Method and Device for Sample Preparation FIELD OF THE INVENTION

Field of the invention is mass spectrometric ionization methods, with special regard to the improvement of efficiency and development of desorption ionization methods. BACKGROUND OF THE INVENTION

Throughout this application, various references are cited in square brackets to describe more fully the state of the art to which this invention pertains. The disclosure of these references is hereby incorporated by reference into the present disclosure.

Mass spectrometric ionization methods have been traditionally developed for the analysis of gaseous or volatile materials. Disadvantage of these ionization methods is that they lack the capability of analysis of non-volatile compounds. This group of compounds includes peptides, proteins, nucleic acids and carbohydrates; that is approx. 90% of biologically relevant molecules.

Recent development of the group of ionization methods termed "direct ionization" or "ambient ionization" has opened a number of novel areas of application for mass spectrometric analysis. Ambient techniques such as thermal desorption/ APCI were first described over 20 years ago, but most of them were never in widespread use. [Van Berkel, G. J., et al., O. J. Mass Spectrom. (2008) 43, 1 161 ; Rommers, P., et al., J. Anal. Chem. (1996) 355] The recent renaissance of the ambient techniques started with the development of desorption electrospray ionization (DESI) [Takats, Z., et al., Science 2004, 306, 471; Takats, Z., et al., Mass Spectrom. (2005) 40, 1261 ], which was quickly followed by "direct analysis in real time" (DART) [Cody, R. B., et al., Anal. Chem. (2005) 77, 2297; Petucci, C, et al., Anal. Chem. (2007) 79, 5064], desorption atmospheric pressure chemical ionization (DAPCI) [Williams, J. P., et al., Rapid Commun. Mass Spectrom. (2006) 20, 1447] and a number of other methods. The common feature of these methods is the lack of the imperative need for sample preparation prior to analysis, which dramatically simplifies and thus accelerates the analytical scheme. [Cooks, R. G., et al., J. M. Science (2006) 31 1 , 1566; Harris, G. A., et al., Analyst 2008, 133, 1297]

The importance of high-throughput (HT) analytical methods has emerged with the advent of the combinatorial chemistry era in the 80s pharmaceutical industry. Semiquantitative methods were developed using mainly mass spectrometric methods with no or minimal chromatographic separation. [Kassel, D. B. Chem. Rev. (2001 ) 101 , 255] The area has gotten further momentum from the development of modern HPLC-MS methods utilizing ESI and APCI ionization techniques.

[Fenn, J. B., et al., Science (1989) 246, 64; Horning, E. C, et al., Anal. Chem. (1973) 45, 936; Kyranos, J. N., et al., Curr. Opin. Biotechnol. (2001) 12, 105; Alford, A. Biomed. Mass Spectrom. (1978) 5, 259; Koh, H.-L., et al., Drug Discov. Today (2003) 8, 889; Lim, C-K.; Lord, G. Biol. Pharm. Bull. (2002) 25, 547; Richardson, S. D. Anal. Chem. (2008) 80, 4373; Vukelic, Z., et al., Chromatogr. A (2006) 1 130, 238; Zollner, P.; Mayer-Helm, B. J. Chromatogr. A (2006) 1 136, 123] The development of HT bioanalytical methods based on electrophoresis, [Garza, S., et al., Chromatogr. A (2007) 1 159, 14] chromatography, and mass spectrometry was the basis of modern genomics, proteomics, metabolomics, and lipidomics. HT analytical techniques have gained high importance in medical diagnostics and clinical chemistry. The rapidly emerging field of personalized medicine also requires HT techniques for therapeutic drug monitoring, genetic and enzyme assays. [Jain, K. K. Curr. Opin. MoI. Ther. (2003) 5, 548; Nunn, A. D. Cancer Biother. Radiopharm. (2007) 22, 722]

Although ambient mass spectrometric techniques are claimed to provide a "sample preparation-free" analytical scheme, this statement does not necessarily stand for regular analytical applications. On one hand, ambient ionization techniques do give mass spectrometric information on untreated, native samples, on the other hand, it is seldom the analytical task to obtain unspecified mass spectrometric data characteristic of the sample. Real life analytical problems generally tackle the qualitative and/or quantitative determination of certain, pre-determined species of interest, and ambient ionization techniques often fail to accomplish this task. An ideal "sample-preparation free" technique should be able to detect and quantify species of interest in native samples in one step, at relevant concentrations. So far this has been accomplished only for a few combinations of analytes and matrixes, and no generally applicable MS-based technique has been described which fulfills these requirements. Furthermore, practically any treatment of sample prior to analysis may qualify as sample preparation (e.g., drawing of blood sample, preparation of plasma and deposition of plasma onto glass slide prior to DESI analysis) so complete elimination of sample preparation is often impossible, and even "sample preparation free" methods employ multiple steps of sample treatment prior to analysis. Complete elimination of sample preparation is necessary only, when in situ, real-time analysis is required, for example, in the case of airport security applications. The lack of proper sample preparation is usually reflected in poor sensitivity and interference from isobaric matrix peaks. The qualitative analysis of biological fluids by ambient mass spectrometry, similarly to traditional methods, requires sample cleanup to eliminate background interference. Purification is

actually more critical for ambient techniques than for LC-MS methods, since the former intrinsically do not involve chromatographic separation of the analytes prior to mass spectrometric detection.

Traditional desorption ionization techniques have a dynamic range of only 1-2 orders of magnitude, while later techniques demonstrated significantly better range (e.g. 2-4 orders of magnitude linear calibration range for DESI) allowing them to be used for quantitative analytics. This ionization efficiency, however, is still 2-4 orders of magnitude lower than those exhibited by competing (but much slower) spray-ionization techniques, practically prohibiting the widespread use of these techniques not withstanding their great potential for high-throughput use.

Cyclosporin A (a ciklopeptite) has an absolute detection limit at the 0,05-0,2 ng/ml range with electorspray-ionization, remaining below 1 ng/ml even when the blood plasma has been deproteinized with acetonitril. DESI exhibits a detection limit of 100-300 ng/ml, and while the use of DESI would eliminate the need for sample preparation (allowing the analysis of the sample directly after having it dried onto the surface of the carrier) this range is well above the upper limit of the detection range used for therapeutic applications (50-70 ng/ml) making the technique inadequate for drug level monitoring in clinical chemistry, as opposed to electrospray-ionization that may be used either directly, or in combination with an HPLC system.

