WO2009144560A1 - Procédé et dispositif pour la préparation d’échantillons - Google Patents

Procédé et dispositif pour la préparation d’échantillons Download PDF

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Publication number
WO2009144560A1
WO2009144560A1 PCT/IB2009/005708 IB2009005708W WO2009144560A1 WO 2009144560 A1 WO2009144560 A1 WO 2009144560A1 IB 2009005708 W IB2009005708 W IB 2009005708W WO 2009144560 A1 WO2009144560 A1 WO 2009144560A1
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target analytes
adsorbent
sample
adsorbent material
assembly
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PCT/IB2009/005708
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English (en)
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Zoltan Takats
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Zoltan Takats
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Publication of WO2009144560A1 publication Critical patent/WO2009144560A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4055—Concentrating samples by solubility techniques
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • B01L3/50255—Multi-well filtration
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0431—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components for liquid samples
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/14—Ion sources; Ion guns using particle bombardment, e.g. ionisation chambers
    • H01J49/142—Ion sources; Ion guns using particle bombardment, e.g. ionisation chambers using a solid target which is not previously vapourised
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
    • H01J49/165—Electrospray ionisation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0851—Bottom walls
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4055—Concentrating samples by solubility techniques
    • G01N2001/4061—Solvent extraction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/84—Preparation of the fraction to be distributed
    • G01N2030/8411—Intermediate storage of effluent, including condensation on surface
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G01N30/724—Nebulising, aerosol formation or ionisation

Definitions

  • Field of the invention is mass spectrometric ionization methods, with special regard to the improvement of efficiency and development of desorption ionization methods.
  • 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.
  • ambient mass spectrometric techniques are claimed to provide a "sample preparation-free" analytical scheme, this statement does not necessarily stand for regular analytical applications.
  • 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.
  • sample preparation e.g., drawing of blood sample, preparation of plasma and deposition of plasma onto glass slide prior to DESI analysis
  • 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.
  • 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.
  • SPEEDI solid phase extraction enhanced desorption ionization
  • 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:
  • 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;
  • 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.
  • 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 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
  • 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.
  • 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:
  • 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:
  • 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 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.
  • matrix interferences are eliminated and analyte molecules are concentrated on the surface of the closing membrane of solid phase extraction (SPE) packing.
  • SPE solid phase extraction
  • 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.
  • 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.
  • 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.
  • elution process using solvent flow rate below and above maximum solvent evaporation rate.
  • 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 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 material 4 can be any solid material with high specific surface area
  • Adsorbent mate ⁇ al 4 include any material suitable for solid phase extraction (SPE)
  • Adsorbent material 4 also refer to as SPE packing, refers to a 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
  • 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.
  • 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 5 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.
  • 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).
  • 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.
  • a housing 5 for high-throughput analysis 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.
  • 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).
  • 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.
  • elution solvent 11 flows through tubing 17.
  • 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.
  • n is an integer larger than 1
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • the liquid sample 1 can just be deposited into adsorbent 4 material, and sample solvent 1 can be let to evaporate.
  • 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.
  • DI-MS methods generally analyze molecules present on the condensed/gas interface; thus molecules "hidden" deep in solid phase adsorbent do not undergo ionization.
  • 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.
  • 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.
  • 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.
  • SPE solid phase extraction
  • matrix interference is eliminated, and the analyte molecules are concentrated on the confined surface of the closing frit of the SPE cartridge.
  • 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.
  • 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.
  • 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.
  • the multi-channel elutor device utilizes external gas source to facilitate the flow of eluting solvent 11 through adsorbent material 4.
  • 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.
  • 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.
  • 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.
  • 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,
  • the rotating stage is mounted on a three dimensional manual moving stage
  • 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 Diego).
  • 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.
  • 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 frame 8.
  • 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.
  • 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.
  • 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.
  • the solvent evaporates from the droplet according to its intrinsic physico-chemical characteristics, resulting in heterogeneously distributed analyte clusters.
  • the eluate seeps through evenly the entire diameter of the closing membrane, and the solvent is evaporated immediately, resulting in homogeneous analyte distribution.
  • initial sample size and hence concentration factor can be arbitrarily chosen for SPEEDI/DESI methodology.
  • saturation of the SPE packing and the usually limited sample volume 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.
  • 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.
  • 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.
  • 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.
  • MER maximum evaporation rate
  • 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.
  • 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.
  • 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
  • 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
  • SPE-cartridges as described in previous examples were used. Surface water sample was applied onto wetted and conditioned cartridges. Sample was spiked with 1 ⁇ g 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 ⁇ l n-pentane/acetone 2: 1 at 60 ⁇ l/min flow rate. During elution, 150 ml/min nitrogen flow at 100 °C was directed onto 3 PE closing frit of cartridge.
  • 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 ⁇ l 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 ⁇ l acetonitrile at 20 ⁇ l/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.
  • DESI analysis was performed by spraying 1 ⁇ l/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.
  • 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
  • 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

Abstract

La présente invention concerne un nouvel ensemble et une application pratique d’un procédé de préparation universel d’échantillons pour une spectrométrie de masse (MS) à ionisation et désorption, en particulier pour une MS à ionisation par électropulvérisation et désorption. Le procédé de préparation d’échantillons se fonde sur l’adsorption des molécules d’analyte sur un boîtier d’extraction liquide-solide (SPE) et sur l’élution de l’analyte sur la membrane qui scelle le boîtier. Ainsi, l’interférence matricielle est éliminée et les molécules d’analyte sont concentrées sur la surface confinée de la membrane de fermeture du boîtier SPE. Le procédé de préparation de la présente invention peut être mis en œuvre pour une analyse à rendement élevée.
PCT/IB2009/005708 2008-05-25 2009-05-25 Procédé et dispositif pour la préparation d’échantillons WO2009144560A1 (fr)

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EP3257066A4 (fr) * 2015-02-10 2018-10-03 Indiana University Research & Technology Corporation Dispositif et procédé pour l'analyse de liquides biologiques par génération d'ions au moyen d'un matériau poreux humide
US10335785B2 (en) 2010-12-17 2019-07-02 Biomerieux, Inc. Methods for the isolation, accumulation, characterization and/or identification of microorganisms using a filtration and sample transfer device

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US10335785B2 (en) 2010-12-17 2019-07-02 Biomerieux, Inc. Methods for the isolation, accumulation, characterization and/or identification of microorganisms using a filtration and sample transfer device
EP3257066A4 (fr) * 2015-02-10 2018-10-03 Indiana University Research & Technology Corporation Dispositif et procédé pour l'analyse de liquides biologiques par génération d'ions au moyen d'un matériau poreux humide
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WO2017210536A1 (fr) 2016-06-03 2017-12-07 Purdue Research Foundation Systèmes et procédés d'analyse d'un analyte extrait d'un échantillon à l'aide d'un matériau adsorbant
CN109564147A (zh) * 2016-06-03 2019-04-02 普度研究基金会 用于分析使用吸附材料从样品中提取的分析物的系统和方法
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US11060959B2 (en) 2016-06-03 2021-07-13 Purdue Research Foundation Systems and methods for analyzing an analyte extracted from a sample using an adsorbent material
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US11680876B2 (en) 2016-06-03 2023-06-20 Purdue Research Foundation Systems and methods for analyzing an analyte extracted from a sample using an adsorbent material

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