessary to include post-column derivatization. Typically, the protocol for derivatization is to introduce a chemical reagent solution that merges with the chromatographic eluent via a Tee mixer. Such a mixing causes an increased time delay for MS ionization, leading to peak broadening resulting from diffusion effects. 
     In addition, integration of electrochemical cells into a LC/MS system will broaden the application of LC/MS. Conventionally, the coupling of EC with MS has been accomplished with ionization methods such as thermospray (TS), fast atom bombardment (FAB), and electrospray ionization (ESI). In particular, the latter method is useful in ionizing non-volatile products or intermediates of electrochemical reactions. However, in coupling EC with ESI, the EC system needs to be electrically floated, or decoupled, from the ionization source to separate the high voltage operation of the ionization source from the low voltage operation of the EC cell. This decoupling increases the complexity of the apparatus and the methods of analysis. Also, in coupling EC with ESI-MS there is a limitation that the solvent for electrolysis in EC must be compatible with ESI ionization. The combination of DESI with the EC system has been shown in U.S. patent application Ser. No. 12/558,819, published as Application Publication No. 2010/0258717, the disclosure of which is incorporated herein by reference in its entirety. 
     Still, there remains a need for improving the performance of LC/MS to tolerate high flow elution rate of LC without a limitation in selecting a solvent for LC mobile phase and electrolysis, online post-column derivatization, and easy integration of EC cells into the LC/MS systems for broader application. 
     SUMMARY OF THE INVENTION 
     The present invention is premised upon the realization that liquid chromatography can be coupled with mass-spectrometry using desorption electrospray ionization (DESI). DESI provides a versatile interface which allows a wide range of elution flow rates online derivatization via reactive DESI and, further, combination with electrochemistry. 
     The objects and advantages of the present invention will be further appreciated in light of the following detailed description and drawings, in which: 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  is a diagrammatic depiction of the present invention; 
         FIG. 2  is a diagrammatic depiction of an alternate embodiment of the present invention; 
         FIG. 3  is a diagrammatic depiction of a second alternate embodiment of the present invention; 
         FIG. 4  is a diagrammatic depiction of a third alternate embodiment of the present invention utilizing an electrochemical cell; 
         FIG. 4A  is a diagrammatic depiction of a variation of the invention shown in  FIG. 4 , showing a commercial Reactorcell; 
         FIG. 5A  is a diagrammatic depiction illustrating an alalytical system for utilizing high sample flow rate DESI-MS; 
         FIGS. 5B-5D  are mass spectra acquired from DESI-MS; 
         FIGS. 6A-6C  are mass spectra obtained in accordance with Example 2; 
         FIG. 7A  is a UV chromatogram made in accordance with Example 3; 
         FIGS. 7B and 7C  are DESI-MS prepared according to the method of Example 3; and 
         FIGS. 8A-8F  are MS spectra of peptide fragments. 
     
    
    
     DETAILED DESCRIPTION 
     The liquid sample desorption electrospray ionization mass-spectrometry (LS-DESI-MS) apparatus was described in detail in U.S. Pat. No. 7,915,579, the disclosure of which is incorporated in its entirety herein by reference. Briefly,  FIG. 1  illustrates the LS-DESI-MS apparatus  10  where an analyte from a liquid sample  12  is ionized by desorption of the analytes by a charged and nebulized solvent  14  generated and emitted from a nebulizing ionizer  16  under ambient conditions. The LS-DESI-MS apparatus  10  forms gas phase ions that can be analyzed by a mass spectrometer  18 . 
     Operation of the LS-DESI-MS apparatus  10 , as shown in  FIG. 1 , begins by providing a sample supplied from a sample supply (labeled as source  22 ) to a High-Performance Liquid Chromatograph (“HPLC  24 ”), which, though not shown, generally includes a pump and at least one liquid chromatography column that is configured to elute the sample as one or more fractions in accordance with the particular stationary phase of the chromatography column, as is known by those of ordinary skill in the art. Generally, the flow rate of the sample through the HPLC  24  ranges from about 0.01 mL/min to about 3 mL/min. 
     The fractions may then be directed into an Ultraviolet-Visible Spectrometer (“UV-Vis  26 ”) where the absorption or transmittance, at a particular wavelength of light in the ultraviolet or visible range of the electromagnetic spectrum, of each fraction may be determined The amount of absorption or transmittance may be related to the presence and the amount of a particular analyte within the fraction. 
     The fractions may then pass into a tube  28  constructed from a non-reactive material, such as silica, stainless steel, or aluminum and that is configured to generate a continuous jet of the liquid sample  12 . While the dimensions of the tube  28  may vary, in some embodiments the tube  28  may have an inner diameter ranging from about 0.05 mm to about 0.1 mm. 
