WO2012170301A1 - Cassettes, systems, and methods for ion generation using wetted porous materials - Google Patents

Abstract

The invention generally relates to cassettes, systems, and methods for ion generation using wetted porous materials.

Description

CASSETTES, SYSTEMS, AND METHODS FOR ION GENERATION USING WETTED

POROUS MATERIALS

Related Applications

The present application claims the benefit of and priority to each of U.S. provisional application serial number 61/593,535, filed February 1, 2012, U.S. provisional application serial number 61/541,460, filed September 30, 2011, and U.S. provisional application serial number 61/493,438, filed June 4, 2011, the content of each of which is incorporate by reference herein in its entirety.

Field of the Invention

The invention generally relates to cassettes, systems, and methods for ion generation using wetted porous materials. Background

Biofluids (e.g., complex mixtures such as blood, saliva, or urine) are routinely separated using chromatography before the MS measurement in order to minimize suppression effects on analyte ionization and to pre-concentrate the analytes. Recently, systems and methods have been developed that allow for sample preparation and pre-treatment to be combined with the ionization process (See Ouyang et al., WO 2010/127059).

These systems and methods use wetted porous material, named paper spray ionization, for direct, qualitative and quantitative analysis of complex biofluids ("paper spray"). Generally, a sample is spotted onto a porous substrate and then solvent is applied to the substrate. Analyte transport is achieved by wicking in the porous material with a macroscopically sharp point and a high electric field is used to perform ionization and chemical analysis of compounds present in biological samples. Pneumatic assistance is not required to transport the analyte: a voltage is simply applied to the wet paper, held in front of a mass spectrometer.

Paper spray has generally been conducted manually in the manner described above. Such a process is not amenable to high-throughput analytical settings, such as hospital, clinical or contract laboratories or security screening check-points. Summary

The invention provides, cassettes, systems, and methods for automating the paper spray process. Cassettes, systems, and methods of the invention streamline and automate the sample preparation and ionization process, allowing for rapid throughput of samples using paper spray and enabling paper spray to be adopted by entities that require robust high-throughput analytical techniques.

In certain aspects, the invention provides a sampling cassette that includes a hollow housing having an inside configured to hold a solid porous substrate, at least one inlet, an outlet, and an electrode, in which the housing is configured such that the inlet is in fluid communication with the substrate held inside the housing and the electrode is in contact the substrate held inside the housing. In certain embodiments, the at least one inlet is a plurality of inlets. In a particular embodiment, a first inlet is located in proximity to the outlet and a second inlet is located distal the first inlet. In particular embodiments, the electrode is located between the first and second inlets. Generally, the first inlet is configured to mate with a fluid injector, such as a pipette tip. The second inlet may further include a solvent reservoir. In certain embodiments, the configuration of the inside of the housing minimizes interference with liquid flow through the substrate, as is further described below.

In certain embodiments, a top of the housing includes at least one transparent section. Generally, the transparent section is located adjacent the first inlet. In particular, embodiments, the top of the housing includes two transparent sections, one on each side of the sample injection inlet. In this manner, the injected sample may be viewed prior to analysis, ensuring that the sample has been properly applied to the substrate. Preferable deposition results in the sample spreading across a width of the substrate, ensuring that there are no flow paths for the solvent to migrate without passing through the sample.

To facilitate drying of the sample prior to analysis, embodiments of the invention may include at least one ventilation hole. In other embodiments, the cassette further includes an identifier so that the cassette can be linked to a particular patient or sample. Any identifier known in the art may be used, such as a barcode or a two-dimensional barcode, in other embodiments, the outlet comprises a shroud. The shroud protects the substrate within the cassette. In other embodiments, the outlet includes a plurality of prongs. In certain

embodiments, the prongs diverge from each other. The cassette can be manufactured with the solid substrate already a part of the cassette. Alternatively, the cassette can be manufactured without the solid substrate and an operator can insert the substrate into the cassette at the desired time. For insertion of the substrate after manufacture of the cassette, the cassette can be made as a two component cassette, a top component and a bottom component that are separable from each other. Generally, the substrate will have a pointed tip.

Another aspect of the invention provides a cassette processing system that includes a cassette holder configured to hold at least one sampling cassette that includes a porous substrate having a sample, at least one solvent reservoir, an analysis stage, an exit port, and a cassette moving component configured to receive the cassette from the holder and move the cassette through the system to interact with the solvent reservoir, the analysis stage, and the exit port. Any component that can hold a cassette can be used with systems of the invention. In certain embodiments, the cassette holder is a magazine. In certain embodiments, the system further includes a camera. Generally, the camera may be positioned to image an identifier on the cassette.

Generally, the solvent reservoir will further include a fluid injector. The fluid injector is configured to mate with an inlet of the sample cassette to thereby inject solvent from the reservoir onto the substrate within the cassette. Generally, the analysis stage may include a voltage supplier. In certain embodiments, a mass spectrometer is operably coupled to the analysis stage. The voltage supplier mates with an electrode in the sample cassette which is in contact with the substrate inside the cassette, thereby producing sample droplets containing analyte that are expelled from the substrate and into the mass spectrometer.

In certain embodiments, the system further includes a controller. The controller can controller any aspects of the cassette processing system, such as the camera, the fluid injector, the voltage supplier, and the cassette moving component. In certain embodiments, the controller controls both the cassette processing system and the mass spectrometer.

Another aspect of the invention provides a mass spectrometer, and an automated apparatus operably coupled to the mass spectrometer, in which the apparatus prepares a sample loaded onto a solid porous substrate for analysis by mass spectrometry and generates sample droplets containing analyte that are expelled from the substrate and into the mass spectrometer.

Another aspect of the invention provides a method for analyzing a sample that involves providing a sampling cassette including a hollow housing having an inside loaded with a solid porous substrate, at least one inlet, an outlet, and an electrode, in which the housing is configured such that the inlet is in fluid communication with the substrate held inside the housing and the electrode is in contact the substrate held inside the housing, loading a sample into the cassette via the inlet so that the sample contacts the substrate, loading a solvent into the cassette via the inlet so that the solvent contacts the substrate, applying a voltage to the substrate via the electrode in the cassette, thereby producing sample droplets containing analyte that are expelled from the substrate via the outlet, and analyzing the analyte. In certain embodiments, the at least one inlet is a plurality of inlets and the sample is loaded via a first inlet and the solvent is loaded via a second inlet.

In certain embodiments, the solvent loading step and the applying step of the method are performed in an automated manner. In these embodiments, the loading step may occur prior to the providing step. In other embodiments, the solvent loading step and the applying step of the method are performed manually. In certain embodiments, prior to solvent loading step, the method further includes drying the substrate. In certain embodiments, prior to the providing step, the method further includes loading the substrate into the cartridge.