The present invention, termed solid phase extraction enhanced desorption ionization (SPEEDI), was developed to overcome the intrinsically poor sensitivity of atmospheric (ambient) pressure desorption ionization methods, and also for the on-line coupling of sample preparation with these analysis methods, and also for the quick and efficient conversion of liquid phase samples into confined areas of solid layer suitable for desorption ionization-MS analysis.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides for an assembly for processing a sample mixture for analytical testing of one or more target analytes in the sample mixture, characterized in that said assembly comprises:

(a) an adsorbent part including an adsorbent material for selectively separating the one or more target analytes from the sample mixture, said adsorbent part having a first outer surface and a second outer surface; and

(b) an elutor part including: (i) means capable of delivering a first drying vehicle and an elutant to the first outer surface for drying the one or more target analytes in the adsorbent material and eluting the one or ore target analytes to the second outer surface; and (ii) means for delivering a

second drying vehicle for evaporating the elutant off the second outer surface, thereby placing the one or more dried target analytes on the second outer surface of said adsorbent part for analytical testing.

In another aspect, the present invention provides for an assembly useful for high-throughput processing of a plurality of sample mixtures for analytical testing of one or more target analytes in the plurality of sample mixtures, characterized in that said assembly comprises:

(a) a solid support part including a plurality of channels disposed thereon, wherein each channel is capable of accommodating one adsorbent part, said adsorbent part comprising an adsorbent material for selectively separating the one or more target analytes in each sample mixture, said adsorbent part having a first outer surface and a second outer surface, each channel having a first open end and a second open end for exposing the first outer surface and the second outer surface of the adsorbent part respectively; and

(b) an elutor part comprising: (i) a first array defining a surface and a plurality of holes for carrying a first drying vehicle and an elutant to the first end of the plurality of channels of the solid support for drying the one or more target analytes in the adsorbent material and eluting the one or more target analytes to the second outer surface, and (ii) a second array defining a surface and a plurality of holes for carrying a second drying vehicle to the second end of the plurality of channels of the solid support for evaporating the elutant off the second outer surface, thereby placing the one or more dried target analytes on the second outer surface in the plurality of channels for high- throughput analytical testing.

In yet another aspect, the present invention provides for a method for analytical testing of one or more target analytes in a sample mixture, characterized in that said method comprises the following steps:

(a) contacting the sample with an adsorbant material capable of selectively separating the one or more target analyte from the sample mixture;

(b) eluting the one or more target analytes from the adsorbent material to an outer surface on the adsorbent material with the use of an eluting solution;

(c) drying the eluting solution from the outer surface with the use of a second drying vehicle, thereby placing the one or more target analytes on the outer surface of the adsorbent material; and (d) performing analytical testing of the one or more target analytes.

In another aspect yet, the present invention provides for a method for high-throughput analytical testing of one or more target analytes in a plurality of sample mixtures, characterized in that said method comprises the following steps:

(a) providing an assembly useful for high-throughput processing of the plurality of sample mixtures according to one aspect of the present invention;

(b) contacting each sample mixture in the plurality of mixtures with the adsorbent material at the first end of the plurality of channels for selectively separating the one or more target analytes in the plurality of sample mixtures;

(c) eluting the one or more target analytes from the adsorbent material in the plurality of channels to second outer surface with the use of an eluting solution;

(d) drying the eluting solution from the second outer surface in the plurality of channels, thereby placing the one or more target analytes on the second outer surface of the adsorbent material; and

(e) performing high-throughput analytical testing on the one or more target analytes.

In a further aspect, the present invention provides for a method for remote analytical testing of one or more target analytes in a sample mixture, characterized in that the method comprises: a) providing a user with a sample holder, said sample holder comprising an adsorbent material for selectively separating the one or more target analytes in the sample mixture, said sample holder having a first outer surface and a second outer surface; b) the user contacting the sample mixture with the adsorbent material of the sample holder; c) the user transferring the sample holder with the sample mixture to a remote location for analytical testing; d) the remote location placing the sample holder in an assembly useful for processing the sample holder for analytical testing of the one or more target analytes, wherein said assembly comprises an elutor part including: (i) means for delivering a first drying vehicle and an elutant to the first outer surface of the sample holder for drying the one or more target analytes in the adsorbent material and eluting the one or more target analytes to the first outer surface; and (ii) means for delivering a second drying vehicle for evaporating the elutant off the second outer surface, thereby placing the one or more target analytes on the second outer surface of said sample holder; and

e) the remote location performing the analytical testing of the one or more target analytes on the second outer surface of the sample holder.

Advantages of the invention include:

(1) the arbitrary increase of sensitivity in the case of mass spectrometric analysis methods utilizing desorption ionization methods to convert analyte molecules into gaseous ions.

(2) enabling the deposition and drying of liquid phase samples onto porous surfaces to form a confined spot where analyte molecules are distributed evenly, providing ideal sample for desorption ionization.

(3) Eliminating matrix interferences in the case of desorption ionization-mass spectrometric analysis of complex samples such as biological fluids. Biological fluids include whole blood, plasma, serum, urine, CSF, interstitial fluid, bile, etc. Analysis of biological fluids carries particular importance on the field of pharmacology, clinical diagnostics, forensic analysis, etc. This advantage results in better reproducibility and accuracy of desorption ionization-mass spectrometric methods.

(4) High throughput analysis of samples. (5) Eliminating the need for long changeover times between different kinds of samples as required by current HPLC-MS methods.

(6) Providing automated sample analysis.

(7) Solves the problem of easy transport for fluid samples designated for analysis.

(8) Enabling sample collection to be carried out by a patient in his/her own home. The method and assembly of the present invention are based on the adsorption of analyte molecules on a solid phase extraction packing and elution of the analyte onto a closing membrane of the packing. Thus, matrix interferences are eliminated and analyte molecules are concentrated on the surface of the closing membrane of solid phase extraction (SPE) packing. Furthermore, in contrast to the given surface characteristics required for dried biological fluids (e.g. blood clot), the present invention offers freedom in choosing optimal type of surface for desorption ionization method used. The method and assembly of the present invention were implemented for high-throughput analysis, and it was demonstrated that sample preparation does not extend time demand of overall analytical procedure.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates a simplified scheme of solid phase extraction enhanced desorption ionization method;

Figure IA shows the application of liquid containing analyte molecules onto SPEEDI cartridge.