     At least a portion of the continuous jet of liquid sample  12  may then be desorbed by the charged and nebulized solvent  14  emitted from the nebulizing ionizer  16 . The nebulizing ionizer  16  may be a desorption electrospray ionization probe (“DESI probe”) that includes a housing  32  having a solvent conduit  34  surrounded by a gas conduit  36 ; however, it would be understood that other ambient ionization apparatus may alternatively be used. An outlet  38  of the gas conduit  36  is positioned about 0.1 mm to about 0.2 mm proximally to an outlet  40  of the solvent conduit  34 . The solvent conduit  34  can be constructed from a fused silica capillary with an inner diameter ranging from about 50 μm to about 100 μm. The gas conduit  36  can also be a fused silica capillary with an inner diameter that is generally larger than the outer diameter of the solvent conduit  34 , i.e., typically about 0.25 mm; however, these dimensions should not be considered limiting. 
     A voltage generator  42  is operable to charge the solvent within the solvent conduit  34 . 
     In using the DESI probe  16 , a solvent  43  is supplied to the solvent conduit  34 . While the particular solvent  43  used is dependent on the chemical nature of the liquid sample  12  in study, one example of an appropriate solvent mixture can be methanol and water with either 0.5% or 1% acetic acid, v/v, which is injected at a rate of about 5 μL/min to about 10 μL/min. A gas, typically an inert gas such as N 2 , is supplied to the gas conduit  36  at pressures ranging from about 8 bar to about 15 bar. The voltage generator  42  is activated and provides a voltage potential, typically ranging from about −5 kV to about 5 kV, to the solvent  43 . This generates an electrically-charged solvent within the solvent conduit  34 . 
     The now electrically-charged solvent traverses the solvent conduit  34  to the solvent conduit outlet  40 . There, the charged solvent is impacted by the surrounding high-pressure gas leaving the gas conduit outlet  38 . This high-pressure gas causes the flow of the charged solvent to be nebulized into a spray of the charged and nebulized solvent  14 , which then impacts the continuous jet of the sample  12 . This impact will cause desorption and ionization of a portion of the sample  12  into the mass spectrometer  18 . It will be readily appreciated that the angle by which the nebulized solvent  14  impacts the sample  12  may be varied to increase the likelihood of the liquid sample  12  entering the mass spectrometer  18 . 
     While not wishing to be bound by theory, it is believed that the mechanism by which the nebulized solvent  14  interacts with the sample  12  and desorbs at least a portion of the sample  12  may be chemical sputtering, charge transfer, or droplet pick-up, with the most likely of these mechanisms being droplet pick-up. During droplet pick-up, the nebulized solvent  14  interacts with the sample  12  to yield desorbed secondary charged droplets containing analyte. The secondary charged droplet will then undergo desolvation to yield ions of the analyte, i.e., gas phase ions. 
     The nebulizing ionizer  16  is interfaced to a cavity of the mass spectrometer  18 , which includes a mass filter  44  and the mass detector  46  maintained at vacuum. This interface can aid in desolving the solvent from the secondary charged droplet to form the ions of analyte. The ions of analyte enter the mass spectrometer  18  through an orifice  48  of a plate  50 , which provides an opening into the mass spectrometer  18  while maintaining vacuum pressures. The ions of analyte are then directed to a skimmer  52 , which can be constructed as a cone-shaped plate  54  having an orifice  56 , and is operable to focus the ions of analyte into a narrow beam (not shown) as it enters the mass spectrometer  18 . This skimmer  52  is typically grounded. In some embodiments, the mass spectrometer  18  can further include a separate focusing lens (not shown) between the skimmer  52  and the mass filter  44  to focus the ion beam and reduce the natural expansion of the ion beam by effusion through the orifice  56  of the skimmer  52 . 
     After passing the skimmer  52 , the ion beam is directed to the mass filter  44 . Conventional mass filters  44  include time-of-flight, quadrupolar, sector, Orbitrap, FT-ICR or ion trap, which are operable to cause ions of analyte having a specified mass-to-charge (m/z) ratio to transverse the mass filter  44  and be quantified at the mass detector  46 . One particularly suitable instrument is a Thermo Finnigan LCQ DECA or DECA MAX ion trap mass spectrometer (San Jose, Calif.). 
