Patent Publication Number: US-7915579-B2

Title: Method and apparatus of liquid sample-desorption electrospray ionization-mass specrometry (LS-DESI-MS)

Description:
FIELD OF THE INVENTION 
     The present invention is related to methods of sample ionization for mass spectrometry. More specifically, the invention relates to the ionization of samples under ambient environmental conditions. 
     BACKGROUND 
     Ambient mass spectroscopy 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, or ionizing, a variety of target compounds of interest under ambient conditions. For example, analytes such as pharmaceuticals, metabolites, drugs of abuse, explosives, chemical warfare agents, and biological tissues have all been studied with these ambient ionization methods. 
     However, DESI analysis has been restricted to solid samples. To analyze a fluid sample, the solution needed to be dried in air. Alternatively, the solution was passed through filter paper or a membrane (collectively “filters”), which captures the analyte, separating it from the solvent. This use of filters or drying sample in air was necessary because the high-velocity nebulizing gas used in direct analysis would blow away the liquid sample from the sample surface and result in a short-lived ion signal. However, these protocols increases the time, complexity, and/or cost for liquid sample analysis and may change the surrounding environment of analytes prior to analysis. 
     Ambient ionization sampling of solids, or liquid samples via filters, by DESI tended to have limited ability to desorb and ionize molecules greater than approximately 25 kDa in molecular weight. This was presumably due to the formation of molecular aggregates by intermolecular interactions within the closely-packed solid sample. 
     One potential method for direct analysis of liquid samples is extractive electrospray ionization (ESSI). ESSI requires two separate nebulizing sprayers: one to nebulize the sample solution and the other to nebulize the ionizing solvent solution. This method is dependent upon liquid-liquid extraction and the collision of microdroplets. Thus, several parameters must be controlled to extract the best possible ion signal for each target sample. This leads to greater complexity of both the method and device. Other existing methods for liquid sample analysis using mass spectrometry include electrospray-assisted laser desertion/ionization (ELDI) and field induced droplet ionization (FIDI). However, these methods require either laser or high electric fields to assist sample desorption thus increasing the protocol complexity. 
     Thus, there remains a need to easily analyze a range of target samples of interest using a simple device, including those of high molecular weights within a liquid matrix environment at ambient conditions. Therefore, it would be beneficial to develop an ambient ionization method, like DESI, for use with liquid samples. Such a method would be particularly useful in bioanalytical, forensic, pharmaceutical, and border security applications where direct and efficient analysis of liquids is needed. 
     SUMMARY OF THE INVENTION 
     According to the present invention, a liquid is ionized for analysis by a mass spectrometer by contacting the liquid sample with charged solvent microdroplets, which desorb and ionize the liquid sample, or analyte. The ionized analyte can then be directed through a mass spectrometer for detection. 
     The present invention further relates to an ionization apparatus, for the analysis of liquid samples. The apparatus includes a sample stage that is adapted to receive a liquid sample and a nebulizing ionizer that is configured to generate a charged and nebulized solvent microdroplets and thereby desorb at least a portion of the liquid sample from the sample stage. 
     The objects and advantages of the present invention will be further appreciated in light of the following detailed description and drawings provided herein. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with a general description of the invention given above and the detailed description given below, serve to explain the principles of the invention. 
         FIG. 1  is a diagrammatic view of an ionization apparatus according to one embodiment of the present invention, with a mass spectrometer shown in cross-section. 
         FIG. 2A  is a diagrammatic view of an alternate embodiment of an ionization apparatus according to the present invention, with a mass spectrometer shown in cross-section. 
         FIG. 2B  is a diagrammatic cross-sectional view of the sample stage of the ionization apparatus of  FIG. 2A . 
         FIG. 3  is a diagrammatic cross-sectional view of a nebulizing ionizer for generating a charged and nebulized solvent according to the present invention. 
         FIG. 4  is a schematic representation of the components of a conventional mass-spectrometer. 
         FIG. 5  is a diagrammatic view of the desorption of the analyte from the liquid sample by an ionization apparatus according to one embodiment of the present invention into the cavity of the mass-spectrometer with a curtain gas interface, shown in cross-section. 