Figure 1 B shows the washing of cartridge with liquid that flows through cartridge and removes residual matrix sample components.

Figure 1C shows drying of samples, which step is accomplished by flowing dry nitrogen gas through cartridge.

Figure ID shows elution of analyte molecules onto surface of porous membrane. Figure IE shows the desorption ionization/mass spectrometric analysis of sample. Figure 2 illustrates a schematic structure of a SPEEDI cartridge.

Figure 3 A and Figure 3 B illustrates schematic designs of SPEEDI elutor device of the present invention.

Figure 4A illustrates a cross section schematic design of SPEEDI card in accordance with one aspect of the present invention. Figure 4B illustrates a schematic design of a multi-layered card in accordance with one aspect of the present invention.

Figure 5 illustrates a simplified multiple-well SPEEDI plate in accordance with one aspect of the invention.

Figure 6 illustrates a simplified multiple-channel elutor in accordance with one aspect of the present invention.

Figure 7 is a schematic working mechanism of SPEEDI procedure. Figure 8 is a schematic working mechanism of SPEEDI card.

Figure 9 is a logarithmic calibration curve for determination of cyclosporine A in human plasma by DESI/MS and SPEEDI/DESI/MS methods. Figure 10 is a calibration curve for determination of atrazine in surface water samples using DART/MS and SPEEDI/DART/MS.

Figure 1 1 is acyl-carnitine profile of whole human blood sample obtained by a) DESI/MS and b) SPEEDI/DESI/MS.

Figure 12 illustrates the schematic workings of a potential business model in accordance with one aspect of the invention, where sample is produced and tagged by home users, GP-s offices, or small healthcare units and are subsequently shipped to the laboratory to be analyzed. Resulting data is sent back to the customers/patients online without human intervention. Figure 13 illustrates a 96-channel DESI ion source on the 2D moving stage. (1) Electro-sonic spray emitter, (2) SPE plate containing 96 individual SPE cartridges (depicted in Figure Ia.) in a 8 x 12 raster, (3) heated capillary, (4) home-built atmospheric interface housing, (5) temperature sensor, (6) 2D moving stage, (7) mass spectrometer. Samples are applied onto a 96 channel SPE plate and analyte is eluted onto top closing frits of individual cartridges. Prepared plate is secured onto aluminum frame (8), and individual cartridges are analyzed sequentially by moving them under sprayer (1). Ion source communicates with data acquisition software of mass spectrometer as an autosampler device.

Figure 14 illustrates LLOD of method depending on sample volume: the line indicates theoretical results with DESI method; points indicate the results of real SPEEDI/DESI measurements. Figure 15 illustrates a comparison of elution process using solvent flow rate below and above maximum solvent evaporation rate. In the case of optimal elution (a) all solvent is evaporated, and analyte is crystallized evenly on frit surface. When evaporation is incomplete, (b) accumulating solvent redissolves analyte crystals, and eventually drying gas flow removes practically all analyte solution from the surface (c). Figure shows schematic representation of the processes, photo of resulting samples, and line scans obtained by DESI analysis.

Figure 16 illustrates (a) Dependence of maximal evaporation rate on gas flow rate and temperature, (b) Dependence of maximal evaporation rate on liquid flow rate and elution time.

Figure 17 illustrates reproducibility as a function of sampling rate. (RSD: relative standard deviation, ISTD: internal standard). DETAILED DESCRIPTION OF THE INVENTION

The invention will be explained in details by referring to the figures.

In the present invention, the Applicant discloses the development, characterization, and practical application of a universal sample preparation method and assembly for analytical testing of one or more target analytes in a sample mixture. More particularly, disclosed herein is the development, characterization, and practical application of a universal sample preparation method and assembly

for desorption ionization mass spectrometry, especially for desorption electrospray ionization-MS The methods and assemblies of the present invention are suitable for high-throughput analytical testing of the one or more target analytes in the sample mixture

The sample extraction assembly in accordance with one aspect of this invention, is shown in figure 1 , and comprises of following elements

Adsorbent The primary function of adsorbent material 4 is to selectively remove or extiact analyte (target) molecules 2 from the sample mixture via adsorption when the sample mixture 1 is passed through the adsorbent 4 or is brought into close contact with it Adsorbent material 4 can be any solid material with high specific surface area Adsorbent mateπal 4 include any material suitable for solid phase extraction (SPE) Adsorbent material 4, also refer to as SPE packing, refers toa particulate adsorbent material (porous or non-porous) which is able to selectively bind certain components of fluid samples when the sample is flown through or mixed with this adsorbent material 4 Examples include granulated active carbon, chemically modified silica particles, and various polymers among many others, or any mixture of the listed adsorbents An important feature of the adsorbent material 4 is the high specific surface area and well-defined interaction between analyte and packing material Adsorbent materials 4 may be used with or without surface modification By "surface modification" what is meant is covalent binding ol molecules onto a surface, application of thin film of molecules, or any other modification for changing the adsorptive character of a surface The function of adsorbent material 4 is the selective adsorption of target molecules from a sample matrix Hence, adsorbent mateπal 4 is chosen with regard to chemical characteristic of analyte and sample matrix Hydrophobic (e g octadecyl-silica) adsorbent may be used for the selective extraction of hydrophobic molecules from water, anionic adsorbent may be used for the selective extraction of cationic components from a non-ionic medium Adsorbent can also be utilized for the selective adsorption of interfering components, e g cationic components may interfere with positive ion desorption ionization due to suppression effects, so additional anionic adsorbent can be used to irreversibly remove certain cationic species

Porous membranes Target membrane 3 has multiple functions Taiget membrane 3 holds the adsorbent 4 in place and serves as sample carrier target for desorption lonization/mass spectrometry analysis Furthermore, membrane 3 also accommodates solvent evaporation process during elution of the analyte molecules Due to its multifunctional character porous membrane 3 can be multi- layered, where top layer is appropriate for solvent evaporation or desorption ionization while bottom layer provides mechanical stability Porous membrane 3 may be made of, without limitation, porous

polymer (porous polyethylene frits, poly-tetrafluoroethylene membranes), fibrous material, glass frit, metal frit or any other porous material having appropriate mechanical rigidity to hold adsorbent in place and appropriately low flow resistance to let sample and elution solvent through. A large effective surface area is desired for efficient evaporation, which is also advantageous for desorption ionization. Membrane 3 may be completely omitted, or combined with adsorbent 4. Bottom membrane 6 functions only for holding adsorbent 4 in place. Membrane 6 can be made of any porous material, which has smaller pore size than particle size of adsorbent 4 in the case of particulate adsorbent.