In methods of the invention, the solvent may function in many different ways. In certain embodiments, the solvent assists in transport of the sample through the porous material. In other embodiments, the solvent includes an internal standard. In other embodiments, the solvent minimizes salt and matrix effects. In other embodiments, the solvent is capable of mixing with the sample.

Analyzing the sample may be by any technique known in the art. In certain

embodiments, analyzing involves providing a mass analyzer to generate a mass spectrum of analytes in the sample. The mass analyzer may be for a mass spectrometer or a handheld mass spectrometer. Exemplary mass analyzers include a quadrupole ion trap, a rectilinear ion trap, a cylindrical ion trap, a ion cyclotron resonance trap, an orbitrap, a time of flight, a Fourier Transform ion cyclotron resonance, and sectors.

The invention may be used to analyze any type of sample, such as chemical samples, biological samples, such as human, animal, plant, bacterium, fungus, or any other cellular organism. Biological samples for use in the present invention include bacteria or viral particles or preparations, or biological fluids that include bacteria or viral particles. In certain embodiments, the sample is a biological fluid, blood, urine, cerebrospinal fluid, seminal fluid, saliva, sputum, stool and tissue. Any tissue or body fluid specimen may be used as a sample.

Brief Description of the Drawings

FIG. 1 is a perspective of a cassette for use with the invention.

FIG. 2 is an exploded view of the cassette of FIG. 1 showing an upper member, a conductive electrode ball, a pointed porous substrate of paper, and a lower member.

FIG. 3 is a top plan view of the upper member of FIG. 2.

FIG. 4 is a bottom plan view of the upper member of FIG. 2.

FIG. 5 is a top plan view of the lower member of FIG. 2.

FIG. 6 is a bottom plan view of the lower member of FIG. 2.

FIG. 7 a side cross section view of the cassette of FIG. 1 taken along its centerline.

FIG. 8 is an upper perspective view of the cassette of FIG. 1 showing a pipette inserted into the sample depositing hole.

FIG. 9 is a closer upper perspective view of the cassette of FIG. 1 with the upper member removed to show the area of deposition of the sample below the pipette shown in FIG. 8.

FIG. 10 is a dryer unit for use in drying cassettes of the type shown in FIG. 1.

FIG. 11 is a perspective of a portion of a conventional mass spectrometer as viewed from the front and left.

FIG. 12 is a perspective of a cassette processing device as viewed from the front and right for attaching to a conventional mass spectrometer such as is in FIG. 11, for sequentially processing an array of cassettes of FIG. 1 to be assayed.

FIG. 13 is a perspective of the cassette processing device of FIG. 12 with its cover removed, and showing a camera on the lower right to image the two dimensional bar code on the underside of the lowest cassette of FIG. 1 in the centrally positioned stack of cassettes.

FIG. 14 is a perspective of the cassette processing device of FIG. 12 as viewed from above and to the left with the cover removed and the display and control panel pivoted away leftwards to illustrate stations 1 (loading and imaging) and 2 (wetting) of the four position indexing table.

FIG. 15 is a similar perspective of FIG. 14 with various covers removed to allow better imaging of the positions of the cartridges in each of the four indexing table positions. FIG. 16 is a perspective of the cassette processing device of FIG. 12 as viewed from the lower front and to the left, with covers removed and the control panel pivoted away leftwards to see both the imaging camera and a portion of the belt arrangement used to maintain the orientation of the cartridges of FIG. 1 in each of the four indexing positions.

FIG. 17 is a perspective from the front upper left side to see on the right the interfacing member on the back side of the cassette processing device that abuts the mass spectrometer of FIG. 11, and showing the discharge chute on the lower left that finished cartridges slide down after use.

FIG. 18 is a cutaway perspective showing the electrical connector contacting the ball that rests on the pointed paper porous paper substrate within the cassette of FIG. 1 (cassette not otherwise shown) and ejecting ions from the point on the paper toward the mass spectrometer inlet shown on the left.

FIG. 19 is a top view of another embodiment of a cassette for use with the invention.

FIG. 20 is a side view of the FIG. 19 cassette.

FIG. 21 is a side sectional view of the FIG. 19 cassette.

FIG. 22 is an exploded view of the FIG. 19 cassette.

FIG. 23 is an enlarged view of the area indicated in FIG. 21 showing detail of the solvent inlet port of the FIG. 19 cassette.

FIG. 24 is an enlarged view of the area indicated in FIG. 22 showing detail of the posts engaging the paper at the outlet of the cassette.

FIG. 25 is a top view of yet another embodiment of a cassette suitable for use with the invention in which the preferred paper substrate is angled downward to engage a lower well of solvent.

FIG. 26 is a side view of a center cross-section of the cassette of FIG. 25.

Detailed Description

The invention generally relates to cassettes, systems, and methods for ion generation using wetted porous materials.

FIG. 1 is a perspective of a cassette 10 for use with the invention. On one end there is a handle 11 for holding cassette 10 while on the other end there is a shroud 12 extending outward to protect a tip 15 of substrate 18. As explained more fully below, the substrate is porous and configured to receive a sample and to have a voltage applied to it while wetted so as to cause sample ions to be ejected from tip 15, which process when used with a paper substrate has been referred to as "paper spray ionization." Further description is provided for example in Ouyang et al., WO 2010/127059, the content of which is incorporated by reference herein in its entirety.

As better seen in the exploded view of FIG. 2, substrate 18 is preferably a planar porous substrate, such as filter paper, having a relatively wide main body section 18a and a tapered section 18b. Sections 18a and 18b are generally symmetric relative to longitudinal axis L with the main body section 18a being rounded at its end and the tapered section 18b tapering continuously from the main body section 18a to the pointed tip 15. Substrate 18 is positioned substantially horizontally inside cassette 10. When used with a mass spectrometer, the pointed end at tip 15 is oriented toward the ion sampling input of the mass spectrometer, as illustrated in FIG. 18.

Cassette 10 has a sloped catch pan 21 which serves as a well which can hold excess solvent being added to the cartridge for flowing into the porous substrate. The top of a ball electrode 22 is near the upper surface of cassette 10 with its bottom resting on the porous substrate 18 within cassette 10. While the shape of electrode 22 is most preferably a ball, it can alternatively have other shapes so long at it provides a conductive path. Preferably for those embodiments where electrodes are incorporated as a part of the cassette, the electrodes will be electrical conductors that are substantially devoid of edges, and more preferably have some symmetry in their shape.