         FIG. 6  is a diagrammatic view of the desorption of the analyte from the liquid sample by an ionization apparatus according to one embodiment of the present invention into the cavity of the mass-spectrometer with a heated capillary interface, shown in cross-section. 
     
    
    
     DETAILED DESCRIPTION 
     According to the present invention, an analyte from a liquid sample is ionized by desorption of the analytes with an ionization apparatus  10 , which generates microdroplets  48  of a charged and nebulized solvent under ambient conditions. This generator in turn forms an ionized sample, which can be analyzed by mass spectrometry. 
     Operation of the ionizing apparatus  10  begins with the preparation of a liquid sample  12 . The liquid sample  12  can be a known entity for generating a calibration curve or an unknown entity for identification. Liquid samples  12  can be prepared by dissolving a solid sample in a nonpolar or polar solvent, such as a 1:1 ratio of water and methanol or a 1:1:0.005 ratio of water, methanol, and acetic acid. Otherwise, liquid samples  12  will generally require little-to-no additional preparation and can include, for example, protein digests or biological fluids. 
     The liquid sample  12  is then pumped via a pump  14 , such as a continuous-flow or syringe pump, onto a surface  18  of a sample stage  16  through a fluid connector  15 . A suitable continuous-flow pump  14  can be a Chemyx Model F100 syringe pump (Houston, Tex.), which is connected to a tube, such as a tubing, a syringe, or a capillary  20 , and moves the liquid sample at flow rates from approximately 0.1 μL/min to approximately 5 μL/min. Other flow pumps and flow rates could also be used. 
     The liquid sample  12  moves continuously by the continuous-flow pump  14  to a capillary  20 . The capillary  20  includes a distally located opening  22 , which is positioned on the sample stage  16 . Though not specifically shown, the capillary  20  can be affixed to the surface  18  of the sample stage  16 , such as by a clamp, which will prevent movement of the opening  22 . The capillary  20  can be constructed from a non-reactive material, such as silica, stainless steel, or aluminum, and can have an inner diameter of approximately 0.1 mm. However, the capillary  20  should not be considered so limited. 
     The sample stage  16  is simply a planar surface. It can be constructed from any nonreactive material, such as polytetrafluoroethylene. The design of the sample stage  16  can vary, but should be suitable to accommodate the capillary  20  and a nebulizing ionizer  38  such that at least a portion of the liquid sample  12  can be desorbed and directed substantially toward a mass analyzer  40  according to methods discussed in detail below. The sample stage  16  can be removably attached to a support structure  42 , which can include a base  44  and a podium  46 . Suitable materials for the support structure  42  can include non-reactive metals, such as aluminum. This support structure  42  can further include the operational mechanics (not shown) within the podium  46  such as those for incorporating a moveable sample stage. 
     The continuous-flow pump  14  supplies the liquid sample  12  to the sample stage  16  at a rate of approximately 0.1 μL/min to approximately 10 μL/min. At these rates an adequate supply of the liquid sample  12  is available on the sample stage  16  for analysis but without excess puddling, which can result in splashing and a short-lived ion signal. 
     Once the liquid sample  12  is supplied to the sample stage  16 , at least a portion of the liquid sample  12  is desorbed by microdroplets  48  of a charged and nebulized solvent discharged from a nebulizing ionizer  38 . The nebulizing ionizer  38  can be an ESSI apparatus  50 , as illustrated in  FIG. 3 . The ESSI apparatus  50  includes a housing  52 , a solvent conduit  56  having a solvent inlet  58  and a solvent outlet  60 , which is surrounded by a gas conduit  64 , or tube, having a gas inlet  66  and a gas outlet  68 . The gas outlet  68  is typically positioned 0.1 mm to 0.2 mm proximally to the solvent outlet  60 . 
     The solvent conduit  56  of the ESSI apparatus  50  can be a fused silica capillary having a tapered tip  57  at the solvent outlet  60  and an inner diameter ranging from approximately 5 μm to approximately 100 μm. The gas conduit  64  can also be a fused silica capillary, but will have an inner diameter larger than the solvent path  56  diameter, i.e. typically about 0.25 mm; however, these dimensions should not be considered limiting. 