Housing. Primary function of housing 5 as shown on figure IA- IE, is to hold the adsorbent material 4, and to provide confined space for the sample mixture in fluid form to flow through the adsorbent material 4. The housing 5 may be provided as a cartridge-type or card-type devices.

The cartridge-type device (shown on figure 2) comprises a housing tube 5, which is packed with adsorbent material 4. Open surfaces of bulk adsorbent material are closed by porous membranes 3, 6. Membranes 3, 6 hold adsorbent material 4 in housing 5, and membrane 3 also serves as carrier surface of analyte 2 molecules during desorption ionization mass spectrometric analysis. In the case of cartridge-type devices the housing 5 is an open tube, which is made of solid material with sufficient mechanical strength and solidity. These materials include plastics, glass, ceramics, and metals. Important feature of tube material is the lack of porosity and inertness, i.e. tube wall is not supposed to interact with any of the components of sample, eluting solvent or drying gas. Housing 5 may also be provided in the form of card-type devices (shown on figures 3, 4A and 4B). In the case of card-type devices (figure 4A), the housing 5 is a sheet piece, such as polypropylene sheets, which embeds a sheet of adsorbent material 4 and, optionally membranes 3, 6. Card-type devices may also have two ore more holes drilled into them, wherein the adsorbent material 4 are embedded into with one or two or more polymeric membranes 3, 6 covering the surfaces. Requirements on characteristics of card-type devices are identical to those of described in the case of cartridge-type devices.

Figure 4B illustrates a multi-layered card-type device comprising the same parts as SPEEDI card shown on figure 4A, however multiple types of adsorbents are applied in separate layers. Different types of adsorbents have different specificity, i.e. adsorb species with different chemical character. Layers may be removable prior to elution in order to eliminate interferences.

With reference to figure 5, there is provided a housing 5 for high-throughput analysis. Figure 5 illustrates a 96-well SPEEDI plate comprising of a solid plate 19 made of plastic, metal, or ceramics with 8 x 12 holes drilled into it, with the possibility of embedding 96 cartridges 5 into the wells, enabling access to the membranes 3 from of the 96 cartridges for analysis. Elutor Device. With reference to figure 3, the elutor is used for the elution of analyte 2 molecules from adsorbent 4 and concentration of them on porous membrane 3 via introduction of solvent 11 through porous membrane 6, desorption of analyte 2 from adsorbent 4, and complete evaporation of solvent 11 from 3 porous membrane.

Elutor device of the present invention comprises tubing 17 which is capable of carrying eluting solvent 11 and a drying vehicle, such as dry gas, and tubing 16 which is capable of carrying a second drying vehicle, such as gas 10. Tubing 16 may be equipped with resistance heater, thermocouple and temperature controller in order to provide control on the temperature of gas 10 (such as dry nitrogen). Tubing 17 is connected to diverter valve V, which is connected to liquid pump and a source of dry gas. During elution solvent 11 flows through tubing 17. When elution step is accomplished, diverter valve V is switched, and dry gas (nitrogen, air) is introduced into cartridge to purge residual solvent 11 from adsorbent. The elutor device may also contain holder elements 19 which provide the user with control over the relative positions of cartridge during elution, fluid delivery tubing 17, and gas emitting nozzle 16.

With reference to figure 6, in the case of multi-n-channel elutor device (wherein "n" is an integer larger than 1) useful for high-throughput analytical testing, the nozzle 16 of figure 3 is changed to heated metal block 16 having n-holes drilled into it. Fluid introduction device 17 may also have an altered function in this case, since it is only used to supply dry gas 10. The plate 19 carrying n number cartridges is positioned between parts 16 and 17.

In figure 6, the multiple-channel (96 channels in this example) device consists of plate 19, an air blower fan, a block 16, frame for a 96 well SPEEDI plate, and fluid introduction device 17. Block 16 may be equipped with heater, thermocouple, temperature controller and 96 holes drilled into it, block 16 is attached to plate 19 via spacer 20. Fluid introduction device 17 may be lined with a soft, elastic material 18 for sealing between fluid introduction device 17 and individual cartridges.

Sample Processing The present invention relates to a method and assembly developed, primarily, for the preparation of non-volatile samples (in fluid or liquid form) for subsequent analytical testing, such as desorption

ionization/mass spectrometric analysis or spectrophotometric methods used in reflexion mode, such as Raman spectroscopy. A general working scheme of method is shown on figure 1.

First step of the method (figure IA) is to bring the liquid phase sample 1 carrying the target molecule 2 into close contact with adsorbent material 4, preferably by flowing sample 1 through tubing 5 packed with adsorbent material 4. Adsorbent material 4 is suitable for selective binding of target molecules 2.

Following application of liquid sample mixture, adsorbent material 4 is washed with a first solvent 1 capable of removing non-binding residues of sample from the adsorbent material 4, but without interfering with the binding of target molecule 2 on adsorbent 4. The washing step is followed by drying of adsorbent material 4 with a drying vehicle such as dry gas 10. Drying is necessary to provide optimal conditions for subsequent elution, since if adsorbent 4 remains wetted with first solvent 1, then, if first solvent 1 is immiscible in a second eluting solvent 11, then 11 solvent cannot be brought into close contact with adsorbent material 4, which results in poor analyte desorption/elution efficiency. The dry adsorbent material 4 is eluted by bringing it into close contact with a second solvent 11 capable of desorption of analyte 2 from surface of adsorbent 4, preferably by flowing solvent 11 through tubing 5 packed with adsorbent 4 carrying analyte 2. As it is shown on figure ID, the second solvent 11 enters adsorbent 4 through porous membrane 6, and leaves it together with target molecules 2 dissolved in it, to porous membrane 3. The second solvent 11 is evaporated from outer surface of porous membrane 3, with the use of a drying vehicle, for example by blowing heated gas 10 onto said outer surface of porous membrane 3. Gas can be blown 10 onto said outer surface of membrane 3 using heated nozzle 16. As second solvent evaporates 11, all analyte molecules are concentrated on gas/solid interface. Evaporation of the second solvent can be implemented by other means, such as laser irradiation, or irradiation of solvent with other appropriate electromagnetic radiation such as microwave radiation.

