Method for determining at least one analyte of interest - Patent application

Abstract

The present invention relates to a method for determining at least one analyte of interest.The present invention further relates to a sample element, an inlet, a composition, a kit and uses thereof for determining at least one analyte of interest.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for determining at least one analyte of interest.The present invention further relates to a sample element, an inlet, a composition, a kit and uses thereof for determining at least one analyte of interest. [Background technology]

[0002] 2. Background of the Invention The ionization process associated with matrix-assisted ionization can be laser-assisted ionization (MALDI / SALDI), matrix-assisted ionization (MAI) and / or ambient voltage-assisted ionization. MAI is an ionization method that uses a liquid / solid support medium to mix a crystalline matrix component(s), such as 3-NBN or 2,2'-azobis(2-methylpropane), with the respective analyte and bring it close to the entrance of a capillary of a mass spectrometer, such as a heated capillary of a mass spectrometer.

[0003] The ionization process is carried out by transferring the matrix components along with the analyte to the mass spectrometer inlet.

[0004] However, these methods are not necessarily fully compatible with automated (magnetic) bead handling workflows. Current MAI compounds are limited, and therefore more substance classes are desirable.

[0005] Therefore, there is an urgent need in the art to overcome the above problems.

[0006] It is an object of the present invention to provide a method for determining at least one analyte of interest. The present invention further relates to a sample element, an inlet, a composition, a kit and uses thereof for determining at least one analyte of interest.

[0007] This object is solved by the subject matter of the independent claims. Further embodiments are subject to the dependent claims. Summary of the Invention

[0008] In the following, the present invention relates to the following aspects:

[0009] In a first aspect, the present invention relates to a method for determining at least one analyte in a sample, comprising the following steps: a) providing a substrate having at least one analyte, at least one microparticle, at least one ionization matrix, and a substrate surface; b) incubating the analyte with microparticles having at least one microparticle surface, whereby the analyte is adsorbed to the surface of the microparticle and an analyte-microparticle complex is formed; c) contacting the analyte-particle complex with an ionization matrix to form a matrix:analyte-particle sample; d) providing a matrix:analyte-particulate sample on a substrate surface; e) ionizing at least the analyte, wherein the ionization is mechanical ionization; f) determining the analyte by ion mobility spectrometry and / or mass spectrometry The present invention relates to a method comprising the steps of:

[0010] In a second aspect, the present invention relates to the use of the method of the first aspect of the invention for determining at least one analyte of interest.

[0011] In a third aspect, the present invention relates to a sample element for determining at least one analyte and suitable for carrying out a method according to any of claims 1 to 6, comprising: a substrate surface; - an ionization matrix disposed on a surface of the substrate for use in matrix-assisted ionization; - an analyte-microparticle complex disposed on a surface of a substrate; the ionization matrix is ​​selected from the group consisting of salsalate, 3-nitrobenzonitrile, 2,2'-azobis(2-methylpropane), 2-nitrobenzonitrile, 5-methyl-2-nitrobenzonitrile, coumarin, methyl-2-methyl-3-nitrobenzoate, methyl-5-nitro-2-furoate, 2-bromo-2-nitropropane-1,3-diol), 3-nitrobenzaldehyde, 6-nitro-o-anisinonitrile, phthalic anhydride, or mixtures thereof; - the ionization matrix and / or the analyte-microparticle complex are crystallized; - the microparticle of the analyte-microparticle complex is magnetic; - for a sample element in which the analyte-particle complex and the ionizing matrix are in contact with each other.

[0012] In a fourth aspect, the present invention relates to the use of the inlet of the third aspect of the invention for determining at least one analyte.

[0013] In a fifth aspect, the present invention relates to an inlet suitable for carrying out the method of the first aspect of the invention and suitable for ion transport into a mass spectrometer or ion mobility spectrometer, or to a detector of a mass spectrometer or ion mobility spectrometer with a truncated sample inlet and filter.

[0014] In a sixth aspect, the present invention relates to the use of the inlet of the fourth aspect of the invention for determining at least one analyte.

[0015] In a seventh aspect, the present invention relates to a composition for vacuum ionization or inlet ionization comprising an ionization matrix, wherein the ionization matrix comprises or consists of salsalate.

[0016] In an eighth aspect, the present invention relates to the use of the composition of the seventh aspect of the invention for determining at least one analyte.

[0017] In a ninth aspect, the present invention relates to a kit according to the seventh aspect of the invention suitable for use in or for carrying out the method of the first aspect of the invention.

[0018] In a tenth aspect, the present invention relates to the use of the kit of the seventh aspect of the invention in the method of the first aspect of the invention. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows a schematic representation of a method for determining at least one analyte in a sample, in particular the matrix ionization particulate workflow. [Diagram 2] FIG. 2 shows a schematic representation of the method for determining at least one analyte in a sample, in particular the matrix ionization particulate workflow. [Figure 3-1] Figures 3a)-d) show the MS spectra (relative abundance vs. time and relative abundance vs. m / z) of 1 μL of the residual liquid after magnetic separation spotted onto a glass plate. [Figure 3-2] Figures 3a)-d) show the MS spectra (relative abundance vs. time and relative abundance vs. m / z) of 1 μL of the residual liquid after magnetic separation spotted onto a glass plate. [Figure 4-1] 4a)-d) show the MS spectra of 1 μL of a mixture of recrystallized 3-NBN as the ionization matrix and analyte-loaded microparticles as the analyte-microparticle-complex. [Figure 4-2] 4a)-d) show the MS spectra of 1 μL of a mixture of recrystallized 3-NBN as the ionization matrix and analyte-loaded microparticles as the analyte-microparticle-complex. [Diagram 5] FIG. 5 shows a schematic illustration of a method for determining at least one analyte in a sample, in particular the matrix ionization particulate workflow. [Figure 6-1] 6a)-d) show MS spectra of analyte-particle complexes (bead-analyte dispersions) aspirated onto a triangular filter with and without ionization matrix added. [Figure 6-2] 6a)-d) show MS spectra of analyte-particle complexes (bead-analyte dispersions) aspirated onto a triangular filter with and without ionization matrix added. [Figure 7] 7a) and b) show the MS spectra of the analyte-microparticle complex (bead-analyte dispersion) triangular filter with and without the addition of ionization matrix. [Figure 8] FIG. 8 shows a schematic illustration of a method for determining at least one analyte in a sample, in particular the matrix ionization particulate workflow. [Figure 9-1] Figures 9a)-d) show the MS spectra of leucine-enkephalin coated microparticles with and without a pre-crystallized ionization matrix (eg, 3-NBN matrix). [Figure 9-2] Figures 9a)-d) show the MS spectra of leucine-enkephalin coated microparticles with and without a pre-crystallized ionization matrix (eg, 3-NBN matrix). [Figure 10] Figures 10a) and b) show the MS spectra of leucine-enkephalin coated microparticles with and without pre-crystallized ionization matrix (eg, 3-NBN matrix). [Figure 11-1] Figures 11a)-d) show the MS spectra of leucine-enkephalin coated microparticles with and without a pre-crystallized ionization matrix (eg, 3-NBN matrix). [Figure 11-2] Figures 11a)-d) show the MS spectra of leucine-enkephalin coated microparticles with and without a pre-crystallized ionization matrix (eg, 3-NBN matrix). [Figure 12] FIG. 12 shows the ionization matrix / analyte ionization of different compounds including the ionization matrix of Salsalate. [Figure 13] FIG. 13 shows different nitrobenzene reaction products as MAI surrogates. [Figure 14]FIG. 14 shows an inlet for ion transport into a mass spectrometer. [Figure 15-1] Figures 15a1)-d2) show the extracted ion mobilograms as well as MS spectra of leucine-enkephalin coated microparticles using an inlet for ion transport into the mass spectrometer with and without the use of a filtration material. [Figure 15-2] Figures 15a1)-d2) show the extracted ion mobilograms as well as MS spectra of leucine-enkephalin coated microparticles using an inlet for ion transport into the mass spectrometer with and without the use of a filtration material. [Figure 15-3] Figures 15a1)-d2) show the extracted ion mobilograms as well as MS spectra of leucine-enkephalin coated microparticles using an inlet for ion transport into the mass spectrometer with and without the use of a filtration material. [Figure 15-4] Figures 15a1)-d2) show the extracted ion mobilograms as well as MS spectra of leucine-enkephalin coated microparticles using an inlet for ion transport into the mass spectrometer with and without the use of a filtration material. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Detailed Description of the Invention Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific embodiments and examples described herein, which may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0021] Several documents are cited herein. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. In the event of a discrepancy between a definition or teaching of such an incorporated reference and a definition or teaching cited herein, the body of the present specification shall control.

[0022] Each element of the present invention will be described below. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed to limit the present invention to only the embodiments explicitly described. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application unless the context indicates otherwise.

[0023] definition It will be understood that the word "comprise", and variations such as "comprises" and "comprising", imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integers or steps or group of integers or steps.

[0024] The terms "have", "comprise" or "include" used below, or any grammatical variants thereof, are used non-exclusively. Thus, these terms may refer both to the situation where no further features are present in the entity described in this context, in addition to the features introduced by these terms, and to the situation where one or more additional features are present. For example, the expressions "A has B", "A comprises B" and "A includes B" may both refer to the situation where, apart from B, no other elements are present in A (i.e., A consists solely and exclusively of B), and to the situation where, apart from B, one or more further elements are present in the entity A, such as element C, elements C and D, as well as further elements.

[0025] Furthermore, it should be noted that the terms "at least one" or "one or more," or similar expressions, indicating that a feature or element may be present one or more times, are typically used only once when introducing each feature or element. In the following, in most cases, when referring to each feature or element, the expressions "at least one" or "one or more" will not be repeated, despite the fact that each feature or element may be present one or more than one time.

[0026] Furthermore, when used below, the terms "preferably", "more preferably", "particularly", "more particularly", "particularly" or "more particularly" or similar terms are used in relation to any feature without limiting the possibility of alternatives. Thus, features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. The invention may be implemented using alternative features, as the skilled person will recognize. Similarly, features introduced by "in an embodiment of the invention" or similar expressions are intended to be optional features without any limitation in relation to alternative embodiments of the invention, without any limitation in relation to the scope of the invention, and without any limitation in relation to the possibility of combining the feature introduced in such a way with other optional or non-optional features of the invention.