Further along the upper portion of cassette 10 there is a sample inlet port 23. Port 23 is designed to receive a fluid injector, such as a pipette 24, as shown in FIGS 8 and 9, for release of a predetermined sample size onto the substrate 18. FIG. 8 is an upper perspective view of the cassette of FIG. 1 showing a pipette inserted into the sample depositing hole. FIG. 9 is a closer upper perspective view of the cassette of FIG. 1 with the upper member removed to show the area of deposition of the sample below the pipette shown in FIG. 8.

In the illustrated embodiment, the top side of port 23 is tapered and positioned above the tapered section 18b of the substrate, as shown in FIG. 7. The pipette 24 is preferably configured and sized such that when fully inserted into the sample inlet port 23, the outlet of the pipette will be aligned between the two sides of porous substrate 18 so the pipette end is below the underside of inlet port opening and yet not so far below that it touches the porous substrate 18. The sample size deposited is sufficiently large so as be analyzed. To promote consistency and accuracy of analysis it is preferable that when deposited onto the porous substrate 18 it spreads across the width of the substrate, for example as shown by the generally circular pattern of the sample 25 in FIG. 2. By spreading across the entire width, there are no paths for solvent to migrate to the tipl5 without being benefited by passing through the sample 25.

Also shown in FIGS. 1 and 2 are two apertures 30 and 31 which allow viewing of the circular pattern to determine that sample 25 has extended to both sides of porous substrate 18 after pipette 24 has inserted the sample. The most common samples envisioned for use with this invention are body fluids, including blood or blood serum, for example.

Cassette 10 is made using an upper housing member 33 shown in top plan view in FIG. 3 and in bottom plan view in FIG. 4, and using a lower housing member 35 shown in top plan view in FIG. 5 and in bottom plan view in FIG. 6. To optimize the performance of the cartridge, the design preferably attempts to minimize interference with the natural wicking or liquid flow or capillary action within porous substrate 18 that is positioned between the two housing members 33 and 35. To accomplish this careful positioning with a minimum of interference with liquid flow by the porous substrate 18, there are several internal posts extending upwardly, as posts 52- 56 as well as three internal posts extending downwardly, as posts 65, 66 and 67 that lightly engage the opposite side of porous substrate 18 where it contacts posts 55 and 56. Additionally there is a larger elongated raised mesa 47 upon which porous substrate 18 rests opposite solvent inlet port 37 which is at the base of sloped catch pan 21 which serves as a solvent well above the top surface of the porous substrate 18. With the shape of the mesa 47 generally corresponding with the shape of the solvent inlet port 37, solvent passing through the port 47 tends to either be absorbed by the porous substrate 18 immediately, or accumulates in the sloped catch pan 21 until it can be later absorbed by the porous substrate 18.

For the preferred manner of solvent wetting, sample inlet port 23 is first placed under the injectors 823 and 824 of FIG. 14, and a minor portion of solvent is applied through the sample inlet port 23 so that the upper surface of the deposited sample is wetted. Then cassette 10 is moved to place well 21 under the injectors 823 and 824, and a major portion of solvent is placed into well 21, which over time allows for more of the solvent to be absorbed by the porous substrate 18. It has been found that by applying solvent to the upper surface of the sample, and then by adding solvent in a separate step from a different location associated with a solvent reservoir, more accurate results can be achieved.

In this most preferred example, about 15 microliters of solvent are first automatically applied from above the sample location, most preferably through the sample inlet port 23. After about 5 seconds, cassette 10 is automatically moved for injectors 823 and 824 to automatically dispense about 75 microliters of solvent into well 21 over the course of about 45 seconds. In other less preferred examples with other cartridge designs, it is likely that the sample wetting will preferably use from 10 to 30 microliters of solvent with the reservoir filling using from 30 to 300 microliters. More generally, it is preferred that a minor portion of solvent is automatically applied to the sample on the substrate from above the sample location and that the major portion of solvent is automatically applied at a different location to a reservoir adjacent the substrate. While two injectors are preferred when a choice between two solvents may be desired, only a single injector is needed be used for applications where only one solvent is used.

While the preferred example dispenses sequentially by automatically moving the cassette from a first solvent dispensing position to a second one, one could also keep the cassette stationary. With the cassette 10 stationary, one could have a first dispensing injector above the sample for wetting its upper surface with solvent and a second injector for adding solvent to a reservoir, or one could have a dispensing injector that is sequentially moved between the two locations.

In addition to the posts 52-56 and mesa 47 from below and posts 65-67 from above that address vertical positioning, there are five side bumpers 81-85 that gently provide both vertical positioning as well as lateral positioning by virtue of their upper inward corners 88 being conical in shape and abutting the edges of porous substrate 18. Thus the porous substrate 18 is positioned within cassette 10 in a manner to provide for a minimal restriction of liquid flow through and on the substrate.

For facilitating drying of samples 25 after being deposited on porous substrate 18 and before they are assayed by the mass spectrometer, there are ventilation holes 60 along with the inherent ventilation provided by the observation apertures 30 and 31 and to a lesser extent by sample inlet port 23 and the narrow path surrounding the tip 15 as it extends toward the shroud 12. While the three ventilation holes are shown in the base, there are alternative placements that can be used as well, such as in each side of the cassette or in other locations in the base that may be more suitable if other label placements are desired. Cassettes may be provided with a unique identifier, and as shown as an example in FIG. 6, cassettes 10 are labeled on their underside, with the labeling include a two dimensional bar code 71 and standard written text 70 as illustrated in FIG. 6. One of skill in the art will recognize that any identifier may be used with cassettes of the invention and that the identifier may be placed anywhere on the cassette.

Further understanding of the details of cassette 10 can be seen in FIG. 7 which is a side cross section view of the cassette of FIG. 1 taken along its centerline, and illustrating how the porous substrate 18 is gently held in place. It can be seen how the cassette has provision for a sample to be applied through sample inlet port 23 using a pipette 24 as illustrated in FIGS. 8 and 9. It can also be seen how fluid flowing into fluid collection well 21 that has a floor sloping toward an elongated opening at solvent inlet port 37 at the base of the well. The elongated opening is in a direction that is generally orthogonal to the discharge direction towards tip 15 along the sectioning shown. Solvent supplied to the cassette can accumulate in well 21 until it passes through inlet port 23 as it becomes absorbed by the porous substrate 18.

Still further as to the details of cassette 10 as can be seen in FIG. 7, the ball electrode 22 contained within the cartridge is positioned to contact the porous substrate 18 between the solvent inlet port 37 and the generally circular sample 25 located on porous substrate. It can be noted that the generally circular sample 25 preferably does not reach the ball electrode 22.