     For example, in operating a conventional quadrupole modality, an ion beam is directed through four parallel electrodes, wherein the four parallel electrodes are comprised of two pairs of electrodes. A radiofrequency field and a DC voltage potential are applied to each of the two pairs of electrodes by a power supply such that the two pairs differ in polarity of the voltage potentials. Only the ions within the ion beam having a particular m/z will continue through the parallel electrodes to the mass detector  56 ; that is, the ions will be equally attracted to and deflected by the two pairs of electrodes while the mean free path induced by the radiofrequency field onto the ion of analyte does not exceed distance between the electrodes. Thus, the ion of analyte having the particular m/z will balance the radiofrequency and DC voltage forces from the parallel electrodes, and will thereby traverse the parallel electrodes and impact the mass detector  46 . 
     The m 1 /z 1  ions that reach the mass detector  46 , typically a Faraday plate coupled to a picoammeter, are measured as a current (I) induced by a total number (n) of ions impacting the mass detector  36  over a period of time (t) and in accordance with n/t=I/e, wherein e is the elementary charge. 
     The method continues with altering the operational conditions of the mass filter  44  such that ions having a second mass-to-charge ratio, m 2 /z 2 , will traverse the mass filter  44  and impact the mass detector  46  in the manner described. A spectrum may then be generated relating the relative abundances with respect to m/z of the ions detected. 
     Operation of the mass filter  44  and the mass detector  46  can be by way of a controller  58 . A suitable controller  58  can be a standard PC computer; however, the present invention should not be considered so limited. 
     The use of the DESI probe  16  enables direct analysis of the continuous jet of the liquid sample  12  eluted from the HPLC  24  at a high flow rate whereas other ionization sources are unable to produce an ion signal due to flooding of the ion source. 
     While the DESI probe  16  is capable of producing an ion signal at high sample flow rates, decreasing the flow rate may provide greater sensitivity.  FIG. 2  illustrates another embodiment of a sample analysis system  59  wherein like reference numbers refer to like features in previous figures. In this embodiment, as the fraction elute from the column of the HPLC  24 , the fractions enter a tube  60  having a splitter  62  therein that diverts a first portion of the fractions (i.e., the liquid sample  12 ) to a first tube  66 , which is directed toward the nebulizing ionizer  16  and a second portion of the fractions to a second tube  68 , which is directed to a waste container  64 . The splitter  62  is adjustable such that the first portion may be adjusted relative to the second portion and in order to maximize the ion signal at the DESI probe  16 . 
     More particularly, the fractions passing through the first tube  66  move to a distally located opening  70  of the tube  66  that is positioned on a sample surface  72 . In the illustrative embodiment, the sample surface  72  includes a planar surface and can be constructed from any nonreactive material, such as polytetrafluoroethylene (PTFE). The design of the sample surface  72  can vary, but should be suitable to accommodate the tube  66  and the DESI probe  16 . In this way, at least a portion of the liquid sample  12  can be desorbed off the planar surface and directed substantially toward the mass spectrometer  18  according to methods discussed in detail above. Though not specifically shown, the tube  66 , which may be constructed in a manner that is similar the tube  28  of  FIG. 1 , may be affixed to the planar surface of the sample surface  72 , such as by a clamp, which will prevent movement of the opening  70 . 
     The second portion of the fractions enters the waste container  64  for proper disposal or other suitable labware for additional off-line analysis. 
       FIG. 3  illustrates another embodiment of an analysis system  73  that is similar to the system  59  of  FIG. 2  except that the second portion enters the UV-Vis  26  instead of the waste container  64 . In this way, the analytes eluted from the columns may be analyzed nearly simultaneously using both UV-Vis and mass spectroscopies. Furthermore, it would be readily understood that the UV-Vis modality is readily adaptable to high sample flow rates that may be associated with the second portion of the fractions. 
     Often times a particular analyte cannot be ionized or may have an m/z value that overlaps with the m/z value of another analyte (which may or may not be of interest). Accordingly, the particular analyte may undergo a derivatization reaction to form a chemical derivative that improves detectability of the particular analyte. Derivatization reactions may include a variety of mechanisms, but should generally form only one product that is stable for at least the detection period, cause the particular analyte to undergo a complete reaction, and should not change the structure of the particular analyte. In some embodiments, a selected chemical reagent may be doped with the DESI spray solvent to allow online derivatization of analyte eluted from the LC column which takes place during DESI ionization. 
     Turning now to  FIG. 4 , wherein like reference numbers refer to like features in previous figures, an analytical system  74  in accordance with another embodiment is shown. Generally, the analytical system  74  is similar to the system  73  of  FIG. 3  but further includes an electrochemical cell  74 , which is positioned at a distal end of the first tube  66 . 