[0027] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0028] Percentages, concentrations, amounts, and other numerical data may be expressed or presented herein in the form of a "range." It is understood that such range formats are used merely for convenience and brevity, and thus should be interpreted flexibly to include not only the numerical values ​​expressly recited as boundaries of the range, but also to include all of the individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were expressly recited. By way of illustration, a numerical range of "4%-20%" should be interpreted not only to include the explicitly recited value of 4%-20%, but also to include each individual value and subrange within the stated range. Thus, this numerical range includes individual values ​​such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20%, and subranges such as 4-10%, 5-15%, 10-20%, etc. This same principle also applies to ranges reciting minimum or maximum values. Moreover, such interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0029] The term "about," when used in connection with a numerical value, is meant to encompass numerical values ​​in a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0030] In the context of this disclosure, the terms "analyte", "analyte molecule" or "analyte(s) of interest" are used interchangeably to refer to a chemical species that is analyzed by mass spectrometry. A chemical species, i.e., an analyte, suitable for analysis by mass spectrometry can be any type of molecule present in an organism, including, but not limited to, nucleic acids (e.g., DNA, mRNA, miRNA, rRNA, etc.), amino acids, peptides, proteins (e.g., cell surface receptors, cytosolic proteins, etc.), metabolites or hormones (e.g., testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (e.g., vitamin D), molecules characteristic of a particular modification of another molecule (e.g., sugar moieties or phosphoryl residues on proteins, methyl residues on genomic DNA), or substances internalized by an organism (e.g., therapeutic drugs, drugs of abuse, toxins, etc.), or metabolites of such substances. Such analytes may serve as biomarkers. In the context of the present invention, the term "biomarker" refers to a substance in a living system that is used as an indicator of the biological state of said system.

[0031] The analyte or analytes of interest may be present in a biological or clinical sample. The term "sample or biological or clinical sample" is used interchangeably herein and refers to a portion or piece of a tissue, organ or individual, usually smaller than such tissue, organ or individual, which is intended to represent the entire tissue, organ or individual. Upon analysis, the biological or clinical sample provides information about the state of the tissue, or the health or disease state of the organ or individual. Examples of biological or clinical samples include, but are not limited to, liquid samples such as blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva and lymphatic fluid, or solid biological or clinical samples such as dried blood spots and tissue extracts. Further examples of biological or clinical samples are cell cultures or tissue cultures.

[0032] As used herein, the term "determining an analyte or determining at least one analyte" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. The term may specifically refer to, but is not limited to, the quantitative and / or qualitative determination of at least one analyte in any sample. The quantitative and / or qualitative determination of an analyte in a sample may be the result or intermediate result of a detection process that may include at least one measurement step, as well as further steps such as at least one preparation step and / or at least one analysis step. As part of the detection process, at least one measurement may be generated, specifically a measurement regarding the presence, concentration or amount of an analyte in a sample.

[0033] As used herein, the term "providing" is a broad term and should be given its ordinary and accustomed meaning to those of skill in the art and should not be limited to a special or customized meaning. The term may specifically refer to, but is not limited to, the process of making available one or more required objects.

[0034] As used herein, the term "microparticle" is a broad term and should be given its common and ordinary meaning to those skilled in the art and should not be limited to a specific or special meaning. The term may specifically refer to, but is not limited to, any particulate matter of microscopic size. Microparticles may have an average diameter ranging from 100 nm to 100 μm, specifically 200 nm to 50 μm. Microparticles may also be referred to as beads. Microparticles may be spherical or globular in shape. However, slight deviations from the spherical or globular shape may be possible. The size of the microparticles may be determined by dynamic light scattering.

[0035] As outlined above, a microparticle has at least one microparticle surface. As used herein, the term "microparticle surface" and / or "substrate surface" is a broad term and should be given its general and ordinary meaning to those skilled in the art and should not be limited to a specific or special meaning. This term may specifically, but not exclusively, refer to the entire area that separates any object from the outside. Thus, a body, such as a microparticle and / or a substrate, may have multiple surfaces. Specifically, a microparticle may have a core surrounded by a surface. The surface and the core may comprise different materials. Furthermore, the surface and the core may have different properties. For example, the core may be magnetic. The surface may be configured to capture molecules, such as non-polar molecules from a wide range of polar molecules, when the microparticle is incubated with a sample containing such molecules.

[0036] As used herein, the term "incubation" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a specific or special meaning. The term may specifically, but not limited to, refer to the mixing of at least two substances and / or the addition of at least one substance to another substance. Specifically, a solid or particulate substance may be added and / or mixed with a liquid sample. Apart from the process of adding and / or mixing, the incubation may further include a period of time called the incubation time. During the incubation time, one of the two substances may be adsorbed on the surface of the other of the two substances. During the incubation time, further conditions may be selected, such as temperature and / or other conditions, for example to promote the desired adsorption. Thus, in step b), microparticles may be added to the sample and, optionally, mixed with the sample. In step b), the sample may be incubated with the microparticles for an incubation time of 1 second to 60 minutes, preferably 1 minute to 30 minutes, most preferably 3 minutes to 12 minutes. However, other durations may be feasible.

[0037] As used herein, the term "adsorbed on a surface" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to any special or customized meaning. The term may specifically, but is not limited to, refer to the result of a process in which atoms, ions or molecules that form part of a gas or liquid accumulate on the surface of a solid or particulate object. The atoms, ions or molecules, which may initially be distributed throughout the gas or liquid, may be attracted by the surface of the solid or particulate material during the process of adsorption.

[0038] As used herein, the term "analyte-particle complex" is a broad term and should be given its usual and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. The term may specifically, but not limited to, refer to an assembly including at least one microparticle and at least one analyte, specifically one microparticle and a plurality of analytes. The microparticles and the analyte, specifically the analyte to be analyzed, which form the complex, may be reversibly associated. Thus, the components of the complex may leave the complex or dissociate from the complex, at least under certain conditions. The analyte-particle complex may be formed based on at least one attractive force between the microparticle and the analyte. In particular, the attractive force may act between the surface of the microparticle and the analyte. Thus, the analyte, which may initially be distributed in the sample, specifically in the liquid phase of the sample, may accumulate in the course of adsorption on the surface of the microparticle. The attractive force may include van der Waals forces and electrostatic attraction. Other attractive forces are also feasible. For example, the attractive force may include a covalent bond, particularly when the immunobead and the analyte form an analyte-particle complex. Specifically, as part of the formation of the analyte-microparticle complex, at least one chemical bond may be formed between the microparticle and the analyte, specifically between the surface of the microparticle and the analyte. The analyte-microparticle complex may also be referred to as an analyte-loaded microparticle.

[0039] As used herein, the term "contacting" is a broad term and should be given its ordinary and customary meaning to one of skill in the art and should not be limited to a specific or special meaning. The term may specifically refer to, but is not limited to, a direct or indirect connection between an analyte-particle complex and an ionization matrix to form a matrix:analyte-particle sample. Contacting may also be described by either co-crystallization and / or mixing with an ionization matrix.

[0040] As used herein, the term "converse" is a broad term and should be given its ordinary and customary meaning to one of skill in the art and should not be limited to any special or customized meaning. The term may refer specifically, but is not limited to, the addition of a dissolved analyte-particle complex to an ionizing matrix. Alternatively, an ionizing matrix may be added to a dissolved analyte-particle complex.

[0041] As used herein, the term "mechanical ionization" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. The term may refer, in particular, but not limited to, to a process of generating ionization energy by a mechanical process. Additionally or alternatively, the term may refer, in particular, but not limited to, to the transfer of energy from a matrix to an analyte that was previously generated by the induction of a mechanical force on the matrix. The mechanical force may be caused by shear forces and / or triboluminescence of the respective crystals.

[0042] As used herein, the term "triboluminescent matrix" is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a specific or special meaning. The term may particularly, but is not limited to, refer to a matrix that generates an electrical discharge or generates an energy discharge or electrical discharge when the matrix is ​​mechanically pulled apart, torn, scratched, crushed, or rubbed.

[0043] As used herein, the term "heterogenic solid liquid phase" is a broad term and should be given its ordinary and customary meaning to those of skill in the art and should not be limited to any special or customized meaning. The term may specifically, but is not limited to, refer to the presence of a crystalline or semi-crystalline phase.

[0044] As used herein, the term "automatically" or "automated" is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to any special or customized meaning. The term may specifically refer to, but is not limited to, a process that is performed entirely by at least one computer and / or computer network and / or machine, particularly without the need for manual action and / or user interaction.

[0045] The term "fully automated" may refer to a process that is performed entirely by at least one computer and / or computer network and / or machine, without manual action and / or user interaction.

[0046] The term "partially automated" may refer to a process performed by at least one computer and / or computer network and / or machine with the help of manual actions and / or user interaction. Preferably, "partially automated" may mean that manual actions and / or user interaction is at most 50% or 40% or 30% or 20% or 10% or 5% of the total process, and the remainder of the process is performed by at least one computer and / or computer network and / or machine. "By at least one computer and / or computer network and / or machine" may mean that the process is performed without manual actions and / or user interaction.

[0047] The term "Mass Spectrometry" ("Mass Spec" or "MS"), or "mass spectrometric determination", or "mass spectrometric analysis" refers to an analytical technique used to identify compounds by their mass. MS is a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio or "m / z". MS techniques generally include (1) ionizing compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating the mass-to-charge ratio. Compounds may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. Generally, one or more molecules of interest are ionized and the ions are then introduced into a mass spectrometry instrument where a combination of magnetic and electric fields causes the ions to follow a path in space that depends on their mass ("m") and charge ("z"). The term "ionization" or "ionization" refers to the process of producing analyte ions having a net charge equal to one or more units. Negative ions are those that have a net negative charge of one or more units, and positive ions are those that have a net positive charge of one or more units. MS methods can be performed in either "negative ion mode," in which negative ions are generated and detected, or "positive ion mode," in which positive ions are generated and detected.