FIG. 10 is a dryer unit 89 for use in drying cassettes 10 of the type shown in FIG. 1. With the dryer unit maintained at about 37°C, samples may be dried in less than five minutes. Alternatively, samples may be left to dry at room temperature in about 30 minutes.

FIG. 11 is a perspective of a portion of a conventional mass spectrometer as viewed from the front and left. The invention can be readily adapted for use with different mass

spectrometers incorporating an ion sampling input suitable for use in ambient conditions by adapting the connection structure. An exemplary spectrometer 100 shown has an ion sampling input 101 shown positioned at the center tip of a conductive cone 102. Two mounting rods 105 and 106 extend horizontally out from the front of spectrometer 100 allowing ready placement of the automated cassette processing device 110 of FIG. 12 around rods 105 and 106.

Automated cassette processing device 110 shown in FIG. 12 can sequentially process an array of cassettes of FIG. 1 to be assayed. A magazine 111 preferably holds at least a dozen cassettes 10 and can be transported from a remote location to the processing device 110 and inserted into it as one unit. Cassettes may also be individually loaded into the open top of magazine one at a time. While there is shown a one dimensional array of stacked cassettes 10, the invention contemplates alternative designs that are either two or three dimensional arrays for applications having higher volume needs. The location where magazine 111 is positioned for use is considered an "incoming sample holding area." Such an area might, for example, store several cassettes 10 each containing porous substrates 18 containing samples 25.

In FIG. 12 one can see the front control panel 115 which is connected to the processing device controller 190 and has buttons 118 for receiving user input and display 117 for displaying messages from controller 190. The controller is coupled to and controls the operation of the index table 120, the camera 129, the fluid injector and the voltage applier. The controller also can interface with the mass spectrometer to control aspects of its operation and receive measurement data from tested samples to correlate with the sample ID information from the camera images of the bottom of the cassettes.

Also reservoirs 113 and 114 are used to hold a first and a second solvent for later automatic application to substrates 18 within cassettes 10. When the automatic mass spectroscopy for a particular cassette 10 is complete, it will then be ejected through the opening for exit chute 116 so as to fall in a container (not shown) positioned below the exit chute 116.

FIG. 13 is a perspective of the processing device 110 of FIG. 12 with its cover removed, and showing a camera 129 on the lower right to image the two dimensional bar code on the underside of the lowest cassette 10 positioned below magazine 111, so that the results of the mass spectrometry can be automatically electronically correlated with the patient identification information as shown in FIG. 6. Camera 129 can be one of any form of optical code reader as well as more conventional cameras.

FIG. 14 is a perspective of the cassette processing device of FIG. 12 as viewed from above and to the left with the cover removed and the display and control panel pivoted away leftwards to illustrate stations 1 (loading and imaging) and 2 (wetting) of the four position indexing table. Position 2 is a first processing stage for the processing device 110 in that at least one solvent from either reservoir 113 or 114 is applied through injectors 823 or 824

respectively, toward the solvent inlet port 37 of a cassette 10. Position 2 is in turn divided into two steps, the first step of which briefly positions the sample inlet port 23 under the injectors for a minor amount of solvent to be applied to the upper portion of the sample, and the second step moves the cassette a small amount to position well 21 under the injectors 823 and 824 for the remainder of the time for dispensing the major amount of solvent into well 21. While all four positions of the indexing table 120 are affected by this two-step repositioning used at Position 2, the first step is brief, and does not interfere with the ability to accomplish the activities needed to be accomplished at the other positions during the second step. Reference to the term "solvent" is intended in a broad sense as a liquid carrier for the ultimately ionized particles to be assayed by the mass spectrometer, and is not intended to be limited to providing a true solution. Transport by the solvent in the form of an emulsion or suspension is equally contemplated for this invention and for the use of the term solvent throughout this written description.

FIG. 14 also shows a part of a round, four-position index table 120 that advances clockwise as viewed from above. Removal of more components, shields and the like from FIG. 14 results in a much clearer illustration of the index table 120 as shown in FIG. 15. In operation, a cassette 10a first enters cassette holder 121 from the cassette magazine 111. In the next stop, cassette 10b is shown in cassette holder 122, at what is the first processing station where solvent is added to the cassette 10b in two sequential steps involving a slight movement between the two steps as described in the previous paragraph. In the next stop after that, cassette 10c is shown in cassette holder 123. At this second processing station a spring biased electrode 151 automatically connects a source of voltage from 150 through electrode 151 and through ball 22 in cassette 10c to the porous substrate that was wetted at the prior station. The spring bias allows the cassette to move into position at the second processing station by passing under the electrode with the cassette lOc's upwardly facing ball effortlessly engaging the downwardly positioned electrode 151 when its processing position is reach. The source of voltage initiates the ionization process to provide ions to the ion sampling input of the mass spectrometer of FIG. 11.

At the final stop, it can be seen that there is an opening at the bottom of the cassette holder 124 that allows cassette lOd to drop and slide down chute 116 to exit the device 110. Movement away from the second processing station towards the final stop can be considered a "sample remover" that automatically removes the substrate contained within a cassette 10 from its position adjacent the ion sampling input 101 after application of the ionizing voltage.

When considering all four stops for the index table, it can further be seen that when processing four or more cassettes sequentially, there will be a time when one of the sample cassettes is at the first processing stage, another one is at the second processing stage, yet another one is being discarded down chute 116, and yet another one is inserted and being photographed, ready to begin the process from the start. As the index table 120 sequentially rotates 90 each step, the four cassette holders 121-124 each in turn move a cassette 10 from the supply of sample cassettes in magazine 111, on to the first and then the second processing stages and thereafter to be discarded down chute 116. The filling and emptying of the cassette holders 121-124 benefit from gravity in that the cassettes are loaded from the top and emptied from the bottom.

It can be still further observed that the indexing mechanism of the invention maintains the same orientation of the discharge end of the cassette relative to an axis defined by the ion- sampling orifice of the mass spectrometer at the loading stage as well as the first and second processing stages. This readily allows the two step solvent dispensing at two different locations at Position 2 with the fixed position solvent injectors 823 and 824. The four cassette holders 121-124 each have a corresponding pivot about point 131-134. Four belts 191-194 in FIG. 16 located under the indexing table 120 are coupled to points 131-134 to maintain the constant orientation of the cassette holders as the indexing table 120 rotates. Due to this feature, for examples, cassette holder 124 actually moves cassette lOd off of the rotating table and over a hole above chute 116, and cassette holder 121 positions the underside of cassette 10a directly over an opening like slot 142, so that a cassette identifier device of camera 129 can image the underside of cassette to obtain bar code and written information as is illustrated in FIG. 6.