     A voltage generator  70  with a voltage supply  72  is attached to the housing  52  as shown and is operable to charge the solvent  58  within the solvent conduit  56 . 
     In operation, the solvent  58  is supplied to the inlet  58  of the solvent conduit  56  at a rate of approximately 0.05 μL/min to approximately 50 μL/min. While the particular solvent used is dependent on 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 approximately 10 μL/min. The gas  62 , typically an inert gas such as N 2 , is supplied to the inlet  66  of the gas conduit  64  at pressures ranging from approximately 8 bar to approximately 25 bar. An electric potential, typically ranging from 4 kV to approximately 5 kV (4.5 V to 5.5 V for positive ion mode), is applied to the solvent  58  through the housing  52  via the voltage generator  70 . This generates an electrically charged solvent  54  within the solvent conduit  56 . 
     The now electrically charged solvent  54  traverses the solvent conduit  56  to the outlet  60 . At the outlet  60 , the charged solvent  54  is impacted by the surrounding high-pressure gas  62  leaving the outlet  68  of the gas conduit  64 . This high-pressure gas  62  causes the flow of the charged solvent  54  to be nebulized into microdroplets  48  of charged and nebulized solvent. 
     The ESSI apparatus  50  is positioned at a spray impact angle, θ, with respect to an x-y plane defined by the surface  18  of the sample stage  16 . This θ will cause the desorption and deflection of the analyte  74  into the mass analyzer  40 , as shown in  FIG. 5 . While θ can range from approximately 30° to approximately 45°, an appropriate value of θ will increase the likelihood of desorbed analyte  74  entering the mass analyzer  40 . As shown in  FIG. 5 , the spray impact angle θ will cause analyte to be desorbed from the surface  18  of the sample stage  16  at a deflection angle, φ. This deflection angle, φ, depends upon the molecular weight of the desorbed analyte  74 , the momentum of the microdroplets  48  of the charged and nebulizing solvent, and θ. Thus, an optimal impact angle θ will exist for each liquid sample  12  that will maximize the amount of desorbed analyte  74  entering the mass analyzer  40  and thus increase the ion signal response. 
     While not wishing to be bound by theory, it is believed that the mechanism by which the microdroplets  48  of the charged and nebulizing solvent interact with the liquid sample  12  and desorbs at least a portion of the liquid sample  12  is chemical sputtering, charge transfer, or droplet pick-up, with the most likely mechanism being droplet pick-up. During droplet pick-up, the microdroplets  48  of the charged and nebulizing solvent interact with the liquid sample  12  to yield desorbed secondary charged droplets  76  of analyte. The secondary charged droplets  76  then undergo desolvation to yield ions of the analyte  78 . Desolvation can occur within the cavity  80  of the mass analyzer  40  and is discussed in greater detail below. 
     The ionizing apparatus  10  can be used with any one of several mass spectrometry instruments. The ionizing apparatus  10  of the present invention is then interfaced to a cavity  80  of a mass spectrometer  82  containing a mass filter  86  and the mass detector  88 , which are maintained at vacuum. This interface typically will also evaporate and remove the solvent from the secondary charged droplet  76 . 
     As shown, the cavity  80  includes a first plate  92 , which is positioned at the opening to the cavity  80 , and a second plate  94 , which defines a space  96  through which a counter-flow curtain gas is supplied, as indicated by arrows  93 . Plates  92  and  94  include aligned orifices  95 ,  97 , respectively, providing inlets for the secondary charged droplets  76  of analyte to enter the mass spectrometer  82 . The curtain gas can be any inert gas, but is typically dry N 2  at slightly above atmospheric pressures. 
     In operation, the curtain gas flows out of the orifice  95  of the first plate  92  and across the secondary charged droplets  76  of analyte causing remaining solvent to be evaporated from the secondary charged droplet  76 . In some instances, a positive voltage potential (ranging from approximately 5 V to approximately 80 V) can be applied to the second plate  94  by a voltage source (not shown). The positive voltage potential will electrostatically decluster the secondary charged droplets  76 . 
     Because the curtain gas exits through the orifice  95  of the first plate  92 , it is possible that the curtain gas may influence the desorption of the secondary charged droplet  76 . Thus, it may be necessary to position the ESSI apparatus  50  approximately 0.5 mm behind the opening  22  of the capillary  20  to overcome this influence. 