[0048] "Tandem mass spectrometry" or "MS / MS" involves selective multiple steps of mass spectrometry where fragmentation of analytes occurs between steps. In a tandem mass spectrometer, ions are generated in an ion source and separated by mass-to-charge ratio in the first stage of mass spectrometry (MS1). Ions of specific mass-to-charge ratios (precursor or parent ions) are selected and fragment ions (daughter ions) are generated by collision-induced dissociation, ion-molecule reactions, or photodissociation. The resulting ions are then separated and detected in the second stage of mass spectrometry (MS2).

[0049] Mass spectrometers separate and detect ions of slightly different masses, and therefore easily distinguish between different isotopes of a given element. Mass spectrometry is therefore an important method for accurate mass determination and characterization of analytes, including but not limited to low molecular weight analytes, peptides, polypeptides or proteins. Its applications include identification of proteins and their post-translational modifications, elucidation of protein complexes, their subunits and functional interactions, and total protein measurement in proteomics. De novo sequencing of peptides or proteins by mass spectrometry can usually be performed without prior knowledge of the amino acid sequence.

[0050] Most sample workflows in MS further include a sample preparation and / or enrichment step, e.g., the analyte(s) of interest are separated from the matrix using gas or liquid chromatography. Typically, for a mass spectrometry measurement, the following three steps are performed: 1. A sample containing the analyte of interest is ionized, for example by matrix assisted ionization (MAI). 2. Ions are sorted and separated according to their mass and charge. For example, a high field asymmetric waveform ion mobility spectrometer (FAIMS) can be used as an ion filter. 3. The separated ions are then detected, for example in multiple reaction mode (MRM), and the results are displayed in a chart.

[0051] The term "matrix-assisted ionization or inlet ionization" can refer to a low-fragmentation (soft) ionization technique that involves the transfer of analyte and matrix sample particles from atmospheric pressure (AP) to a heated inlet tube that connects the AP region to the vacuum of the mass analyzer.

[0052] "Field Asymmetric Waveform Ion Mobility Spectroscopy (FAIMS)" is an atmospheric pressure ion mobility technique that separates gas phase ions according to their behavior in strong and weak electric fields.

[0053] "Multiple reaction mode" or "MRM" is a detection mode of an MS instrument in which a precursor ion and one or more fragment ions are selectively detected.

[0054] The mass spectrometric determination may be combined with additional analytical methods, including chromatographic methods such as gas chromatography (GC), liquid chromatography (LC), particularly HPLC, and / or ion mobility-based separation techniques. In a preferred embodiment, the mass spectrometric determination does not include additional analytical methods, including chromatographic methods such as gas chromatography (GC), liquid chromatography (LC), particularly HPLC, and / or ion mobility-based separation techniques.

[0055] Prior to analysis by mass spectrometry, samples may be pretreated in a manner specific to the sample and / or analyte. In the context of the present disclosure, the term "pretreatment" refers to any means necessary to enable subsequent analysis of the desired analyte by mass spectrometry. Pretreatment means typically include, but are not limited to, elution of solid samples (e.g., elution of dried blood spots), addition of hemolyzing reagents (HR) to whole blood samples, and addition of enzyme reagents to urine samples. Similarly, addition of an internal standard (ISTD) is considered as sample pretreatment.

[0056] The term "hemolytic reagent" (HR) refers to a reagent that lyses cells present in a sample, and in the context of the present invention, refers to a hemolytic reagent, in particular, but not limited to, a reagent that lyses cells present in a blood sample, including red blood cells present in a whole blood sample. A well-known hemolytic reagent is water (H2O). Further examples of hemolytic reagents include, but are not limited to, deionized water, liquids with high osmolarity (e.g., 8M urea), ionic liquids, and various detergents.

[0057] Typically, an "internal standard" (ISTD) is a known quantity of a substance that exhibits similar properties to the analyte of interest when subjected to a mass spectrometry detection workflow (i.e., including any pretreatment, enrichment, and actual detection steps). The ISTD exhibits similar properties to the analyte of interest, but is clearly distinguishable from the analyte of interest. By way of example, during a chromatographic separation, such as gas or liquid chromatography, the ISTD has approximately the same retention time as the analyte of interest from the sample. Thus, both the analyte and the ISTD enter the mass spectrometer at the same time. The ISTD, however, exhibits a different molecular weight than the analyte of interest from the sample. This allows ions from the ISTD and the analyte to be distinguished in mass spectrometry using different mass / charge (m / z) ratios. Both are subjected to fragmentation to obtain daughter ions. These daughter ions can be distinguished by their m / z ratios to each other and their respective parent ions. As a result, separate determinations and quantifications of the signals from the ISTD and the analyte can be performed. Because the ISTD is added in a known amount, the signal intensity of the analyte from the sample can be attributed to a specific quantitative amount of the analyte. Thus, the addition of the ISTD allows for relative comparison of the amount of analyte detected, allowing unambiguous identification and quantification of the analyte(s) of interest present in the sample when the analyte(s) reach the mass spectrometer. Typically, but not necessarily, the ISTD is an isotopically labeled variant of the analyte of interest (e.g., 2 H, 13 C, or 15 (including labels such as N).

[0058] In addition to pretreatment, the sample may also be subjected to one or more enrichment steps. In the context of the present disclosure, the term "first enrichment process" or "first enrichment workflow" refers to an enrichment process that is performed after sample pretreatment and provides a sample with enriched analytes relative to the initial sample. The first enrichment workflow may include chemical precipitation (e.g., using acetonitrile) or the use of a solid phase. Suitable solid phases include, but are not limited to, solid phase extraction (SPE) cartridges, and beads. The beads may be non-magnetic, magnetic, or paramagnetic. The beads may be coated differently to be specific for the analytes of interest. The coating may vary depending on the intended application, i.e. the intended capture molecule. It is well known to those skilled in the art which coating is suitable for which analyte. The beads may be made of a variety of different materials. The beads may have a variety of sizes and may be equipped with a surface that contains or does not contain pores. The beads may be immunofunctionalized.

[0059] In the context of this disclosure, the term "second enrichment process" or "second enrichment workflow" refers to an enrichment process that is performed after sample pretreatment and the first enrichment process to provide a sample containing enriched analytes relative to the initial sample and the sample after the first enrichment process.

[0060] The term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical components as they flow around or over a stationary liquid or solid phase. In an embodiment of the invention, the method or sample element or device or kit does not comprise a chromatographic step and a chromatographic unit, respectively.

[0061] The term "liquid chromatography" or "LC" refers to the process of selectively retarding one or more components of a fluid solution as the fluid permeates uniformly through a column or capillary passage of finely divided material. Retardation results from the distribution of mixture components between one or more stationary phases and the bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal phase liquid chromatography (NPLC), and methods in which the stationary phase is less polar than the mobile phase (e.g., water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) are called reversed phase liquid chromatography (RPLC).

[0062] "High performance liquid chromatography" or "HPLC" refers to a method of liquid chromatography in which the degree of separation is increased by passing the mobile phase under pressure through a stationary phase, typically a densely packed column. Typically, the column is packed with a stationary phase composed of irregular or spherical particles, a porous monolithic layer, or a porous membrane. HPLC has historically been divided into two different subclasses based on the polarity of the mobile and stationary phases. Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal phase liquid chromatography (NPLC), and the opposite (e.g., water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) are called reversed phase liquid chromatography (RPLC). Micro LC refers to an HPLC method that uses a column with a narrow internal diameter, typically less than 1 mm, e.g., about 0.5 mm. "Ultra High Performance Liquid Chromatography" or "UHPLC" refers to an HPLC method using a pressure of 120 MPa (17,405 lbf / in2) or about 1200 atmospheres. Rapid LC refers to an LC method using a short column with an internal diameter as above and a length of less than 2 cm, e.g., 1 cm, at a flow rate as above and a pressure as above (micro LC, UHPLC). A short rapid LC protocol involves trapping / washing / elution steps using a single analytical column, achieving LC in a very short time of less than 1 minute.

[0063] Additionally, hydrophilic interaction chromatography (HILIC), size-exclusion LC, ion-exchange LC, and affinity LC are well known.

[0064] The LC separation may be single-channel LC or multi-channel LC, comprising multiple LC channels arranged in parallel. In LC, analytes may be separated according to their polarity or log P value, size, or affinity, as is commonly known to those skilled in the art.

[0065] As used herein, the term "ion mobility spectrometry" is a broad term and should be given its ordinary and accustomed meaning to one of ordinary skill in the art and should not be limited to any particular or special meaning. The term may particularly, but not exclusively, refer to a device for separating ions in an electric field and in the presence of at least one buffer gas based on the mobility characteristics of analyte ions.

[0066] As used herein, the term "crystallized" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to any special or customized meaning. The term may specifically refer to, but is not limited to, the formation of highly organized solid structures of molecules from supersaturated liquid solutions that may further include different analyte molecules in their structures.

[0067] As used herein, the term "pre-crystallized" is a broad term and should be given its ordinary and customary meaning to one of skill in the art and should not be limited to any special or customized meaning. The term may specifically, but is not limited to, refer to the process of crystallization of the ionization matrix prior to addition to the analyte-bead mixture.

[0068] In embodiments, the terms "crystallized" and "pre-crystallized" may be used interchangeably.