The solvents used are highly dependent on the solvent of the original sample, the type of adsorbent and the nature of analyte molecule. For example, when cholesterol is determined in a biological fluid using octadecyl-silica adsorbent, then the adsorbent can be washed with water or methanol. In general, the solvent of original sample can always be used as solvents, however there are many other solvents which can also be used.

The final step of method is the desorption ionization/mass spectrometric analysis, as shown on figure Ie. Analytical beam 12 of desorption ionization source 13 is directed onto said outer surface of porous membrane 3, which carries analyte molecules 2. Analyte molecules 2 are converted to gaseous ions 14 on the impact of analytical beam 12. Gaseous ions 14 sampled then by atmospheric inlet 15 of mass spectrometer, and subjected to mass spectrometric analysis.

Figure 8 illustrates another embodiment of the present invention, wherein an analogous method was is presented for the analysis of samples adsorbed on sheet geometry adsorbent material. With reference to figure 8, in this second embodiment, the liquid sample 1 can just be deposited into adsorbent 4 material, and sample solvent 1 can be let to evaporate. Particularly important example for such case is when liquid sample 1 is blood or other biological fluid and adsorbent 4 material is filter paper.

Sheet adsorbents include solid phase extraction (SPE) discs and various filter papers. SPE discs are particularly widespread in environmental analysis, especially for the extraction of surface water samples, while filter paper-type sample collection devices are widely used in medical diagnostics for handling biological fluid samples. The analysis of samples present on sheet adsorbents by the means of desorption ionization mass spectrometry has been demonstrated in the case of dried spots of whole blood for drug monitoring and diagnostics of metabolic diseases. [Takats, Z., et al., Science (2004) 306, 471 ] While the synergism between this sample format and desorption ionization (DI, especially DESI) mass spectrometry is evident, direct DI analysis of samples does not provide full availability of analyte molecules present in the bulk material of the adsorbent. DI-MS methods generally analyze molecules present on the condensed/gas interface; thus molecules "hidden" deep in solid phase adsorbent do not undergo ionization. Furthermore, in the case of DESI analysis, spray solvent tends to diffuse into the adsorbent material, which process decreases the availability of the analyte even further, and also hinders the formation of continuous liquid film on the surface. (Continuous liquid film has been considered being prerequisite for ion formation in DESI. [Costa, A. B.; Cooks, R. G. Chem. Commun. (2007) 3915]

Surface elution combined with sample card format (figures 4A and 4B) provides a solution for the problems associated with DI-MS analysis of sheet adsorbents. To eliminate disadvantageous lateral diffusion effects, confined filter paper discs were embedded into a plastic card and were lined with hydrophobic PTFE membranes on one side. The sample format was termed SPE card. The PTFE membrane has a dual role in the depicted setup; it keeps the aqueous sample in the filter paper when the sample is applied and also provides an optimal surface for DESI analysis. Both application and

elution of the sample occurs from the exposed surface of filter paper; hence eluted analyte molecules are concentrated on PTFE membrane prior to DESI analysis.

Figure 4 B illustrates an aspect of the present invention in which multiple types of adsorbents are used 4. Use of different adsorbent 4b from adsorbent 4a may be useful for the extraction of target analyte molecules 2 from a sample, when other molecules in the sample interfere with desorption ionization/mass spectrometric analysis of target analyte molecules 2. Adsorbent 4b then ideally does not show any affinity to bind target analyte molecules 2, but strongly adsorbs other non-target molecules in the sample. Different types of adsorbents can be mixed to together, or used in separate layers. If different adsorbents are used in separate layers, then different types of adsorbents are preferably isolated from each other by using appropriate 3a and 3b porous membranes. Adsorbent 4b together with membrane 3b can preferably be removed from extraction device after application of sample and washing. In this case surface concentration and desorption ionization analysis occurs on porous membrane 3a.

The sample preparation method in accordance with the different aspects of the present invention, is based on the adsorption of the analyte molecules on solid phase extraction (SPE) packing and the elution of the analyte onto the membrane which seals the cartridge. Thus, matrix interference is eliminated, and the analyte molecules are concentrated on the confined surface of the closing frit of the SPE cartridge. Furthermore, instead of accepting the intrinsic surface characteristics of dried biological fluids (e.g., blood clot), the method offers a possibility to choose the optimal surface type for the desorption ionization method used. The sample preparation method was implemented for high-throghput analysis, and it was demonstrated that the proposed sample preparation does not extend the overall time demand of the analytical procedure.

High throughput sample processing

In one aspect, the present invention is a method and device for high/throughput applications, and multiple samples, e.g. 96 can samples can be prepared in parallel fashion. For high throughput experiments, samples are pipetted into an array of tubes packed with adsorbent material. Figure 7 and 7b shows horizontal and vertical cross section of device according to this aspect of the invention.

Following application of samples, the whole device is dried in one step, eluting solvent 11 is pipetted into tubes and the plate is positioned into an appropriate elutor device shown on figure 6.

The multi-channel elutor device utilizes external gas source to facilitate the flow of eluting solvent

11 through adsorbent material 4. In a parallel fashion, dry gas 10 is flown over porous membranes 3 using an outer gas source (gas cylinder, compressor, gas pump, fan) through holes drilled into metal block 16. Metal block 16 is equipped with heater elements, temperature sensor and these are connected to temperature controller unit. Eluting solvent 11 flows through adsorbents 4, desorbs analyte molecules 2 from adsorbent 4 and leaves extraction devices through porous membranes 3. Resulting solution of analyte 2 in solvent 11 is completely evaporated from porous membranes 3, concentrating analyte molecules 2 on gas/solid interface between porous membrane material 3 and atmosphere.