FIG. 17 is a perspective from the front upper left side to see on the right the interfacing member on the back side of the cassette processing device that abuts the mass spectrometer of FIG. 11, and showing the discharge chute 116 on the lower left that finished cartridges slide down after use. Mounting rods 105 and 106 extending from the mass spectrometer 100 of FIG. 11 fit into corresponding tubular members 155 and 156 and are locked into position by handled latches 161 and 162 with mounting plate 160 adjacent spectrometer 100 to position samples in the second processing stages to be adjacent the ion sampling port 101 of mass spectrometer 100.

FIG. 18 is a cutaway perspective showing the a spring biased electrode 151 contacting ball 22 that rests on the pointed paper porous paper substrate 18 within cassette 10c (cassette not otherwise shown) and ejecting ions from the tip 15 of substrate 18 toward the mass spectrometer inlet shown 101 on the left. Turning now to FIGS. 19-24, another embodiment of a cassette for use with the invention is disclosed. Cassette 200 is similar to cassette 10 except for three features.

First, the ion ejection end of cassette 200 is provided with a plurality of divergent prongs 201-204, rather than a protective shroud 12 to improve the electric field characteristics while limiting risk of damage to the point on the substrate. Prongs extend from the cassette portions 33, 35 beyond the suspended tip 15 a sufficient distance to prevent inadvertent touching of the tip 15 during handling. Prongs 201-204 are also preferably divergently angled and spaced from the tip so as to limit interference with the electric field created during ionization.

Second, the upper portion 33 of cassette 200 is also provided with ramped portions 214, 215 on either side of the ball electrode 22. As the cassette is moved into contact with the biased electrode 151, the electrode 151 may ride up at least a part of one ramped portion to reach the ball electrode 22 and then after use, can ride down at least part of the other ramped portion This can smooth the engagement and disengagement of the cassette into its ion ejecting position on the processing machine.

Third, the sample inlet port 223 of cassette 200 is made larger to encompass the area shown in FIGS. 1 and 2 as two apertures 30 and 31. This larger sample inlet port allows for even better viewing of a sample's circular pattern on the substrate, and allows delivery of a sample to the substrate with a reduced risk that the surface tension of liquid samples might cause part of a sample to cling to the sides of the inlet port if touched, rather than being transferred to the substrate.

With reference to FIG. 23, it can be seen that the substrate 18 is supported by the mesa 47 such that the top of substrate 18 gently touches the underside of the solvent injection port 37. By the mesa being preferably under the entire well opening above, and in direct contact with the substrate 18, the solvent cannot drip or accumulate underneath substrate 18 in that area, but rather it flows by within the substrate toward the tip. By contrast, the posts 67, 54 which support the substrate 18 at the discharge end, where there is no weight of liquid above it, are sized to be small in area and apply slight compression to the substrate, as seen in FIG. 24. Like posts 67 and 54, the interior supporting post 56 also has a relatively small contact area with the substrate 18, whereas supporting post 52 that is opposite electrode 22 and supporting post 53 that is opposite the sample inlet 23 are preferably flat with relatively larger contact areas with the substrate. Another embodiment of a cassette is depicted in FIGS. 25 and 26. In this embodiment, cassette 300 is provided with a solvent well 321 containing solvent 360 in which a rear portion 350 of substrate 318 is immersed by being bent downwardly at its end. Solvent 360 is added to cassette 300 through the solvent inlet port 337. The bottom of solvent well 321 is positioned below the upper surface of substrate 318, and thus substrate 318 is wetted by the wicking of the solvent 360 using capillary action, rather than being assisted by gravity as with the other cassette designs disclosed above. A rectangular electrode 322 extends above the main body of cassette 300 for connection to high voltage, and extends below to contact rear portion 350 of substrate 318.

As with the other cassettes disclosed, cassette 300 has a pointed tip 315 on substrate 318 near the ion sampling input 101 of the mass spectrometer at the center tip of conductive cone 102. While not specifically shown in this drawing of cassette 300, the majority of substrate 318 is preferably suspended with small posts in the manner shown with cassette 200. A generally circular sample 325 is shown on the preferably paper substrate 318. While substrate 318 is shown in a triangular configuration, it could alternatively be configured with only the tip 315 being pointed, and the other points of the triangle being modified by rounding, or by another non-triangular design that retained the single pointed tip near the ion sampling inlet 101.

Projecting portions 301 and 302 at the top and 303 and 304 (not shown, but positioned behind projecting portion 303 and below projecting portion 302) at the bottom of cassette 300 help to protect the pointed tip 315.

Ion generation using wetted porous material

Cassettes, systems, and methods of the invention allow for automation of paper spray. For paper spray generally, porous materials, such as paper (e.g. filter paper or chromatographic paper) or other similar materials are used to hold and transfer liquids and solids, and ions are generated directly from the edges of the material when a high electric voltage is applied to the material. In certain embodiments, the porous material is kept discrete (i.e., separate or disconnected) from a flow of solvent, such as a continuous flow of solvent. Instead, sample is either spotted onto the porous material or swabbed onto it from a surface including the sample. In other embodiments, the paper substrate is directly connected to a continuous flow of solvent. Further description is provided for example in Ouyang et al., WO 2010/127059, the content of which is incorporated by reference herein in its entirety. With cassettes of the invention, the cassettes are generally configured such that porous substrate is kept in continuous contact with a reservoir of solvent. However, cassettes of the invention can also be configured such that the substrate is kept discrete from the solvent reservoir.

In certain embodiments, the porous material is any cellulose-based material. In other embodiments, the porous material is a non-metallic porous material, such as cotton, linen wool, synthetic textiles, or plant tissue. In still other embodiments, the porous material is paper.

Advantages of paper include: cost (paper is inexpensive); it is fully commercialized and its physical and chemical properties can be adjusted; it can filter particulates (cells and dusts) from liquid samples; it is easily shaped (e.g., easy to cut, tear, or fold); liquids flow in it under capillary action (e.g., without external pumping and/or a power supply); and it is disposable.

In particular embodiments, the porous material is filter paper. Exemplary filter papers include cellulose filter paper, ashless filter paper, nitrocellulose paper, glass microfiber filter paper, and polyethylene paper. Filter paper having any pore size may be used. Exemplary pore sizes include Grade 1 (Ιΐμιη), Grade 2 (8μιη), Grade 595 (4-7μιη), and Grade 6 (3μιη), Pore size will not only influence the transport of liquid inside the spray materials, but could also affect the formation of the Taylor cone at the tip. The optimum pore size will generate a stable Taylor cone and reduce liquid evaporation. The pore size of the filter paper is also an important parameter in filtration, i.e., the paper acts as an online pretreatment device. Commercially available ultrafiltration membranes of regenerated cellulose, with pore sizes in the low nm range, are designed to retain particles as small as 1000 Da. Ultra filtration membranes can be commercially obtained with molecular weight cutoffs ranging from 1000 Da to 100,000 Da.