[0069] A "clinical diagnostic system" is a laboratory automation instrument dedicated to the analysis of samples for in vitro diagnosis. A clinical diagnostic system may have different configurations as needed and / or according to a desired laboratory workflow. Additional configurations can be obtained by coupling several instruments and / or modules together. A "module" is a work cell, typically smaller in size than the entire clinical diagnostic system, with a dedicated function. This function may be analytical, but also pre-analytical or post-analytical, or a supplementary function to either the pre-analytical, analytical or post-analytical functions. In particular, a module can be configured to cooperate with one or more other modules for performing a dedicated task of a sample processing workflow, for example by performing pre-analytical and / or analytical and / or post-analytical steps of the sample. In particular, a clinical diagnostic system can comprise one or more analytical instruments designed to perform respective workflows optimized for a specific type of analysis, for example clinical chemistry, immunochemistry, coagulation, hematology, liquid chromatography separation, mass spectrometry, etc. Thus, a clinical diagnostic system may comprise one analytical instrument or any combination of such analytical instruments with their respective workflows, and pre-analytical and / or post-analytical modules may be combined with individual analytical instruments or shared by several analytical instruments. Alternatively, pre-analytical and / or post-analytical functions may be performed by units integrated into the analytical instruments. A clinical diagnostic system may also comprise functional units such as liquid handling units for pipetting and / or pumping and / or mixing of samples and / or reagents and / or system fluids, as well as functional units for sorting, storage, transport, identification, separation, detection. A clinical diagnostic system may comprise a sample preparation station for the automated preparation of samples containing analytes of interest, a liquid chromatography (LC) separation station, optionally including several LC channels, and / or a sample preparation / LC interface for inputting the optionally prepared samples into any one of the LC channels.The clinical diagnostic system may further comprise a controller programmed to assign samples to predefined sample preparation workflows, each of which includes a predefined sequence of sample preparation steps and requires a predefined time for completion depending on the analyte of interest. The clinical diagnostic system may further comprise a mass spectrometer (MS) and an LC / MS interface for connecting the LC separation station to the mass spectrometer.

[0070] A "sample preparation station" is a pre-analytical module coupled to one or more analytical devices or units within an analytical device, designed to perform a series of sample processing steps aimed at removing or at least reducing interfering matrix components in the sample and / or concentrating the analytes of interest in the sample. Such processing steps may include one or more of the following processing operations performed in a sequential, parallel or staggered manner on a sample or multiple samples: pipetting (aspirating and / or dispensing) fluids, pumping fluids, mixing with reagents, incubation at a specific temperature, heating or cooling, centrifugation, separation, filtering, sieving, drying, washing, resuspension, sorting, transport, storage.

[0071] The clinical diagnostic system, e.g. a sample preparation station, may also comprise a buffer unit for receiving multiple samples before a new sample preparation start sequence is initiated, and the samples may be individually randomly accessible, the preparation of each of which may be initiated according to the sample preparation start sequence.

[0072] Clinical diagnostic systems utilize mass spectrometry, which is more convenient and reliable and therefore more suitable for clinical diagnosis. In particular, high throughput, e.g., up to 100 samples / hour or more, can be obtained using random access sample preparation and LC separation, while allowing on-line coupling to mass spectrometry. Furthermore, the process can be fully automated, improving yields and reducing the level of skill required.

[0073] A "kit" is any article of manufacture (e.g., package or container) that includes at least one reagent, such as a drug for the treatment of a disorder, or a probe for specifically detecting a biomarker gene or protein of the invention. The kit is preferably promoted, distributed, or sold as a unit for carrying out the method of the invention. Typically, the kit may further include a carrier means compartmentalized to receive in close confinement one or more container means, such as vials, tubes, and the like. In particular, each of the container means comprises one of the separate elements used in the method of the first aspect. The kit may further include one or more other reagents, including, but not limited to, reaction catalysts. The kit may further include one or more other containers containing additional materials, including, but not limited to, buffers, internal standards, diluents, filters, needles, syringes, and a package insert with instructions for use. Labels may be presented on the container to indicate that the composition is to be used in a particular application, and may also indicate instructions for either in vivo or in vitro use. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., compact disc), or directly on a computer or data processing device. Additionally, the kits may contain standard amounts of biomarkers, as described elsewhere herein, for calibration purposes.

[0074] Embodiment In a first aspect, the present invention relates to a method for determining at least one analyte in a sample, comprising the following steps: a) providing a substrate having at least one analyte, at least one microparticle, at least one ionization matrix, and a substrate surface; b) incubating the analyte with microparticles having at least one microparticle surface, whereby the analyte is adsorbed to the surface of the microparticle and an analyte-microparticle complex is formed; c) contacting the analyte-particle complex with an ionization matrix to form a matrix:analyte-particle sample; d) providing a matrix:analyte-particulate sample and / or a matrix:analyte sample on a substrate surface; e) ionizing at least the analyte, the ionization being under mechanical ionization conditions; f) determining the analyte by ion mobility spectrometry and / or mass spectrometry The present invention relates to a method comprising the steps of:

[0075] The inventors have surprisingly found that the subject matter of the present invention, and in particular the method according to the first aspect of the invention, represents a simple and robust way to overcome the above mentioned drawbacks.

[0076] The principle of matrix ionization also works when a solid dual-support workflow is applied. Surprisingly, the inventors have found that the ionization process still works for a wide variety of analytes, even when the analyte is adsorbed on a solid support, in particular a microparticle, and the ionization matrix is ​​on a solid, preferably substrate, such as a paper tissue, together with the microparticle (e.g., in the case of paper, the analyte-loaded microparticle and the crystalline ionization matrix component are simultaneously attracted).

[0077] This method has the advantage that no elution step is required to desorb the analytes from the microparticles. The analyte-loaded microparticles can be washed and the analyte-loaded microparticles are absorbed into the paper tissue together with the (pre-crystallized) ionization matrix. The microparticles on the paper tissue can be dried and stored for later analysis. This can be called a dried bead spot.

[0078] In a first aspect of the present invention, a method for determining at least one analyte in a sample is disclosed.

[0079] According to step a), a substrate is provided having at least one analyte, at least one microparticle, at least one ionizing matrix, and a substrate surface.

[0080] Step b) incubates the analyte with microparticles having at least one microparticle surface. Thus, the analyte is adsorbed to the surface of the microparticles, and an analyte-microparticle complex is formed. In this context, the expression can be understood as forming a plurality of analyte-microparticle complexes. This means that in step b), the sample can be incubated with microparticles having at least one surface, and thus, the analyte is adsorbed to the surface of the microparticles, and an analyte-microparticle complex is formed.

[0081] In an embodiment of the first aspect of the present invention, the microparticles may be modified with a chemical compound selected from the group consisting of a hydrophobic compound, a hydrophilic compound, an immunochemical compound.

[0082] In an embodiment of the first aspect, the hydrophobic compound is, for example, a compound having a carboxylic acid group and / or an alkyl group.

[0083] In an embodiment of the first aspect, the hydrophilic compound is, for example, a compound having a hydroxy functional group.

[0084] In an embodiment of the first aspect, the immunochemical compound is, for example, a compound for which a specific antibody has been developed.

[0085] In an embodiment of the first aspect, the microparticle is a magnetic particle.

[0086] In an embodiment of the first aspect, the microparticles are coated magnetic particles, wherein the coating is a glass coating or a polymer coating.

[0087] In an embodiment of the first aspect, the microparticles are immunobeads for immobilizing antibodies.

[0088] In an embodiment of the first aspect, the microparticles are protein-coated, for example streptavidin-coated, magnetic beads.

[0089] In an embodiment of the first aspect, the microparticles are selected from the group consisting of magnetic microparticles; silica microparticles; melamine resin microparticles; poly(styrene)-based microparticles; and poly(methyl methacrylate) microparticles.

[0090] In particular, the microparticle or microparticles may be selected from the group consisting of magnetic microparticles, specifically magnetic microparticles having a magnetic core and a modified surface; silica microparticles, specifically silica microparticles having a silica core and a modified surface; melamine resin microparticles, specifically melamine resin microparticles having a melamine resin core and a modified surface; poly(styrene)-based microparticles, specifically poly(styrene)-based microparticles having a poly(styrene) core and a modified surface; poly(methyl methacrylate) microparticles, specifically poly(methyl methacrylate) microparticles having a poly(methyl methacrylate) core and a modified surface. However, other particles may also be feasible. The melamine resin microparticles may have an average diameter of 500 nm to 20 μm, preferably 2 μm to 4 μm, most preferably 3 μm. The poly(styrene)-based microparticles may have an average diameter of 500 nm to 50 μm, preferably 2 μm to 4 μm, most preferably 3 μm. The poly(methyl methacrylate) microparticles may have an average diameter of 500 nm to 50 μm, preferably 2 μm to 4 μm, and most preferably 3 μm. The modified surface of the magnetic microparticles may be a modified poly(styrene) surface, and the magnetic microparticles may have an average diameter of 5 μm to 50 μm, preferably 10 μm to 30 μm, and most preferably 20 μm. The modified surface of the magnetic microparticles may be a silica surface, and the magnetic microparticles may have an average diameter of 100 nm to 1000 nm, preferably 200 nm to 500 nm, and most preferably 300 nm. The modified surface of the silica microparticles may be a cyanopropylsilane functionalized surface, and the silica microparticles may have an average diameter of 5 μm to 100 μm, preferably 20 μm to 80 μm, and most preferably 40 μm. Other dimensions may also be feasible.

[0091] In an embodiment of the first aspect, the microparticle is a magnetic particle.

[0092] In an embodiment of the first aspect, the microparticles are magnetic particles comprising a polymer surface and at least one magnetic core, the polymer surface comprising a highly crosslinked polymer, and the magnetic particles having a particle size in the range of 5 to 40 micrometers as determined according to ISO 13320. For the term "polymer surface", "polymer matrix" can also be used.

[0093] In an embodiment of the first aspect, the polymer surface comprises pores having a pore size smaller than 100 nm, preferably less than or equal to 50 nm, as determined according to ISO 15901-3.

[0094] In an embodiment of the first aspect, the particles have a BET specific surface area, as determined according to ISO 9277, in the range of 50 to 2500 m / g.

[0095] In an embodiment of the first aspect the magnetic particles have a saturation magnetization of at least 1 Am / kg, preferably at least 10 Am / kg.

[0096] In an embodiment of the first aspect, the at least one magnetic core comprises at least one magnetic nanoparticle, preferably at least one iron oxide nanoparticle, more preferably an Fe3O4 nanoparticle.

[0097] In an embodiment of the first aspect, the magnetic core comprises at least one nanoparticle and a coating C1, more preferably consists of at least one nanoparticle and a coating C1.