The array of extraction devices is then subjected to desorption ionization/mass spectrometric analysis using appropriate desorption ionization method. Desorption ionization of samples dried onto porous membranes 3 is implemented sequentially. Individual samples are moved into appropriate relative position to analytical beam 12 emitter and atmospheric inlet 15 of mass spectrometer using a moving stage system, preferably using a computer controlled moving stage system. When porous membranes 3 carrying analyte molecules 2 reach appropriate position for analysis, analytical beam 13 is directed onto surface of porous membranes 3, and impact of analytical beam on surface converts analyte 2 molecules into corresponding gaseous ions 14.

Gaseous ions 14 are sampled by mass spectrometer and subjected to mass analysis.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

EXAMPLES The examples are described for the purposes of illustration and are not intended to limit the scope of the invention.

Example 1 : Materials and Instrumentation

Materials. Rhodamine 1 16 and Rhodamine 123, Sudan Red 6 B, Cyclosporine A (from

Tolypocladium inflatum, g95%), Atrazine (PESTANAL, analytical standard), and Simazine (PESTANAL, analytical standard) were obtained from Sigma-Aldrich (St. Louis, MO).

Cyclosporine D was obtained from BioMarker Ltd. (Godollo, Hungary). All solvents (HPLC-grade) were purchased from Merck (Nottingham, U.K.).

SPE Cartridge. Custom built SPE cartridges consisting of polypropylene tubes, polymer frits, and SPE packing were used. The scheme of the cartridge is shown in Figure I a. Polypropylene tubes were obtained from B. Braun (Melsungen, Germany), PTFE frits were purchased from Supelco (Bellefonte, PA), and PE frits were purchased from Biotage (Uppsala, Sweden). Various SPE packings were obtained from Biotage, Supelco, and Varian (Palo Alto, CA).

The scheme of the sampling card is shown in Figure Ib. The cards consist of polypropylene sheets, with 5 mm diameter holes drilled into them. Five millimeter diameter disks of SPE material were embedded into polypropylene with one or two polymeric membranes covering the surfaces. SPE disks were obtained from Biotage (Uppsala, Sweden) and 3 M (St. Paul, MN); porous polymeric membranes were purchased from Millipore (Billerica, MA). Filter paper (Whatman 903) was also used in the sampling cards.

Instrumentation. SPE cartridges were eluted using a purposemade elutor device, consisting of a stainless steel cartridge holder, a gas heater unit, and a fluid delivery unit. The 96-channel elutor device employs an 8 x 12 array of individual SPE cartridges inserted into a PPS plate. Application of samples and elution is carried out in parallel fashion on all cartridges, using custom-made vacuum manifold and elutor devices.

The 96-channel DESI ion source (Figure 13) consists of an electrosonic spray 1, the primary electrospray emitter 1 mounted on a PTFE holder, which is secured onto a rotating stage (Parker,

Cleveland, OH). The rotating stage is mounted on a three dimensional manual moving stage

(Parker). The aluminum frame 8 for the SPE plate 2 is mounted on a computer controlled 2D moving stage 6 (Newmark System, Mission Viejo, CA). Z dimensional (up-down) relative movement is implemented by a moving sprayer and a heated capillary inlet of the mass spectrometer in the z direction. Both moving stage systems are built onto a common aluminum platform, which is secured onto the mass spectrometer through a welded, square hollow section steel frame. Mass spectrometric experiments were performed on a TSQ Quantum Discovery (ThermoFinnigan, San

Jose, CA) triple quadrupole mass spectrometer equipped with a slightly modified OmniSpray DESI ion source (Prosolia Inc., Indianapolis, IN) and on a modified API 4000 Q-TRAP hybrid triple quadrupole/ion trap mass spectrometer 7 (Applied Biosystems/MDS Sciex, Concorde, Ontario,

Canada). The atmospheric interface of the API 4000 mass spectrometer was modified; the original

interface was replaced by a home-built heated capillary-type interface unit. With reference to figure 13, electro-sonic spray emitter 1, SPE plate 2, heated capillary 3, home-built atmospheric interface housing 4, temperature sensor 5, 2D moving stage 6, mass spectrometer 7 and frame 8.

Sample Preparation. C18 cartridges were wetted with methanol and conditioned with methanol/water 5:95. Sample was applied directly (Figure IA); sample reservoirs connected to the cartridges were used when sample volume exceeded 0.5 niL. Following the application of the sample, the cartridges were washed with 0.5 mL methanol/water 5:95, and then were allowed to air- dry. The cartridges were transferred to the elutor device and were eluted with 150 μL of organic solvent at 40-200 μL/min flow rate (figure IB). The organic solvent content of the eluate was evaporated from the closing frit of the cartridge using a 0.05-1 L/min hot (50-200 ⁰ C) nitrogen gas flow. The elution was concluded by replacing the organic solvent flow with 2 mL/min air flow by switching the 6-port valve to remove all residual solvent from cartridge. Skipping the latter step results in back-diffusion of the analyte into the wet SPE packing, which compromises the sensitivity and the reproducibility of the DESI method (figure I E). The 96-channel SPE plate was constructed by combining 96 individual cartridges in a 8 x 12 format, keeping the standard pattern and raster of 96-well microtiter plates. Samples were pipetted into the equilibrated cartridges using an 8-channel pipettor. Application of the samples was carried out using a homebuilt vacuum manifold. However, since the handling of 96 individual solvent lines is troublesome, an alternative approach was chosen for elution. In this case the cartridges are upside down compared to the single channel elutor. Appropriate volumes of eluent were pipetted into cartridges, and the eluting solvent was forced through the SPE packings by pressurized nitrogen (300-400 mbar). The eluate was evaporated from the closing frits by hot (100-150 ⁰ C) airflow provided by the elutor device.

Analysis. Cartridges were placed into the DESI source and were analyzed using either DESI or DAPCI ionization. The ion source parameters and instrumental settings are summarized in Table 1. Geometrical parameters and flow rates of DESI were set to obtain a spray fingerprint size in the range of 0.1-1 mm2, to analyze cartridges in a single step, without moving during analysis.