In certain embodiments, the substrate includes a material that substantially prevents diffusion of a sample into the substrate. The material for the porous substrate will depend on the properties of the sample to be analyzed. For example, when the sample is hydrophilic, the substrate is less hydrophilic than the sample. Alternatively, when the sample is hydrophobic, the substrate is less hydrophobic than the sample. Thus the sample remains substantially on top of the substrate until an appropriate solvent is applied to the substrate. The solvent is capable of diffusing into the substrate. The solvent interacts with the sample, causing the sample or components of the sample to diffuse into the substrate. In certain embodiments, the solvent is capable of mixing with the sample and both the solvent and the sample diffuse into the substrate. In other embodiments, the solvent is not capable of mixing with the sample but is capable of extracting the components from the sample that diffuse into the substrate along with the solvent.

In other embodiments, the porous material includes a drying agent in order to rapidly dry a sample that is applied to the substrate. Any drying agent that is compatible with the sample and does not interfere with analysis by mass spectrometry may be used. An exemplary drying agent is an anhydrous salt, such as magnesium sulfate, sodium sulfate, sodium carbonate, or calcium chloride. Other exemplary drying agents include blood coagulants, such as alum powder. In certain embodiments, the porous substrate also includes an internal standard. Rapid drying decreases waiting time and allows for sample analysis within approximately two to five minutes of applying a sample to the substrate. With respect to analysis of biological fluids, such a substrate allows for a point-of-care device for rapid and convenient use.

In other embodiments, the substrate includes a material that modifies an interaction between the sample and the substrate. The material may be any material that modifies the interaction between the sample and the substrate. In certain embodiments, the material modifies the interaction between the sample and substrate during sample deposition. In other

embodiments, the material modifies the interaction between the sample and substrate during sample elution. The material may coat at least a portion of the substrate. Alternatively, the material may impregnate at least a portion of the substrate. In certain embodiments, the material is silica. In particular embodiments, the silica coats a surface of the substrate.

The substrate is connected to a high voltage source to produce sample droplets containing analyte which are subsequently mass analyzed. The sample is transported through the porous material without the need of a separate solvent flow. Pneumatic assistance is not required to transport the analyte; rather, a voltage is simply applied to the porous material that is held in front of a mass spectrometer.

In certain embodiments, the porous material is integrated with a solid tip having a macroscopic angle that is optimized for spray. In these embodiments, the porous material is used for filtration, pre-concentration, and wicking of the solvent containing the analytes for spray at the solid type.

Probes of the invention work well for the generation of micron scale droplets simply based on using the high electric field generated at an edge of the porous material. In particular embodiments, the porous material is shaped to have a macroscopically sharp point, such as a point of a triangle, for ion generation. Probes of the invention may have different tip widths. In certain embodiments, the probe tip width is at least about 5μιη or wider, at least about ΙΟμιη or wider, at least about 50μιη or wider, at least about 150μιη or wider, at least about 250μιη or wider, at least about 350μιη or wider, at least about 400μ or wider, at least about 450μιη or wider, etc. In particular embodiments, the tip width is at least 350μιη or wider. In other embodiments, the probe tip width is about 400μιη. In other embodiments, probes of the invention have a three dimensional shape, such as a conical shape.

As mentioned above, no pneumatic assistance is required to transport the droplets.

Ambient ionization of analytes is realized on the basis of these charged droplets, offering a simple and convenient approach for mass analysis of solution-phase samples.

Sample solution is directly applied on the porous material held in front of an inlet of a mass spectrometer without any pretreatment. Then the ambient ionization is performed by applying a high potential on the wetted porous material. In certain embodiments, the porous material is paper, which is a type of porous material that contains numerical pores and

microchannels for liquid transport. The pores and microchannels also allow the paper to act as a filter device, which is beneficial for analyzing physically dirty or contaminated samples.

In other embodiments, the porous material is treated to produce microchannels in the porous material or to enhance the properties of the material for use as a probe of the invention. For example, paper may undergo a patterned silanization process to produce microchannels or structures on the paper. Such processes involve, for example, exposing the surface of the paper to tridecafluoro-l,l,2,2-tetrahydrooctyl-l-trichlorosilane to result in silanization of the paper. In other embodiments, a soft lithography process is used to produce microchannels in the porous material or to enhance the properties of the material for use as a probe of the invention. In other embodiments, hydrophobic trapping regions are created in the paper to pre-concentrate less hydrophilic compounds.

Hydrophobic regions may be patterned onto paper by using photolithography, printing methods or plasma treatment to define hydrophilic channels with lateral features of 200-1000 μιη. See 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 et al. (Anal. Chem. 2008, 80, 9131-9134), the content of each of which is incorporated by reference herein in its entirety Liquid samples loaded onto such a paper-based device can travel along the hydrophilic channels driven by capillary action.

Another application of the modified surface is to separate or concentrate compounds according to their different affinities with the surface and with the solution. Some compounds are preferably absorbed on the surface while other chemicals in the matrix prefer to stay within the aqueous phase. Through washing, sample matrix can be removed while compounds of interest remain on the surface. The compounds of interest can be removed from the surface at a later point in time by other high-affinity solvents. Repeating the process helps desalt and also concentrate the original sample.

In certain embodiments, methods and systems of the invention use a porous material, e.g., paper, to hold and transport analytes for mass spectral analysis. Analytes in samples are pre- concentrated, enriched and purified in the porous material in an integrated fashion for generation of ions with application of a high voltage to the porous material. In certain embodiments, a discrete amount of transport solution (e.g., a droplet or a few droplets) is applied to assist movement of the analytes through the porous material. In certain embodiments, the analyte is already in a solution that is applied to the porous material. In such embodiments, no additional solvent need be added to the porous material. In other embodiments, the analyte is in a powdered sample that can be easily collected by swabbing a surface. Systems and methods of the invention allow for analysis of plant or animal tissues, or tissues in living organisms.

Methods and systems of the invention can be used for analysis of a wide variety of small molecules, including epinephrine, serine, atrazine, methadone, roxithromycin, cocaine and angiotensin I. All display high quality mass and MS/MS product ion spectra from a variety of porous surfaces. Methods and systems of the invention allow for use of small volumes of solution, typically a few μί, with analyte concentrations on the order of 0.1 to 10 μg/mL (total amount analyte 50 pg to 5 ng) and give signals that last from one to several minutes.