[0098] In an embodiment of the first aspect, at least one magnetic core comprises, preferably consists of, superparticles and optionally comprises a coating C1.

[0099] In an embodiment of the first aspect, the at least one coating C1 is selected from the group consisting of tensides, silicas, silicates, silanes, phosphates, phosphonates, phosphonic acids and mixtures of two or more thereof.

[0100] In an embodiment of the first aspect, the polymeric surface comprises a copolymer obtained or obtainable by a process comprising copolymerizing suitable monomeric components in the presence of at least one monomeric component which is a crosslinker, preferably 5-90% by volume of all monomeric components is the crosslinker, more preferably divinylbenzene.

[0101] In an embodiment of the first aspect, the microparticles are supermagnetic.

[0102] In an embodiment of the first aspect, the highly crosslinked polymer may be prepared by highly crosslinking, the highly crosslinking being carried out in the presence of a catalyst selected from the group consisting of Lewis acids, preferably selected from the group consisting of FeCl3, ZnCl2, AlCl3, BF3, SbCl5, SnCl4, TiCl4, SiCl4 and mixtures of two or more thereof, more preferably FeCl3 or ZnCl2, or mixtures thereof.

[0103] In an embodiment of the first aspect, the method comprises the steps of: b1) separating the analyte-particle complex, in particular the analyte-particle complex, from further components of the sample; and b2) removing further components of the sample from the analyte-microparticle complex, in particular from the analyte-microparticle complex; It may further include.

[0104] In an embodiment of the first aspect, the method comprises the steps of: b3) washing the analyte-microparticle complex, in particular the analyte-microparticle complex; may include.

[0105] Specifically, the analyte-particle complex may be washed with a solvent or a washing solvent. The composition of the washing solvent may be selected such that the analyte remains bound to the microparticles. The washing solvent may be or may include deionized water. Furthermore, the washing solvent may include a mixture of water, one or more buffer salts, one or more pH adjusting additives and / or one or more organic solvents. The organic solvent may be selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile. The content of the organic solvent may be 0% to 10% by volume. Step b3) may be repeated at least twice, preferably at least three times.

[0106] According to step c), the analyte-particle complex is contacted with an ionizing matrix to form a matrix:analyte-particle sample.

[0107] In an embodiment of the first aspect of the present invention, step c) comprises c1) providing an analyte-microparticle complex dissolved in a solvent; and c2) adding an ionizing matrix to the dissolved analyte-particle complex, or vice versa, to form a matrix:analyte-particle sample; and c3) applying the matrix:analyte-particulate sample to a substrate surface, where the ionization matrix of step c2) is crystallized or dissolved in a further solvent, the solvent and the further solvent may be the same or different; Includes.

[0108] In an embodiment of the first aspect of the present invention, step c) comprises c4) providing an analyte-microparticle complex dissolved in a solvent; and c5) applying the dissolved analyte-microparticle complex to a substrate surface; and then c6) adding an ionization matrix to the dissolved analyte-particle complexes to form a matrix:analyte-particle sample, where in step c6) the ionization matrix is ​​crystallized or dissolved in a further solvent, where the solvent and the further solvent can be the same or different. Includes.

[0109] In an embodiment of the first aspect of the present invention, the ionizing matrix is ​​crystallized at least in step c).

[0110] In an embodiment of the first aspect of the invention, the analyte-microparticle complex is in a fluid state in step c) and / or in a solid state by carrying out step f).

[0111] According to step d), a matrix:analyte-particulate sample is provided on the substrate surface.

[0112] Step e) results in at least the analyte being ionized, the ionization being mechanical ionization.

[0113] In an embodiment of the first aspect of the present invention, the ionization in step e) is induced by a mechanical force comprising or consisting of a shear force and / or the mechanical ionization is induced by a mechanical stimulus, preferably the mechanical stimulus is triboluminescence.

[0114] In an embodiment of the first aspect of the present invention, the ionization in step e) is mechanical ionization, wherein the mechanical ionization is induced by shear forces and / or triboluminescence.

[0115] In an embodiment of the first aspect of the present invention, the mechanical ionization is induced by a mechanical force, preferably comprising or consisting of a shear force. Preferably, the mechanical force is caused by shear forces and / or triboluminescence of the respective crystals.

[0116] In an embodiment of the first aspect of the present invention, the mechanical ionization is induced by a mechanical stimulus, preferably the mechanical stimulus is triboluminescence.

[0117] In an embodiment of the first aspect of the present invention, the mechanical ionization is induced by a mechanical force, preferably comprising or consisting of a shear force, and / or the mechanical ionization is induced by a mechanical stimulus, preferably the mechanical stimulus is triboluminescence.

[0118] In an embodiment of the first aspect of the invention, the ionization in step e) is matrix-assisted ionization (MAI), preferably solid dual support matrix-assisted ionization. For example, the analyte is adsorbed to a solid support, in particular a microparticle, and the ionization matrix is ​​on a solid, preferably a substrate, such as a paper tissue, together with the microparticle (e.g., in the case of paper, the analyte-loaded microparticles and the crystalline ionization matrix components are simultaneously attracted).

[0119] In an embodiment of the first aspect of the present invention, step e) is not laser induced.

[0120] In an embodiment of the first aspect of the present invention, the ionizable matrix is ​​a triboluminescent matrix.

[0121] In an embodiment of the first aspect of the present invention, the ionizing matrix is ​​selected from the group consisting of salsalate, 3-nitrobenzonitrile, 2,2'-azobis(2-methylpropane), 2-nitrobenzonitrile, 5-methyl-2-nitrobenzonitrile, coumarin, methyl-2-methyl-3-nitrobenzoate, methyl-5-nitro-2-furoate, 2-bromo-2-nitropropane-1,3-diol), 3-nitrobenzaldehyde, 6-nitro-o-anisinonitrile, phthalic anhydride, or mixtures thereof.

[0122] In an embodiment of the first aspect of the invention, the ionization matrix is ​​in a heterogeneous solid-liquid phase at room temperature and pressure.

[0123] In an embodiment of the first aspect of the invention, the ionizable matrix preferably undergoes a phase transition from a solid phase to a gas phase under subatmospheric pressure.

[0124] In an embodiment of the first aspect of the invention, the ionizable matrix preferably undergoes a phase transition from a solid phase to a gas phase when placed under subatmospheric pressure at a temperature below 120°C.

[0125] In an embodiment of the first aspect of the invention, the ionisable matrix preferably undergoes a phase transition from a solid phase to a gas phase when placed under sub-atmospheric pressure at a temperature below 70°C.

[0126] In an embodiment of the first aspect of the invention, step d) is carried out by disposing the matrix:analyte-particulate sample as a spot on the substrate surface.

[0127] In an embodiment of the first aspect of the invention the analyte comprises a biological tissue, a biological material, a foodstuff, a polymer, a painting, an archaeologic artefact, an artificial bone, skin, urine or blood.

[0128] In an embodiment of the first aspect of the present invention, the sample comprises formic acid (FA).

[0129] In an embodiment of the first aspect of the invention, the analyte of interest is selected from the group consisting of nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids, molecules characterized by a specific modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

[0130] In an embodiment of the first aspect of the present invention, the solvent and / or further solvent is water, methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, chloroform, dimethylformamide, dimethylsulfoxide, acetone, or a mixture thereof.

[0131] In an embodiment of the first aspect of the invention, the analyte and the microparticle are covalently linked to form an analyte-microparticle complex.

[0132] In an embodiment of the first aspect of the invention, the analyte is vitamin D and the microparticle is an immunobead.

[0133] In an embodiment of the first aspect of the invention, a mass spectrometer or ion mobility spectrometer comprises an inlet and a region proximate to said inlet, said region proximate to said inlet being maintained at sub-atmospheric pressure.

[0134] In an embodiment of the first aspect of the invention, the inlet is a system where at least one analyte and / or matrix:analyte-particulate sample is injected or inserted into the chamber under vacuum and, optionally, heated to achieve vaporization.

[0135] In an embodiment of the first aspect of the invention, the inlet comprises a disconnected sample inlet and a filter.

[0136] In an embodiment of the first aspect of the present invention, the filter is a nylon mesh, a membrane, a metal grid. In principle, other polymeric materials for the filter can be used, such as polyester mesh, poly(tetrafluoroethylene) filter membrane, polypropylene filter membrane, poly(ether ether ketone) filter membrane.

[0137] In an embodiment of the first aspect of the invention, the filter is part of the truncated sample inlet.

[0138] In an embodiment of the first aspect of the invention, the filter is replaceable.

[0139] In an embodiment of the first aspect of the invention, the filter is coated with an ionizing matrix and / or matrix:analyte-particle sample and / or analyte-particle complex.

[0140] In an embodiment of the first aspect of the invention, the sample is a biological sample, the biological sample being selected from the group consisting of blood, serum, plasma, saliva, ocular lens fluid, cerebrospinal fluid, sweat, urine, milk, peritoneal fluid, mucus, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cells.

[0141] In an embodiment of the first aspect of the present invention, the substrate is selected from the group consisting of metal, paper, fabric, ribbon, glass, plastic, polymer, sodium dodecyl sulfate gel, agarose gel, paper chromatography plate, silica plate and woven fabric.

[0142] In an embodiment of the first aspect of the invention, the substrate is a plate, such as a glass plate, or a filter, such as a triangular filter.

[0143] In an embodiment of the first aspect of the invention, the method is automated, preferably fully or partially automated.

[0144] According to step f), the analytes are determined by ion mobility spectrometry and / or mass spectrometry.

[0145] In an embodiment of the first aspect of the present invention, the method comprises the steps of: g) providing a filter disposed between the analyte-particle complex according to step d) and the ion mobility spectrometry or mass spectrometry to prevent the particles from entering the ion mobility spectrometry or mass spectrometry; and / or h) washing the analyte-microparticle complexes, preferably using water as a washing reagent, at least after step b). The present invention further includes at least one of the following:

[0146] In an embodiment of the first aspect of the present invention, the filter has a mesh size smaller than the particle size of the particulates.