Example 2 - Monitoring of cvclosporine A in human clinical samples

The calibration curves for the determination of Cyclosporine A in human plasma by direct deposition DESI and SPE/DESI are shown in figure 9. In the case of direct deposition, 1 μL deproteinized plasma sample was dried onto a porous PTFE target, while in the case of SPE/DESI

100 μL sample was extracted. The SPE-based sample preparation results in the deposition of 2

orders of magnitude more analyte; thus, 2 orders of magnitude higher signal intensity is expected. In the range where both curves are linear (3-30 μg/mL), the ratio of signal intensities is close to the expected value (figure 9). However, the dynamic range of the direct deposition DESI is limited to this concentration range, while SPEEDI/DESI is linear from 10 ng/mL to 30 μg/mL. At lower and higher concentrations, direct deposition DESI gives unreliable, practically qualitative infoπnation on the presence of Cyclosporine A in the samples. At low concentrations, the better linearity of SPE/DESI data can primarily be attributed to higher signal intensity and the partial elimination of the matrix-interference. The standard deviation of the SPEEDI/DESI data is remarkably lower throughout the entire investigated concentration range. The observed better reproducibility was most likely due to the more even distribution of the analyte on the surface. Following direct deposition, the solvent evaporates from the droplet according to its intrinsic physico-chemical characteristics, resulting in heterogeneously distributed analyte clusters. During the elution step of SPEEDI/DESI the eluate seeps through evenly the entire diameter of the closing membrane, and the solvent is evaporated immediately, resulting in homogeneous analyte distribution. In theory, initial sample size and hence concentration factor can be arbitrarily chosen for SPEEDI/DESI methodology. In practice, though, saturation of the SPE packing and the usually limited sample volume (e.g., blood samples) set limits on the enhancement of sensitivity. Solution of atrazine in tap water was used to determine the LLOD of the method at different initial sample volumes. LLOD of the method improves as a function of initial sample volume in the case of tap water samples. At concentration factors higher than 10000, signal intensities tend to reach a maximum value (Figure 14). It has to be noted that in the case of spiked HPLC-grade water samples no saturation effects were observed in this range; the curve was linear throughout 6 orders of magnitude. Analytical parameters of DESI and SPE/DESI are summarized in Table 1.

Table 1. Comparison of Analytical Parameters of DESI and SPEEDI/DESI

DESI SPEEDI/DESI absolute LLOD lO pg-1 ng lO pg-1 ng

LLOQ 10-1000 ng/mL lOO fg/mL-lOO pg/mL time of the analysis 5 s 10 s linear range 1-100 μg/mL 10 ng- 100 μg/mL volume of the sample limited unlimited

Example 3 - Systematic Characterization of SPEEDI /DESI,

The effect of various elution parameters was investigated by using dilute aqueous solutions of dyes (Rhodamine 1 16, Rhodamine 123, and Sudan Red 6 B) as samples and different eluting solvents including pentane, hexane, acetone, acetonitrile, dichloromethane, and methanol. Since elution volume for the dyes with water eluent is practically infinite on the octadecyl-silica packing used, the effects of sample volume and concentration were not studied. Investigated parameters included physico-chemical character of the eluent, elution flow rate, drying gas flow rate, drying gas temperature, and geometric parameters of the elutor device. Buffered aqueous solution (10 mL) of 100 ng dye was applied onto equilibrated SPE cartridges. The cartridges were dried and eluted with the appropriate solvent. Elution volumes depend on the polarity and H-bonding characteristics of solvents and analytes; minute deviations were associated with longitudinal diffusion effects.

Since the elution volumes are determined by intrinsic characteristics of the solvent, the analyte, and the cartridge, optimal elution flow rate equals to the maximum evaporable solvent rate from the surface. This latter value was defined as the maximum flow rate, which does not result in the formation of a continuous liquid film on the closing frit of the cartridge (figure 15a). If a continuous liquid film is formed on the frit surface, the eluted analyte is redissolved (figure 15b) and crystallized in a circular pattern around the rim of the closing frit (figure 15c). This phenomenon occurs at a well-defined solvent flow rate under given elutor geometry, drying gas flow rate, and gas temperature.

The result of DESI line scan through the frit clearly indicates that the concentration of the analyte into a ring around the frit yields poor overall sensitivity. This effect was associated with the high local surface concentration of analyte, which exceeds the dynamic range of the DESI analysis.

Since the general objective of systematic characterization was to find a global maximum for solvent evaporation rate, maximum evaporation rate (MER) values (defined above) were studied as a function of various parameters. Elutor geometry was found to have a well-defined effect on MER, since even analyte distribution was achieved only when drying gas emitter nozzle was in-line (in coaxial position) with the cartridge.

The dependence of MER on gas flow rate and temperature using methanol as solvent is depicted in Figure 16a. The figure shows that MER is a non-linear function of both gas flow rate and temperature. Under the given conditions MER shows a global maximum indicated on the figure,

corresponding to ~340 μL/min. This in the case of Rhodamine 123 and methanol gives a minimum elution time of ~ 15 s (Figure 16b). MER values give an inversely proportional function of evaporation enthalpy of the solvents used. In the case of more volatile solvents (isopentane, dichloromethane) MER as high as ~800 μL/min is achievable, which results in 4-6 s elution times. Elution time under the above conditions is comparable to DESI analysis time, which means that sample preparation is not the rate-limiting step of the analytical procedure.

Example 4 - Multiplexing

SPEEDI/DESI was also designed in a 96-channel format for HT application. DESI-MS analysis of individual cartridges was carried out by moving the plate under the spray along the x axis, keeping the spray tip-surface distance constant. [Kertesz, V.; Van Berkel, G. J. Anal. Chem. (2008) 80, 1027] An analysis time of 2 s/sample was routinely achieved without compromising sensitivity. Reproducibility was found to highly depend on the sampling rate (Figure 17). Sampling rates as high as 5 sample/s are theoretically achievable based on the linear motion parameters of the moving stage. Example 5 - Sampling Card

Performance of the sampling device and the sample preparation method was tested using whole blood samples spiked with 10-1000 ng/mL Cyclosporine A. Dried blood spots (DBS) on Whatman 903 filter paper were used with and without surface elution. Samples were applied directly onto filter paper, and excess blood was removed from card surface. In the case of surface elution of filter paper, eluted area was confined using a stainless steel clamp. Cyclosporine A was eluted using acetone/isopropanol 1 : 1. Results are summarized in Table 2.