     After the desolvation of the secondary charged droplet  76 , the now ions of analyte  78  enter the mass analyzer  40  through an orifice  94  of the second plate  94 , which provides an opening into the mass analyzer  40  of the mass spectrometer  82  while maintaining a vacuum within the mass analyzer  40 . Once the ions of analyte  78  are within the mass analyzer  40 , the ions of analyte  78  are directed to a skimmer  106  before entering the mass filter  86 . The second plate  94  encloses the mass analyzer  40  and is connected to a vacuum pump (not shown), which creates the vacuum. A skimmer  106  includes a plate  105  and an orifice  104 , which is usually cone-shaped. The skimmer  106  is operable to focus the ions of analyte  78  into a narrow beam (not shown) of ion current as it enters the mass analyzer  40 . This skimmer is typically grounded. Additionally, a separate focusing lens (not shown) can be included between the skimmer  106  and the mass filter  86  to further focus the beam containing the ions of analyte  78  and reduce the natural expansion of the beam by effusion through the orifice  104  of the skimmer  106 . 
     After passing the skimmer  106 , the ions of analyte  78  are directed to the mass filter  86 . Conventional mass filters include time-of-flight, quadrupolar, sector, or ion trap, which are operable to cause ions of analyte  78  having a specified mass-to-charge (m/z) ratio to transverse the mass filter  86  and be quantified at the mass detector  88 . Those ions of analyte  78  having a m/z value that differs from a specified m/z value will impact the mass filter  86 . One particularly suitable instrument is the hybrid triple-quadrupole-linear ion trap mass spectrometer, Q-trap 2000, by Applied Biosystems/MDS Sciex (Concord, Canada). 
     In operation of a conventional quadrupole modality of a mass spectrometer  82 , the ions of analyte  78  are 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. In operation, only the ions of analyte  78  having a particular m/z will continue through the parallel electrodes to the mass detector  88 . That is, the ion of analyte  78  with the particular m/z 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  78  does not exceed distance between the electrode. Thus, the ion of analyte  78  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  88 . 
     Those ions of analyte  78  that reach the mass detector  88 , typically a Faraday plate coupled to a picoammeter, are measured as a current (l) induced by a total number (n) of ions of analyte  78  impacting the mass detector  88  over a period of time (t) and in accordance with n/t=l/e, wherein e is the elementary charge. 
     The controller  90  operates the four parallel electrodes and the mass detector  88  such that the current measured at the mass detector  88  can be correlated to the radiofrequency field and the DC voltage potential applied to the four parallel electrodes. A suitable controller  90  can be a standard PC computer; however, the present invention should not be considered so limited. The controller  90  may further include a memory for storing data related to operation of the mass spectrometer  82  for later chemical analysis. The memory can be internal, such as a hard-drive ROM, or a removable ROM for off-site, off-line chemical analysis. Additionally, the controller  90  can include a data transmission means for sending the stored data to another suitable workstation. Said data transmission means can be a wireless device or hard-wired. 
     Typically, the controller  90  will further include a chemical analysis software for on-site and immediate analysis of a liquid sample  12 . This chemical analysis software is operable to generate a calibration curve, generated in a known manner with liquid samples  12  containing known chemical analytes, and is operable to extrapolate the m/z value for an unknown chemical analyte based upon the calibration and in a known manner. 
     While the ionization apparatus  10  and method of using the ionization apparatus  10  have been provided in some detail above, various other embodiments of the present invention are envisioned and will now be explained. 
     In one embodiment, this LS-DESI-MS can be coupled to conventional separation techniques, such as HPLC, electrophoresis, or microfluidics. In this regard, the liquid sample  12  is prepared according to the particular needs of the separation techniques. The liquid sample  12  flowing out of the separation device will be loaded into the LS-DESI-MS. Because of the flexible nature of the ionizing apparatus  10  of the present invention, and the reduced affects thereon by the liquid matrix, the liquid sample  12  can be prepared with a high salt matrix, surfactants, or other solvents and solutes not traditionally used with mass spectroscopy analysis. 