[0147] In a second aspect, the present invention relates to the use of the method of the first aspect of the invention for determining at least one analyte of interest. All embodiments mentioned for the first aspect of the invention apply to the second aspect of the invention and vice versa.

[0148] In a third aspect, the present invention relates to a sample element for determining at least one analyte and suitable for carrying out a method according to the first aspect of the invention, comprising: a substrate surface; - an ionization matrix disposed on a surface of the substrate for use in matrix-assisted ionization; - an analyte-microparticle complex disposed on a surface of a substrate; the ionization matrix is ​​selected from the group consisting of salsalate, 3-nitrobenzonitrile, 2,2'-azobis(2-methylpropane), 2-nitrobenzonitrile, 5-methyl-2-nitrobenzonitrile, coumarin, methyl-2-methyl-3-nitrobenzoate, methyl-5-nitro-2-furoate, 2-bromo-2-nitropropane-1,3-diol), 3-nitrobenzaldehyde, 6-nitro-o-anisinonitrile, phthalic anhydride, or mixtures thereof; - the ionization matrix and / or the analyte-microparticle complex are crystallized or pre-crystallized; - the microparticle of the analyte-microparticle complex is magnetic; - relating to a sample element, in which the analyte-microparticle complex and the ionization matrix are in contact with each other. All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention apply to the third aspect of the invention and vice versa.

[0149] In an embodiment of the third aspect of the invention, the ionization matrix is ​​not induced by a laser or laser ionization technique, such as MALDI or SALDI.

[0150] In a fourth aspect, the present invention relates to an inlet for ion transport into a mass spectrometer or ion mobility spectrometer comprising a truncated sample inlet and a filter. All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention apply to the fourth aspect of the invention and vice versa.

[0151] In a fifth aspect, the present invention relates to the use of the inlet of the fourth aspect of the invention for determining at least one analyte of interest. All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention and / or the fourth aspect of the invention apply to the fifth aspect of the invention and vice versa.

[0152] In an embodiment of the fifth aspect of the invention, the inlet is part of a device, preferably the device is a clinical diagnostic system, which may mean that the device comprises the inlet.

[0153] In an embodiment of the fifth aspect of the invention, the clinical diagnostic system comprises a sample preparation station.

[0154] In a sixth aspect, the present invention relates to the use of the inlet of the fifth aspect of the invention for determining at least one analyte of interest. All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention and / or the fourth aspect of the invention and / or the fifth aspect of the invention apply to the sixth aspect of the invention and vice versa.

[0155] In a seventh aspect, the present invention relates to a composition for vacuum ionization or inlet ionization comprising an ionization matrix, wherein the ionization matrix comprises or consists of salsalate. All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention and / or the fourth aspect of the invention and / or the fifth aspect of the invention and / or the sixth aspect of the invention apply to the seventh aspect of the invention and vice versa.

[0156] In an embodiment of the seventh aspect of the present invention the composition is for matrix assisted ionization (MAI), preferably for solid dual support matrix assisted ionization.

[0157] In an embodiment of the seventh aspect of the present invention, the salsalate has the CAS number 552-94-3.

[0158] In an embodiment of the seventh aspect of the present invention, salsalate has the following formula: [ka] has.

[0159] In an embodiment of the seventh aspect of the present invention the composition further comprises at least one analyte.

[0160] In an embodiment of the seventh aspect of the present invention the composition further comprises coated microparticles, preferably magnetic particles, wherein the coating is a glass coating or a polymer coating.

[0161] In an embodiment of the seventh aspect of the invention the composition further comprises microparticles, preferably immunobeads.

[0162] In an embodiment of the seventh aspect of the present invention, the molar ratio of ionization matrix to analyte is 5:1 to 1x10 7 :1.

[0163] In an embodiment of the seventh aspect of the invention, the composition comprises a matrix:analyte-particulate sample or a matrix:analyte sample, wherein the ionized matrix:analyte sample or matrix:analyte-particulate sample is in a solid phase when exposed to subatmospheric pressure.

[0164] In an embodiment of the seventh aspect of the present invention, the ionization matrix is ​​crystallized when performing vacuum or inlet ionization.

[0165] In an embodiment of the seventh aspect of the present invention, the ionisation matrix:analyte-particulate sample or matrix:analyte sample is preferably arranged on the substrate as a spot.

[0166] In an embodiment of the seventh aspect of the present invention the substrate is selected from the group consisting of metal, paper, cloth, ribbon, glass, plastic, polymer, sodium dodecyl sulfate gel, agarose gel, paper chromatography plate, silica plate or woven fabric.

[0167] In an embodiment of the seventh aspect of the present invention the composition comprises a solvent.

[0168] In an embodiment of the seventh aspect of the present invention the solvent is water, methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, chloroform, dimethylformamide, dimethylsulfoxide, acetone, or a mixture thereof.

[0169] In an embodiment of the seventh aspect of the invention, the matrix:analyte-particulate or matrix:analyte sample is prepared by mixing or grinding together the analyte and ionization matrix, and any optional particulates.

[0170] In an embodiment of the seventh aspect of the invention the matrix:analyte-particulate sample or matrix:analyte sample is a solid.

[0171] In an embodiment of the seventh aspect of the present invention the solid sample is in a frozen state.

[0172] In an embodiment of the seventh aspect of the invention, the matrix:analyte-particulate sample or the matrix:analyte sample further comprises an ammonium salt, a metal salt, an acid, a base or a buffer.

[0173] In an eighth aspect, the present invention relates to the use of a composition according to the seventh aspect of the invention, preferably in a method according to the first aspect of the invention. All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention and / or the fourth aspect of the invention and / or the fifth aspect of the invention and / or the sixth aspect of the invention and / or the seventh aspect of the invention apply to the eighth aspect of the invention and vice versa.

[0174] In a ninth aspect, the present invention provides a kit suitable for carrying out a method according to the first aspect of the invention, comprising: (A) an ionization matrix; (B) a solvent or further solvent; (C) fine particles; (D) optionally, at least one internal standard; Equipped with.

[0175] All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention and / or the fourth aspect of the invention and / or the fifth aspect of the invention and / or the sixth aspect of the invention and / or the seventh aspect of the invention and / or the eighth aspect of the invention apply to the ninth aspect of the invention and vice versa.

[0176] In a tenth aspect, the present invention relates to the use of a kit according to the ninth aspect of the invention in a method according to the first aspect of the invention. All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention and / or the fourth aspect of the invention and / or the fifth aspect of the invention and / or the sixth aspect of the invention and / or the seventh aspect of the invention and / or the eighth aspect of the invention and / or the ninth aspect of the invention apply to the tenth aspect of the invention and vice versa.

[0177] In further embodiments, the present invention relates to the following aspects: 1. A method for determining at least one analyte in a sample, comprising the following steps: a) providing a substrate having at least one analyte, at least one microparticle, at least one ionization matrix, and a substrate surface; b) incubating the analyte with microparticles having at least one microparticle surface, whereby the analyte is adsorbed to the surface of the microparticle and an analyte-microparticle complex is formed; c) contacting the analyte-particle complex with an ionization matrix to form a matrix:analyte-particle sample; d) providing a matrix:analyte-particulate sample and / or a matrix:analyte sample on a substrate surface; e) ionizing at least the analyte, wherein the ionization is mechanical ionization; f) determining the analyte by ion mobility spectrometry and / or mass spectrometry A method comprising:

[0178] 2. The method of embodiment 1, wherein the mechanical ionization is induced by a mechanical force, preferably comprising or consisting of a shear force.

[0179] 3. The method of any of the preceding aspects, wherein mechanical ionization is induced by a mechanical stimulus, preferably the mechanical stimulus is triboluminescence.

[0180] 4. The method of any of the preceding aspects, wherein the mechanical ionization is not induced by evaporation or sublimation.

[0181] 5. The method of any of the preceding aspects, wherein the method is automated.

[0182] 6. The method according to any of the preceding embodiments, wherein the ionization in step e) is matrix assisted ionization (MAI), preferably solid dual support matrix assisted ionization.

[0183] 7. The method of any of the preceding aspects, wherein step e) is not induced by a laser.

[0184] 8. The method of any of the preceding embodiments, wherein the ionization matrix is ​​crystallized at least in step c).

[0185] 9. The method of any of the previous aspects, wherein the ionizable matrix is ​​a triboluminescent matrix.

[0186] 10. Step c) c1) providing an analyte-microparticle complex dissolved in a solvent; and c2) adding an ionizing matrix to the dissolved analyte-particle complex, or vice versa, to form a matrix:analyte-particle sample; and c3) applying the matrix:analyte-particulate sample to a substrate surface, where the ionization matrix of step c2) is crystallized or dissolved in a further solvent, the solvent and the further solvent may be the same or different; 4. The method of any preceding aspect, comprising:

[0187] 11. Step c) c4) providing an analyte-microparticle complex dissolved in a solvent; and c5) applying the dissolved analyte-microparticle complex to a substrate surface; and then c6) adding an ionization matrix to the dissolved analyte-particle complexes to form a matrix:analyte-particle sample, where in step c6) the ionization matrix is ​​crystallized or dissolved in a further solvent, where the solvent and the further solvent can be the same or different. 4. The method of any preceding aspect, comprising:

[0188] 12. A method comprising the steps of: g) providing a filter disposed between the analyte-particle complex according to step d) and the ion mobility spectrometry or mass spectrometry to prevent the particles from entering the ion mobility spectrometry or mass spectrometry; and / or h) washing the analyte-microparticle complexes, preferably using water as a washing reagent, at least after step b).

[0023] 3. The method of any of the preceding aspects, further comprising at least one of:

[0189] 13. The method of any of the previous aspects, wherein the filter has a mesh size smaller than the particle size of the particulate.

[0190] 14. The method of any of the preceding aspects, wherein the microparticles may be modified by a chemical selected from the group consisting of a hydrophobic compound, a hydrophilic compound, an immunochemical compound.

[0191] 15. The method of any of the preceding aspects, wherein the microparticles are magnetic particles.

[0192] 16. The method of any of the previous aspects, wherein the microparticles are coated magnetic particles, and the coating is a glass coating or a polymer coating.