     Electrochemical cells are devices that are used to generate oxidized or reduced species via electrochemical reactions. In the illustrative embodiment, the electrochemical cell  74  is a thin-layer electrochemical flow cell, such as a commercially-available REACTORCELL ( FIG. 4A ) or μ-PREPCELL ( FIG. 4 ) brands of thin-layer electrochemical cells available from Antec Leyden (Zoeterwoude, Netherlands); however, other types of electrochemical cells may also be used. The thin-layer electrochemical flow cell  74  includes a sample inlet  80  and a sample outlet  82  and is operably coupled to a potentiostat  84 . The potentiostat  84  provides and controls the electrical voltage levels supplied to a working electrode  86 , a reference electrode  88 , and a counter electrode  90  of the thin-layer electrochemical flow cell  74 . The working electrode  86  may be constructed from glass carbon or it may be a magic diamond (“MD”) electrode Antec Leyden (Zoeterwoude, Netherlands), and the counter electrode  90  may be a titanium block. 
     In use, the fractions elute from the column of the HPLC  24  and are pumped into the splitter  62 . The second portion may enter the UV-Vis  26  as shown herein, or the waste container  64  as shown in  FIG. 2 . The first portion (i.e., the liquid sample  12 ) enters the sample inlet  80  of the electrochemical cell  74  where the voltage potential applied by the working electrode  86  induces reduction or oxidation of a chemical species within the liquid sample  12 . The liquid sample  12  with the reduced/oxidized species flows through the sample outlet  82  and into another third tube  92 , which, as shown, may be angled (Ψ) with respect to horizontal (indicated as axis  92 ). The liquid sample  12  may be emitted from the third tube  92  may be directly desorbed from an opening  94  of the third tube  92 . The analytes within the liquid sample  12  may then be analyzed as described previously. 
     Accordingly, various embodiments have been shown that a DESI probe may be configured to produce a suitable ion signal at very high sample flow rates, such as those associated with an HPLC. Additionally, derivatization of the liquid sample, such as by online reaction accompany DESI ionization (i.e., by reactive DESI), may be implemented with the DESI probe without time delays, which greatly reduces the occurrence of peak broadening in mass spectroscopy detection. Finally, the combination of liquid chromatography with a DESI probe and an electrochemical cell provides particular analytical value for achieving fast structural analysis, such as disulfide bond analysis of in peptides from enzymatic digestion. 
     The invention will be further appreciated in light of the following examples: 
     Example 1 
       FIG. 5A  illustrates an analytical system for demonstrating high sample flow rate DESI-MS that was similar to the embodiment of  FIG. 1 . More specifically, the system included a Thermo Finnigan LCQ DECA or DECA MAX ion trap mass spectrometer (San Jose, Calif.) and a Perkin Elmer HPLC system (Perkin Elmer, Shelton, Conn.) with an Agilent C18 column (250 mm×4.6 mm i.d.) The DESI spray voltage was set at +5 kV and the nebulizing gas (N 2 ) pressure used was 170 psi. Unless specified otherwise, the DESI spray solvent was 1% acetic acid in acetonitrile or in methanol/water (1:1 by volume) and sprayed at 10 μL/min. 
     A sample comprising a 10 μL mixture consisting of three neurotransmitter compounds (3 mg/mL each), norepinephrine (“NE”), normetanephrine (“NM”), and dopamine (“DA”), underwent liquid chromatography separation using an isocratic elution with the mobile phase being 50 mM aqueous ammonium formate (pH 3.0 adjusted with formic acid), flowed through the UV-Vis (detection wavelength was set at 266 nm) and then was subject to ionization by the DESI probe. 
       FIGS. 5B-5D  illustrate the acquired DESI-MS mass spectra of the three separated NE, NM, and DA, and clearly show the corresponding protonated ions at m/z 170, 184 and 154, respectively. In addition, the extracted ion chromatograms (XICs) of m/z 170, 184 and 154 are shown in the Figure insets. These XICs agree well with the UV chromatogram and there is less than 3 s delay between the UV and DESI-MS detection, owing to the high elution flow rate used. It can also be seen that m/z 152 came from background. In  FIG. 1C , m/z 166 arose from the protonated NM (m/z 184) by loss of one water molecule. 
     In this experiment, the aqueous eluent containing no organic solvent was ionized directly by DESI. 