     In another embodiment, the LS-DESI-MS apparatus can be used for remote detection of dangerous liquid substances, such as explosives and chemical/biological warfare agents. The dangerous liquid, located in a remote site, can be introduced by a peristaltic pump and an extended tube into the LS-DESI-MS apparatus. In this way, only a small aliquot of the dangerous liquid will be introduced to the proximately-located detection device, i.e. the mass analyzer. This embodiment can be useful in providing personnel safety in airports and the battle fields while a potentially dangerous liquid substance is analyzed. 
     In yet other embodiments, a reactant can be added to the solvent  58  of the DESI apparatus  50 . This is particularly applicable in instances wherein an ionic or molecular reaction is required during the sampling process or to enhance the selectivity of the chemical analysis. For example, zinc complexes (Zn 2 +) have been shown to aid in the ionization of phosphate-containing compounds. For example, [Zn(DPA)] 2+  is a known phosphate binding motif. In this way, an aqueous solution of Zn(NO 3 ) 2  and 2,2′-dipicolylamine (DPE) can be added to the solvent  58  entering the solvent conduit  56  of the DESI apparatus  50 . Thus, the microdroplets  48  of the charged and nebulized solvent will include the [Zn(DPA)] 2+  complex, which can then react with an analyte of the sample. The product of the [Zn(DPA)] 2+  and analyte reaction can then be desorbed in a manner described above. 
     Alternatively, the selective nature of zinc complex chemistry can lead to selective ionization. That is, the zinc complex can be selected based upon its selective reactivity with a first analyte over a second analyte, wherein the first and second analytes are in the liquid sample  12 . In this way, the first analyte will react with the zinc complex and can then be desorbed while the second analyte remains in the liquid sample  12 . 
     In yet other embodiments, the ionizing apparatus  10  includes a modified sample stage  24  having a microfluid channel  26  as shown in  FIG. 2A . In this way, the continuous-flow pump  14  delivers the liquid sample  12  to a capillary  28 , which terminates at an inlet  30  of the microfluid channel  26 . The inlet  30  can further include a sealant, such as an O-ring  32 , for providing a fluid-tight seal between the capillary  28  and the microfluid channel  26  (see  FIG. 2B ). The liquid sample  12  will traverse the microfluid channel  26  and exit the microfluid channel  26  at an outlet  34  upon the surface  36  of the sample stage  24 . The microfluid channel  26 , which can be formed during the sample stage  24  molding process or created thereinafter by drilling or similar method and will be substantially similar in size as compared to the capillary  20 . Other arrangements for delivery of the liquid sample  12  would be appropriate and may depend on the nature of the analyte or the liquid matrix. 
     In yet another embodiment, as shown in  FIG. 6 , the plates  92  and  94  and the gas  93  can be eliminated by interfacing the ionizing apparatus  10  with the cavity  80 , which includes a heated capillary interface  98 . This interface  98  includes a capillary  100  positioned in a wall  102  of the cavity  80 , wherein the capillary  100  is aligned with the orifice  104  of the skimmer  106 . The capillary  100  can be constructed of metal or glass, which is resistively heated to a range from about 100° C. to about 200° C. by an energy source (not shown). As the secondary charged droplets  76  are desorbed toward, and then enter, the capillary  100 , the secondary charged droplets  76  are heated and any remaining solvent within the secondary charged droplet  76  is evaporated. An energy source (not shown) can apply a positive voltage potential to the capillary  100 , which will decluster the secondary charged droplets  76 . 
     In yet another embodiment, the ionizing apparatus  10  may be enclosed within a chamber (not shown) and operate under a carrier gas environment, such as nitrogen. While it is not necessary for the carrier gas to alter the local pressures significantly from ambient conditions, the N 2  environment can decrease the likelihood of an undesired reaction occurring between the liquid sample  12  and a component within the air. 
     As provided for herein, the ionizing apparatus  10  of the present invention can operate under ambient conditions while ionizing analytes of interest from a liquid sample  12  and without the use of filters or by air drying the samples. The ionizing apparatus  10  is capable of desorbing various analytes of interest, including those with high molecular weights (above 60 kDa), from the liquid sample, does not require additional sample preparation, and operates with minimal adjustment by the user. 
     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.