[0193] 17. The method of any of the previous aspects, wherein the microparticles are immunobeads for immobilizing antibodies.

[0194] 18. The method of any of the previous aspects, wherein the microparticles are protein-coated, e.g. streptavidin-coated, magnetic beads.

[0195] 19. The method of any of the previous aspects, wherein the analyte-microparticle complex is in a fluid state in step c) and / or in a solid state by carrying out step f).

[0196] 20. The method of any of the preceding aspects, wherein the microparticles are selected from the group consisting of magnetic microparticles; silica microparticles; melamine resin microparticles; poly(styrene)-based microparticles; and poly(methyl methacrylate) microparticles.

[0197] 21. The method of any of the preceding aspects, wherein the microparticles are magnetic particles.

[0198] 22. The method of any of the preceding aspects, wherein the microparticles are magnetic particles comprising a polymeric surface (P) and at least one magnetic core (M), the polymeric surface comprising a highly crosslinked polymer, and the magnetic particles having a particle size in the range of 5 to 40 micrometers, as determined according to ISO 13320.

[0199] 23. The method of any of the previous embodiments, wherein the polymer surface comprises pores having a pore size smaller than 100 nm, preferably equal to or smaller than 50 nm, as determined according to ISO 15901-3.

[0200] 24. The method of any of the preceding embodiments, wherein the particles have a BET specific surface area, as determined according to ISO 9277, in the range of 50 to 2500 m / g.

[0201] 25. The method according to any of the previous embodiments, wherein the magnetic particles have a saturation magnetization of at least 1 Am / kg, preferably at least 10 Am / kg.

[0202] 26. The method according to any of the previous embodiments, wherein the at least one magnetic core (M) comprises at least one magnetic nanoparticle, preferably at least one iron oxide nanoparticle, more preferably Fe304 nanoparticle.

[0203] 27. The method according to any of the previous aspects, wherein the magnetic core (M) comprises at least one nanoparticle and a coating C1, more preferably consists of at least one nanoparticle and a coating C1.

[0204] 28. The method according to any of the preceding embodiments, wherein at least one magnetic core (M) comprises, preferably consists of, a superparticle and optionally comprises a coating C1.

[0205] 29. The method according to any of the previous aspects, wherein the at least one coating C1 is selected from the group consisting of tensides, silicas, silicates, silanes, phosphates, phosphonates, phosphonic acids, and mixtures of two or more thereof.

[0206] 30. The method of any of the preceding embodiments, wherein the polymeric surface comprises a copolymer obtained or obtainable by a process comprising copolymerizing suitable monomeric components in the presence of at least one monomeric component that is a crosslinker, preferably 5-90% by volume of all monomeric components is a crosslinker, more preferably divinylbenzene.

[0207] 31. The method of any of the preceding aspects, wherein the microparticles are supermagnetic.

[0208] 32. The method of any of the preceding aspects, wherein the highly crosslinked polymer can be produced by highly crosslinking, the highly crosslinking being carried out in the presence of a catalyst selected from the group consisting of Lewis acids, preferably selected from the group consisting of FeCl3, ZnCl2, AlCl3, BF3, SbCl5, SnCl4, TiCl4, SiCl4 and mixtures of two or more thereof, more preferably FeCl3 or ZnCl2, or a mixture thereof.

[0209] 33. The method of any of the preceding aspects, wherein the sample is a biological sample, and the biological sample is selected from the group consisting of blood, serum, plasma, saliva, ocular lens fluid, cerebrospinal fluid, sweat, urine, milk, peritoneal fluid, mucus, synovial fluid, peritoneal fluid, amniotic fluid, tissue, and cells.

[0210] 34. The method of any of the preceding aspects, wherein the substrate is selected from the group consisting of metal, paper, cloth, ribbon, glass, plastic, polymer, sodium dodecyl sulfate gel, agarose gel, paper chromatography plate, silica plate, and woven fabric fiber.

[0211] 35. The method of any of the preceding aspects, wherein the substrate is a plate, such as a glass plate, or a filter, such as a triangular filter.

[0212] 36. The method of any of the preceding aspects, wherein the ionization matrix is ​​selected from the group consisting of salsalate, 3-nitrobenzonitrile, 2,2'-azobis(2-methylpropane), 2-nitrobenzonitrile, 5-methyl-2-nitrobenzonitrile, coumarin, methyl-2-methyl-3-nitrobenzoate, methyl-5-nitro-2-furoate, 2-bromo-2-nitropropane-1,3-diol), 3-nitrobenzaldehyde, 6-nitro-o-anisinonitrile, phthalic anhydride, or a mixture thereof.

[0213] 37. The method of any of the preceding aspects, wherein the ionization matrix is ​​in a heterogeneous solid-liquid phase at room temperature and pressure.

[0214] 38. The method of any of the preceding embodiments, wherein the ionization matrix undergoes a phase transition, preferably from solid to gas phase, under subatmospheric pressure.

[0215] 39. The method of any of the preceding embodiments, wherein the ionizable matrix preferably undergoes a phase transition from solid to gas phase when placed under subatmospheric pressure at a temperature below 120°C.

[0216] 40. The method of any of the preceding embodiments, wherein the ionizing matrix preferably undergoes a phase transition from solid to gas phase when placed under subatmospheric pressure at a temperature below 70° C.

[0217] 41. The method of any of the previous aspects, wherein the analyte comprises a biological tissue, a biological material, a foodstuff, a polymer, a painting, an archaeal artefact, an artificial bone, skin, urine or blood.

[0218] 42. The method of any of the preceding aspects, wherein the sample comprises formic acid (FA).

[0219] 43. The method of any of the preceding aspects, wherein in step d) the method is carried out by disposing the matrix:analyte-particulate sample as a spot on the substrate surface.

[0220] 44. The method of any of the preceding aspects, wherein the analyte of interest is selected from the group consisting of nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids, molecules characterized by a specific modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

[0221] 45. The composition according to any of the preceding embodiments, wherein the solvent and / or further solvent is water, methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, chloroform, dimethylformamide, dimethylsulfoxide, acetone, or mixtures thereof.

[0222] 46. ​​The method of any of the preceding embodiments, wherein the analyte and the microparticle are covalently bonded to form an analyte-microparticle complex.

[0223] 47. The method of any of the previous embodiments, wherein the analyte is vitamin D and the microparticle is an immunobead, or wherein the analyte is testosterone and the microparticle is a bead, e.g. an immunobead.

[0224] 48. The method of any of the preceding aspects, wherein the mass spectrometer or ion mobility spectrometer includes an inlet and a region proximate to the inlet, the region proximate to the inlet being maintained at or below atmospheric pressure.

[0225] 49. The method of any of the preceding aspects, wherein the inlet is a system in which at least one analyte and / or matrix:analyte-particulate sample is injected or inserted into the chamber under vacuum and, optionally, heated to achieve vaporization.

[0226] 50. The method of any of the preceding aspects, wherein the inlet comprises a disconnected sample inlet and a filter.

[0227] 51. The method of any of the preceding aspects, wherein the filter is a nylon mesh, membrane, metal grid.

[0228] 52. The method of any of the preceding aspects, wherein the filter is part of a disconnected sample inlet.

[0229] 53. The method of any preceding aspect, wherein the filter is replaceable.

[0230] 54. The method of any of the previous aspects, wherein the filter is coated with an ionization matrix and / or matrix:analyte-microparticle sample and / or analyte-microparticle complex.

[0231] 55. Use of the method according to any of the preceding aspects for determining at least one analyte of interest.

[0232] 56. A sample element suitable for determining at least one analyte and for carrying out a method according to any of the preceding embodiments, comprising: a substrate surface; - an ionization matrix disposed on a surface of the substrate for use in matrix-assisted ionization; - an analyte-microparticle complex disposed on a surface of a substrate; the ionization matrix is ​​selected from the group consisting of salsalate, 3-nitrobenzonitrile, 2,2'-azobis(2-methylpropane), 2-nitrobenzonitrile, 5-methyl-2-nitrobenzonitrile, coumarin, methyl-2-methyl-3-nitrobenzoate, methyl-5-nitro-2-furoate, 2-bromo-2-nitropropane-1,3-diol), 3-nitrobenzaldehyde, 6-nitro-o-anisinonitrile, phthalic anhydride, or mixtures thereof; - the ionization matrix and / or the analyte-microparticle complex are crystallized; - the microparticle of the analyte-microparticle complex is magnetic; - a sample element in which the analyte-microparticle complex and the ionization matrix are in contact with each other.

[0233] 57. The sample element of embodiment 56, wherein ionization is not induced by a laser.

[0234] 58. Use of a sample element according to any of the preceding embodiments for determining at least one analyte of interest, preferably in a method according to any of the preceding embodiments.

[0235] 59. An inlet suitable for carrying out a method according to any of the preceding aspects and suitable for ion transport into a mass spectrometer or ion mobility spectrometer, or into a detector of a mass spectrometer or ion mobility spectrometer with a truncated sample inlet and filter.

[0236] 60. Use of the inlet of embodiment 59 for determining at least one analyte of interest, preferably in a method according to any of the preceding embodiments.

[0237] 61. A composition for vacuum ionization or inlet ionization comprising an ionization matrix, the ionization matrix comprising or consisting of salsalate.

[0238] 62. Salsalate has the formula: [ka] 62. The composition of embodiment 61, having the following formula:

[0239] 63. The composition of any of the preceding aspects, further comprising at least one analyte.

[0240] 64. The composition of any of the previous embodiments, further comprising coated microparticles, preferably magnetic particles, wherein the coating is a glass coating or a polymer coating.

[0241] 65. The composition of any of the proceeding embodiments, further comprising a microparticle, preferably an immunobead.

[0242] 66. The composition of any of the previous aspects, wherein the composition is used for matrix assisted ionization (MAI).

[0243] 67. The molar ratio of ionization matrix to analyte is 5:1 to 1x10 7 5. The composition of any of the preceding embodiments, wherein the R 1 -R 2 ratio is 1:1.