Methods and systems of the invention can be used also for analysis of a wide variety of biomolecules, including proteins and peptides. Methods of the invention can also be used to analyze oligonucleotides from gels. After electrophoretic separation of oligonucleotides in the gel, the band or bands of interest are blotted with porous material using methods known in the art. The blotting results in transfer of at least some of the oligonucleotides in the band in the gel to the porous material. The porous material is then connected to a high voltage source and the oligonucleotides are ionized and sprayed into a mass spectrometer for mass spectral analysis.

Methods and systems of the invention can be used for analysis of complex mixtures, such as whole blood or urine. The typical procedure for the analysis of pharmaceuticals or other compounds in blood is a multistep process designed to remove as many interferences as possible prior to analysis. First, the blood cells are separated from the liquid portion of blood via centrifugation at approximately 1000 x g for 15 minutes (Mustard, J. R; Kinlough-Rathbone, R. L.; Packham, M. A. Methods in Enzymology; Academic Press, 1989). Next, the internal standard is spiked into the resulting plasma and a liquid-liquid or solid-phase extraction is performed with the purpose of removing as many matrix chemicals as possible while recovering nearly all of the analyte (Buhrman, D. L.; Price, P. I.; Rudewicz, P. J. Journal of the American Society for Mass Spectrometry 1996, 7, 1099-1105). The extracted phase is typically dried by evaporating the solvent and then resuspended in the a solvent used as the high performance liquid

chromatography (HPLC) mobile phase (Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M., Ithaca, New York, Jul 23-25 1997; 882-889). Finally, the sample is separated in the course of an HPLC run for approximately 5-10 minutes, and the eluent is analyzed by electrospray ionization-tandem mass spectrometry (Hopfgartner, G.; Bourgogne, E. Mass Spectrometry Reviews 2003, 22, 195-214).

Methods and systems of the invention avoid the above sample work-up steps. Methods and systems of the invention analyze a dried blood spots in a similar fashion, with a slight modification to the extraction procedure. First, a specialized device is used to punch out identically sized discs from each dried blood spot. The material on these discs is then extracted in an organic solvent containing the internal standard (Chace, D. H.; Kalas, T. A.; Naylor, E. W. Clinical Chemistry 2003, 49, 1797-1817). The extracted sample is dried on the paper substrate, and the analysis proceeds as described herein.

Methods and systems of the invention can directly detect individual components of complex mixtures, such as caffeine in urine, 50 pg of cocaine on a human finger, 100 pg of heroin on a desktop surface, and hormones and phospholipids in intact adrenal tissue, without the need for sample preparation prior to analysis. Methods and systems of the invention allow for simple imaging experiments to be performed by examining, in rapid succession, needle biopsy tissue sections transferred directly to paper. Analytes from a solution are applied to the porous material for examination and the solvent component of the solution can serve as the electrospray solvent. In certain embodiments, analytes (e.g., solid or solution) are pre-spotted onto the porous material, e.g., paper, and a solvent is applied to the material to dissolve and transport the analyte into a spray for mass spectral analysis.

In certain embodiments, a solvent is applied to the porous material to assist in

separation/extraction and ionization. Any solvents may be used that are compatible with mass spectrometry analysis. In particular embodiments, favorable solvents will be those that are also used for electrospray ionization. Exemplary solvents include combinations of water, methanol, acetonitrile, and THE The organic content (proportion of methanol, acetonitrile, etc. to water), the pH, and volatile salt (e.g. ammonium acetate) may be varied depending on the sample to be analyzed. For example, basic molecules like the drug imatinib are extracted and ionized more efficiently at a lower pH. Molecules without an ionizable group but with a number of carbonyl groups, like sirolimus, ionize better with an ammonium salt in the solvent due to adduct formation.

In certain embodiments, a multi-dimensional approach is undertaken. For example, the sample is separated along one dimension, followed by ionization in another dimension. In these embodiments, separation and ionization can be individually optimized, and different solvents can be used for each phase.

In other embodiments, transporting the analytes on the paper is accomplished by a solvent in combination with an electric field. When a high electric potential is applied, the direction of the movement of the analytes on paper is found to be related to the polarity of their charged forms in solution. Pre-concentration of the analyte before the spray can also be achieved on paper by placing an electrode at a point on the wetted paper. By placing a ground electrode near the paper tip, a strong electric field is produced through the wetted porous material when a DC voltage is applied, and charged analytes are driven forward under this electric field.

Particular analytes may also be concentrated at certain parts of the paper before the spray is initiated.

In certain embodiments, chemicals are applied to the porous material to modify the chemical properties of the porous material. For example, chemicals can be applied that allow differential retention of sample components with different chemical properties. Additionally, chemicals can be applied that minimize salt and matrix effects. In other embodiments, acidic or basic compounds are added to the porous material to adjust the pH of the sample upon spotting.

Adjusting the pH may be particularly useful for improved analysis of biological fluids, such as blood. Additionally, chemicals can be applied that allow for on-line chemical derivatization of selected analytes, for example to convert a non-polar compound to a salt for efficient

electrospray ionization.

In certain embodiments, the chemical applied to modify the porous material is an internal standard. The internal standard can be incorporated into the material and released at known rates during solvent flow in order to provide an internal standard for quantitative analysis. In other embodiments, the porous material is modified with a chemical that allows for pre- separation and pre-concentration of analytes of interest prior to mass spectrum analysis.

The spray droplets can be visualized under strong illumination in the positive ion mode and are comparable in size to the droplets emitted from a nano -electro spray ion sources (nESI).

In the negative ion mode, electrons are emitted and can be captured using vapor phase electron capture agents like benzoquinone. Without being limited by any particular theory or mechanism of action, it is believed that the high electric field at a tip of the porous material, not the fields in the individual fluid channels, is responsible for ionization.

The methodology described here has desirable features for clinical applications, including neotal screening, therapeutic drug monitoring and tissue biopsy analysis. The procedures are simple and rapid. The porous material serves a secondary role as a filter, e.g., retaining blood cells during analysis of whole blood. Significantly, samples can be stored on the porous material and then analyzed directly from the stored porous material at a later date without the need transfer from the porous material before analysis. Systems of the invention allow for laboratory experiments to be performed in an open laboratory environment.

Incorporation by Reference

References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Equivalents

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

Claims

What is claimed is:

1. A sampling cassette, the cassette comprising: a hollow housing comprising an inside configured to hold a solid porous substrate, at least one inlet, an outlet, and an electrode, wherein the housing is configured such that the inlet is in fluid communication with the substrate held inside the housing and the electrode is in contact the substrate held inside the housing.