     Example 2 
     Post-column derivatization in LC/MS was demonstrated using an analytical system that is similar to the embodiment shown in  FIG. 3 . The sample was a neurotransmitter mixture with adopted selective boronic acid chemistry for derivatization. It is known that phenylboronic acid can selectively bind cis-diol to form a stable cyclic boronate via complexation reactions. In this study, 0.1 mM N-methyl-4-pyridineboronic acid iodide in acetonitrile was used as the DESI spray solution to provide a reagent ion of the positively charged N-methyl-4-pyridineboronic acid. The permanent charge carried by the reagent ion is helpful in enhancing the sensitivity of DESI-MS analysis. Operational conditions were similar to those of Example 1 but for including an ASI adjustable splitter to reduce the flow rate to 4.5 μL/min. 
       FIGS. 6A and 6C  illustrate the product ions of m/z 271 and 255, arising from the complexation reactions of the reagent ions with NE and DA, respectively, via losses of two molecules of water. In contrast, no such product ion was seen for the compound NM in  FIG. 6B  as one of its cis-diol was methylated. Only [NM+H] +  (m/z 184), [NM−H 2 O+H] +  (m/z 166) and [NM+CH 3 CN+H] +  (m/z 225) were observed. It is evident that both NE and DA containing the cis-diol functionality react with the reagent ion selectively while NM with one protonated hydroxyl group does not. 
     Example 3 
     LC/MS in conjunction with electrochemical conversion has unique applications in proteomics, such as in the fast structural eludication of disulfide-containing peptides via online electrochemical reduction. Conventionally, disulfide linkages increase the complexity for the protein/peptide structure elucidation by MS. Thus the cleavage of disulfide bonds is often essential as dissociation of a reduced protein/peptide ion can give rise to more structurally informative fragment ions than that of the intact counterpart. 
     Using an analytical system that is similar to the embodiment provided in  FIG. 4 , a disulfide-containing peptide somatostatin 1-14 (MW 1637.9 Da) was digested by trypsin to produce a peptide mixture: AGCK/TFTSC (this peptide has two chains of AGCK and TFTSC linked by a disulfide bond), and NFFWK, the sequences of each shown in  FIG. 7A . This digest mixture was examined using the analytical system. The separation of the mixture was carried out using a gradient elution. As displayed in  FIG. 7A , the two peptides eluted at 15.51 min and 17.46 min, respectively (these assignments were confirmed by MS/MS spectra, shown in  FIGS. 8A and 8B ). Other peaks in  FIG. 7A  were also observed, probably originating from the trypsin used in digestion.  FIG. 7B  shows the DESI-MS of the peptide AGCK/TFTSC eluted at 15.51 min, when no potential applied to the cell. The singly, doubly, and triply charged peptide ions were detected at m/z 933.2, 467.1, and 311.7, respectively. 
     When a −1.5 V potential was applied to the cell for reduction, these peptide ion peaks disappeared, as shown in  FIG. 7C , indicating a 100% reduction yield (probably due to a large MD electrode of 12×30 mm 2  used). Instead, two new ions of m/z 378.1 and 558.0 were observed. As the electrochemical reduction occurs after chromatographic separation, it is reliable to conclude that the two ions are from the reduction products of the AGCK/TFTSC. Indeed, they correspond to the protonated ions of two chains AGCK and TFTSC, respectively. Further, the sum of the MWs of the two products (378.1 Da+558.0 Da−2 Da=934.1 Da) is higher than that of precursor peptide (932.2 Da) by 1.9 Da, suggesting that the precursor peptide has one disulfide bond. In addition, tandem MS analysis was performed to elucidate the electro-generated ion structures, for establishing the disulfide bond connectivity as well as the sequence of the peptide AGCK/TFTSC. Upon collision induced dissociation, the m/z 378.1 gives rise to b 2 , b 3 , b 4 , y 1 , and y 2  fragment ions (refer to  FIG. 8C ), from which its sequence can be determined as either AGCK or GACK (cysteine is in 3rd position). Likewise, the m/z 558.0 dissociates into a 2 , b 2 , b 3 , b 4 -H 2 O, b 5 -H 2 O, and y 3 -H 2 O ions (refer to  FIG. 8D ), which reveals its sequence to be either TFTSC or FTTSC and the cysteine residue to be the 5th position in chain. Thus it can be seen that the sole disulfide bond of AGCK/TFTSC bridges the 3rd residue of one chain with the 5th residue of the other chain. These results show that, by using the LC/EC/DESI-MS, one can obtain useful sequence information of the examined peptide and explicitly establish the connectivity of the disulfide bond. Also, the protonated peptide NFFWK containing no disulfide bond remained unchanged once the cell potential was applied, as shown in  FIGS. 8E and 8F . This suggests that LC/EC/DESI can also be used to differentiate disulfide-bond containing peptides from others in an enzymatic digest. 
     This has been a description of the present invention along with the various methods of practicing the present invention. However, the invention itself should only be defined by the appended claims.

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