Table 2 ^a

Native DBS on Surface elution/ Surface filter paper filter paper eliit ion/sampling card

LLOQ (S/N = 3) 880000 120 50 ng/mL

RSD no ISTD 45% 18% 12%

Cyclosporine 26% 12% 4.2%

D ISTD

^a DBS, dried blood spots; LLOQ, lower limits of quantitation; RSD, relative standard deviation; ISTD, internal standard; S/N, signal to noise ratio

As it is shown in Table 2, surface elution gives almost an order of magnitude lower LLOD, which is further improved in the case of sampling card. There was also a considerable difference between the reproducibility of the different experimental setups. This latter feature was associated with the reproducibility of the sampling volume. In the case of unprocessed filter paper the distribution of the analyte molecules on the surface depends on a number of factors including inhomogenity of the filter paper, exposure to environmental effects, and blood clotting pattern among others. When a confined area (d = 2 mm) of dried blood spot undergoes surface elution, most of these factors are eliminated; however, the uneven distribution of the blood in the paper still results in poor reproducibility. The sampling card eliminates this effect, since in that case the adsorbent is completely saturated with the biological fluid, and the excess blood is simply wiped off from the card surface.

Strictly quantitative analysis of dried blood spot samples is usually not feasible because of poor reproducibility of sample volume discussed above. The use of internal standard (ISTD) also involves problems, since internal standard can only be added to liquid phase sample prior to application onto the filter paper; however, in that case the simplicity of the sampling procedure is compromised. The well-defined adsorbed volume in case of the sampling card offers a solution for this problem. Since the volume of biological fluid retained by the well-defined filter paper disk is reproducible (standard deviation of blood volume was found to be less than 5% by weight measurement) internal standard solution can be dried onto filter paper prior to analysis. This technology, using Cyclosporine D as internal standard and heparinized blood containing known amounts of Cyclosporine A, resulted in highly reproducible results. Standard deviation varied between 3.1 and 6.8% for different concentration levels, compared to 10-15% SD of direct DESI analysis of the blood spots.

The described sampling card offers a robust, portable sample format, especially in the case of clinico-chemical applications. The sampling cards combined with described surface elution technology make a proper basis for a new type of centralized clinical diagnostics scheme.

Example 6: Determination of atrazine in surface water samples For this example, SPE-cartridges as described in previous examples were used. Surface water sample was applied onto wetted and conditioned cartridges. Sample was spiked with 1 μg simazine

internal standard. Cartridges were washed with 10 ml water and 1 ml water/methanol 9: 1 , then were dried in air. Solvent free cartridges were placed into elutor device shown on figure 3 B. cartridges were eluted with 120 μl n-pentane/acetone 2: 1 at 60 μl/min flow rate. During elution, 150 ml/min nitrogen flow at 100 °C was directed onto 3 PE closing frit of cartridge. Following elution 1 ml nitrogen was also passed through cartridge to remove residual eluting solvent, while hot nitrogen stream was still on. Eluted samples were placed into home built DAPCI ion source mounted on a Thermo TSQ Quantum Triple quadrupole mass spectrometer. DAPCI analysis was performed by intoducing 3 μl/min nhexane into nitrogen gas jet at nominal linear velocityof 350 m/s. Discharge current of corona discharge was kept at 3 μA during experiments. Jet carrying primary ions was directed onto 3 closing frit of cartridge and data was collected in selective ion monitoring mode for 5 s for each sample. Protonated molecular ions of atrazine and simazine were monitored. Calibration was performed using relative integrated signal intensity of ions.

Calibration curve obtained is shown on figure 10.

Example 7: Screening for inborn disorders of fatty acid beta-oxidation in infants Inborn disorders of fatty acid beta-oxidation generally cause considerable deviations in whole blood acyl-carnitine profile. High throughput (population level) screening for these disorders is performed by ESI-MS/MS method worldwide. Annually approx. 5 M test of this type are performed.

Polypropylene cards (5) with 5 mm discs of Whatman 903 filter paper (4) spiked with isotope labeled acyl-carnitine standards were used for these experiments. Blood was spotted onto filter paper discs, and excess blood was wiped off using tissue paper. Cards were let dry, and were processed 3 days after samples were taken. Firstly, 10 μl freshly prepared acetyl-chloride/n-butanol was pipetted onto disks and cards were dried in microwave oven. Prepared samples were placed into elutor device and were eluted with 50 μl acetonitrile at 20 μl/min flow rate. During elution, 50 ml/min nitrogen flow at 1 10 ⁰ C was directed onto paper discs. Following elution 1 ml nitrogen was also passed through card to remove residual eluting solvent, while hot nitrogen stream was still on.

Following elution, cards were placed into home-built DESI source for mass spectrometric analysis. DESI analysis was performed by spraying 1 μl/min methano I/water 1 : 1 and using nebulizing nitrogen gas at nominal linear velocityof 350 m/s. Spray was directed onto filter paper discs and data was collected in precursor ion scan mode for m/z 85. Since acyl-carnitines give predominantly m/z 85 fragment ion in MS/MS experiments, this scan mode selectively detects carnitine esters. For control experiment, butyl esterified sample was directly analyzed by DESI-MS/MS. Resulting

spectra are shown on figure 1 1 A (SPEEDI/DESI) and 1 I B (DESI) It is clearly shown on figures 1 1 A and 1 1 B that while the spectrum of 1 1 A is practically free ot noise, in the case of the spectrum 1 1 B large part of spectrum disappear in baseline noise

Example 8 Business model - Centralized clinical diagnostics One aspect of the present invention envisages the possibility for easy home use of sample provision This aspect of the invention is illustrated in figure 12 In this aspect, the present invention makes it possible for various applications including, without limitation, drug level monitoring, population screening (both full or statistical iemote area screening) and standard health checks, that patients receive (paid by the hospital or insurance company etc ) or buy a kit containing a specially designed sample holder onto which the patients themselves place the sample to be tested (drop of own blood, urine or other specified body liquid) in their own homes The same or a slightly modified kit makes it possible for smaller healthcare units, or 3rd world locations to easily, cheaply and safely provide analytical services for patients The sample is posted and taken (postage may be included in the price of the kit) to a central laboratory, where it is analyzed and the iesults sent either directly to the patient, or to a healthcare professional dealing with the patient This method would allow patients to have certain services available at or close to their homes, saving time, money an inconvenience lor the patient and relieving the healthcare and insurance companies of cost and patient visits

While the present invention has been described with reference to what are presently considered to be examples, it is to be understood that the invention is not limited to the disclosed examples To the contrary, the invention is intended to cover various modifications and equivalent aπangements included within the scope of the appended claims