2. The cassette according to claim 1, wherein the at least one inlet is a plurality of inlets and a first inlet is located in proximity to the outlet and a second inlet is located distal the first inlet.

3. The cassette according to claim 2, wherein the electrode is located between the first and second inlets

4. The cassette according to claim 3, wherein the first inlet is configured to mate with a pipette tip.

5. The cassette according to claim 4, wherein the second inlet further comprises a solvent reservoir.

6. The cassette according to claim 5, wherein the configuration of the inside of the housing minimizes interference with liquid flow through the substrate.

7. The cassette according to claim 6, wherein a top of the housing comprising at least one transparent section.

8. The cassette according to claim 7, wherein the transparent section is located adjacent the first inlet.

9. The cassette according to claim 7, further comprising at least one ventilation hole.

10. The cassette according to claim 9, further comprising an identifier.

11. The cassette according to claim 10, wherein the identifier is a barcode.

12. The cassette according to claim 11, wherein the barcode is a two-dimensional barcode.

13. The cassette according to claim 10, wherein the outlet comprises a shroud.

14. The cassette according to claim 10, wherein the outlet comprises a plurality of prongs.

15. The cassette according to claim 14, wherein the prongs diverge from each other.

16. The cassette according to claim 1, further comprising the solid porous substrate.

17. The cassette according to claim 16, wherein the substrate comprises a pointed tip.

18. A cassette processing system, the system comprising:

a cassette holder configured to hold at least one sampling cassette that comprises a porous substrate comprising a sample;

at least one solvent reservoir;

an analysis stage;

an exit port; and

a cassette moving component configured to receive the cassette from the holder and move the cassette through the system to interact with the solvent reservoir, the analysis stage, and the exit port.

19. The system according to claim 18, wherein the cassette holder is a magazine.

20. The system according to claim 19, wherein the solvent reservoir further comprises a fluid injector.

21. The system according to claim 20, wherein the analysis stage comprises a voltage supplier.

22. The system according to claim 21, further comprising a mass spectrometer operably coupled to the analysis stage.

23. The system according to claim 22, further comprising a camera.

24. The system according to claim 23, wherein the camera is positioned to image an identifier on the cassette.

25. The system according to claim 24, wherein the fluid injector is configured to mate with an inlet of the sample cassette to thereby inject solvent from the reservoir onto the substrate within the cassette.

26. The system according to claim 25, wherein the voltage supplier mates with an electrode in the sample cassette which is in contact with the substrate inside the cassette, thereby producing sample droplets containing analyte that are expelled from the substrate and into the mass spectrometer.

27. The system according to claim 26, further comprising a controller.

28. The system according to claim 27, wherein the controller controls both the cassette processing system and the mass spectrometer.

29. A sample analysis system, the system comprising:

a mass spectrometer; and

an automated apparatus operably coupled to the mass spectrometer, wherein the apparatus prepares a sample loaded onto a solid porous substrate for analysis by mass spectrometry and generates charged sample droplets containing analyte that are expelled from the substrate and into the mass spectrometer.

30. The system according to claim 29, wherein the automated apparatus comprises:

a cassette holder configured to hold at least one sampling cassette that comprises the porous substrate comprising the sample;

at least one solvent reservoir;

an analysis stage;

an exit port; and

a cassette moving component configured to receive the cassette from the holder and move the cassette through the system to interact with the solvent reservoir, the analysis stage, and the exit port.

31. The system according to claim 30, wherein the cassette holder is a magazine.

32. The system according to claim 31, wherein the solvent reservoir further comprises a fluid injector.

33. The system according to claim 32, wherein the analysis stage comprises a voltage supplier.

34. The system according to claim 33, further comprising a camera.

35. The system according to claim 34, wherein the camera is positioned to image an identifier on the cassette.

36. The system according to claim 34, wherein the fluid injector is configured to mate with an inlet of the sample cassette to thereby inject solvent from the reservoir onto the substrate within the cassette.

37. The system according to claim 36, wherein the voltage supplier mates with an electrode in the sample cassette which is in contact with the substrate inside the cassette, thereby producing sample droplets containing analyte that are expelled from the substrate and into the mass spectrometer.

38. The system according to claim 37, further comprising a controller.

39. The system according to claim 38, wherein the controller controls both the cassette processing system and the mass spectrometer.

40. A method for analyzing a sample, the method comprising:

providing a sampling cassette comprising: a hollow housing comprising an inside loaded with a solid porous substrate, at least one inlet, an outlet, and an electrode, wherein the housing is configured such that the inlet is in fluid communication with the substrate held inside the housing and the electrode is in contact the substrate held inside the housing;

loading a sample into the cassette via the inlet so that the sample contacts the substrate; loading a solvent into the cassette via the inlet so that the solvent contacts the substrate; applying a voltage to the substrate via the electrode in the cassette, thereby producing sample droplets containing analyte that are expelled from the substrate via the outlet; and

analyzing the analytes.

41. The method according to claim 40, wherein the at least one inlet is a plurality of inlets and the sample is loaded via a first inlet and the solvent is loaded via a second inlet.

42. The method according to claim 40, wherein the solvent assists in transport of the sample through the porous material.

43. The method according to claim 40, wherein the solvent comprises an internal standard.

44. The method according to claim 40, wherein the solvent minimizes salt and matrix effects.

45. The method according to claim 40, wherein the solvent is capable of mixing with the sample.

46. The method according to claim 40, wherein prior to solvent loading step, the method further comprises drying the substrate.

47. The method according to claim 40, wherein prior to the providing step, the method further comprises loading the substrate into the cartridge.

48. The method according to claim 40, wherein analyzing comprises providing a mass analyzer to generate a mass spectrum of analytes in the sample.

49. The method according to claim 48, wherein the mass analyzer is for a mass spectrometer or a handheld mass spectrometer.

50. The method according to claim 49, wherein the mass analyzer is selected from the group consisting of: a quadrupole ion trap, a rectilinear ion trap, a cylindrical ion trap, a ion cyclotron resonance trap, an orbitrap, a time of flight, a Fourier Transform ion cyclotron resonance, and sectors.

51. The method according to claim 40, wherein the sample is a biological fluid.

52. The method according to claim 51, wherein the biological fluid is blood.

53. The method according to claim 40, wherein the solvent loading step and the applying step of the method are performed in an automated manner.

54. The method according to claim 40, wherein the solvent loading step and the applying step of the method are performed manually.

55. The method according to claim 53, wherein the loading step occurs prior to the providing step.