[0244] 68. The composition of any of the preceding aspects, comprising a matrix:analyte-particulate sample or matrix:analyte sample, wherein the ionized matrix:analyte sample or matrix:analyte-particulate sample is in a solid phase when exposed to subatmospheric pressure.

[0245] 69. The composition of any of the previous aspects, wherein the ionization matrix is ​​crystallized when subjected to vacuum or inlet ionization.

[0246] 70. The composition of any of the preceding aspects, wherein the ionization matrix:analyte-particulate sample or matrix:analyte sample is disposed on the substrate, preferably as a spot.

[0247] 71. The composition of any of the preceding aspects, wherein the substrate is selected from the group consisting of metal, paper, cloth, ribbon, glass, plastic, polymer, sodium dodecyl sulfate gel, agarose gel, paper chromatography plate, silica plate, or woven fabric fiber.

[0248] 72. The composition of any of the preceding embodiments, wherein the composition comprises a solvent.

[0249] 73. The composition of any of the preceding embodiments, wherein the solvent is water, methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, chloroform, dimethylformamide, dimethylsulfoxide, acetone, or a mixture thereof.

[0250] 74. The composition of any of the preceding aspects, wherein the matrix:analyte-particulate sample or matrix:analyte sample is prepared by mixing or grinding together the analyte and ionization matrix, and any optional particulates.

[0251] 75. The composition of any of the preceding aspects, wherein the matrix:analyte-particulate sample or matrix:analyte sample is a solid.

[0252] 76. The composition of the preceding embodiment, wherein the solid sample is in a frozen state.

[0253] 77. The composition of any of the preceding aspects, wherein the matrix:analyte-particulate sample or matrix:analyte sample further comprises an ammonium salt, a metal salt, an acid, a base, or a buffer.

[0254] 78. Use of the composition of the preceding embodiment for determining at least one analyte of interest, preferably in a method according to any of the preceding embodiments.

[0255] 79. (A) an ionization matrix; (B) a solvent or further solvent; (C) fine particles; (D) optionally, at least one internal standard; A kit suitable for carrying out the method according to any of the preceding aspects, comprising:

[0256] 80. Use of the kit of embodiment 79 in a method according to any of the preceding embodiments. EXAMPLES

[0257] The following examples are offered to illustrate, but not to limit, the invention claimed herein.

[0258] Example 1 As a first example, a method for determining at least one analyte in a sample was carried out.

[0259] FIG. 1 shows a schematic description of the method for determining at least one analyte in a sample, in particular the matrix ionization microparticle workflow. A model analyte, for example leucine-enkephalin, is pipetted into a horse serum matrix (for example, 150 μl bulk volume). Thus, for a blank measurement, the model analyte is pipetted into a solution of H2O / ACN (90 / 10, 150 μL bulk volume). Microparticles, for example magnetic bead particles, are added to the sample containing the horse serum matrix and mixed appropriately. Analyte-microparticle complexes are formed. After an incubation time of 10 minutes, the analyte-microparticle complexes are washed twice with a solvent, for example water. Thus, a certain amount of the remaining analyte-microparticle complexes, for example as a dispersion, is transferred to a glass plate and a solution of ionization matrix (for example 3-nitrobenzonitrile, 3-NBN) is added. Subsequently, at least the analyte and / or the mixture of ionization matrix and analyte-microparticle complexes is measured by MS.

[0260] Example 2 As a second example, a method for determining at least one analyte in a sample was performed.

[0261] FIG. 2 shows a schematic description of the method for determining at least one analyte in a sample, in particular the matrix ionization microparticle workflow. A model analyte is pipetted into a horse serum matrix (e.g., bulk volume 150 μl, H2 / ACN=90 / 10). Thus, for a blank measurement, a model analyte is pipetted into a solution of H2O / ACN (90 / 10, bulk volume of 150 μL). Microparticles, e.g., magnetic bead particles, are added to the sample containing the horse serum matrix and mixed appropriately. Analyte-microparticle complexes are formed. After an incubation time of 10 minutes, the analyte-microparticle complexes are washed twice with a solvent, e.g., water. Then, an amount of ionization matrix solution (e.g., 3-nitrobenzonitrile, 3-NBN), e.g., as a dispersion, is pipetted into the washed analyte-microparticle complexes. The ionization matrix solution and the analyte-microparticle complexes are co-crystallized in a reaction vessel to form a matrix:analyte-microparticle sample. On the one hand, the analyte molecules are extracted from the analyte-particle complex, and on the other hand, the analyte is co-crystallized with an ionization matrix, e.g., 3-NBN. The analyte is measured by MS in the remaining extract and co-crystallization matrix: the analyte-particle sample or the analyte-particle complex.

[0262] Figure 3a)-d) show the MS spectra (relative abundance vs. time and relative abundance vs. m / z) in positive ionization mode of 1 μL of the residual liquid after magnetic separation spotted on a glass plate. The relative abundance of the total ion current of the blank experiment (a) and the analyte experiment (b) is shown. The corresponding mass spectrum of the blank (c) shows various background signals with relatively low signal intensity. The analyte experiment (d) shows a clear signal of leucine-enkephalin at m / z 556. Thus, with the addition of an ionization matrix (e.g., 3-nitrobenzonitrile, 3-NBN), the model analyte showed a clear MS signal without the use of further ionization energy. Samples without spiked model analyte showed no signal after the addition of the ionization matrix.

[0263] Figure 4a)-d) show the MS spectra of 1 μL of a mixture of recrystallized 3-NBN as the ionization matrix and analyte-loaded beads as the analyte-microparticle-complex. The spectrum of the blank experiment on the left shows no Cyclosporine A D10 signal. The spectrum of the analyte experiment shows the [M+H] of Cyclosporine A D10. + The signal at m / z 1213 corresponds to the [M+Na] signal of cyclosporine A D10. + Magnetic microparticles were used for sample clean-up and analyte / matrix separation. Crystallization of the analyte:microparticle complexes followed by magnetic separation and measurement of the analyte / matrix mixture results in different analyte signals.

[0264] FIG. 5 shows a schematic description of the method for determining at least one analyte in a sample, in particular the matrix ionization microparticle workflow. A model analyte is pipetted into a horse serum matrix. Magnetic bead particles as microparticles are added to the sample containing the horse serum matrix and mixed properly. After an incubation time of 10 min, the analyte-microparticle complexes are washed twice with a solvent, e.g., water. After the last washing step, a triangular filter is placed in the remaining analyte-microparticle complexes (bead-analyte dispersion) and the analyte-microparticle complexes are aspirated into the filter tissue. An ionization matrix (e.g., 3-nitrobenzonitrile, 3-NBN, 100 mg / mL) is added to the filter tip and the mixture of the ionization matrix and the analyte-microparticle complexes is measured by MS.

[0265] Figure 6a)-d) show the MS spectra of analyte-particle complexes (bead-analyte dispersions) aspirated into triangular filters with (a) and (b)) added ionization matrix and without (c) and (d)). The spectrum of the analyte experiment on the left shows a strong signal at m / z 556.3, corresponding to the [M+H]+ signal of leucine-enkephalin. The blank experiment on the right shows no corresponding signal of leucine-enkephalin and almost no background signal. Solid-phase microparticle sample extraction of the analyte and subsequent addition of the ionization matrix results in different MS signals of the analyte. MS ionization is performed directly from the solid microparticles on the solid subtract. The blank experiment without ionization matrix shows no analyte signal.

[0266] Figures 7a) and 7b) show the MS spectra of the triangular filter with and without the ionization matrix added analyte-microparticle complex (bead-analyte dispersion). The blank experiment without the analyte-microparticle complex (bead-analyte dispersion) shows no corresponding signal for leucine-enkephalin and almost no background signal. Both blank experiments (without analyte (Figures 6c and 6d) and without ionization matrix (Figures 7a and 7b)) showed no background analyte signal and very low background noise.

[0267] Figure 8 shows a schematic description of the method for determining at least one analyte in a sample, in particular the matrix ionization microparticle workflow. A model analyte is pipetted into the horse serum matrix. Magnetic bead particles as microparticles are added to the sample containing the horse serum matrix and mixed properly. After an incubation time of 10 minutes, the analyte-microparticle complex is washed with a solvent, e.g. water.

[0268] In the first embodiment, after the final washing step, the triangular filter is placed in the remaining analyte-particle complexes (bead-analyte dispersion) and the analyte-particle complexes are aspirated into the filter tissue. An ionization matrix (e.g., 3-nitrobenzonitrile, 3-NBN, 100 mg / mL) is then added to the filter tip and the mixture of ionization matrix and analyte-particle complexes is measured by MS.

[0269] In a second embodiment, after the final washing step, an ionization matrix is ​​pipetted onto the washed analyte-particle complexes, a triangular filter is placed into the analyte-particle sample containing the matrix:analyte-particle complexes and the ionization matrix, and the matrix:analyte-particle sample is aspirated into the filter tissue. The mixture of ionization matrix and analyte-particle complexes is then measured by MS.

[0270] Figure 9a)-d) show the MS spectra of leucine-enkephalin coated microparticles with and without pre-crystallized ionization matrix (e.g., 3-NBN matrix). Crystallization of ionization matrix, e.g., 3-NBN, was performed by mixing 20 μL of ionization matrix, e.g., 3-NBN (100 mg / mL in ACN+0.1% formic acid)+10 μL H2O. An aliquot of 10 μL of pre-crystallized ionization matrix, e.g., 3-NBN, was transferred to the first washed analyte-microparticle complex and loaded into the triangular filter. The spectrum of the analyte experiment on the left shows the [M+H] of leucine-enkephalin. + The blank experiment on the right shows no corresponding signal for leucine-enkephalin and very little background signal.

[0271] Figure 10a) and b) show the MS spectrum of leucine-enkephalin coated microparticles with pre-crystallized ionization matrix (here 3-NBN). Crystallization of 3-NBN was performed by mixing 20 μL of 3-NBN (100 mg / mL in ACN + 0.1% formic acid) + 10 μL of HO. An aliquot of 10 μL of pre-crystallized 3-NBN matrix was transferred to the triangular filter loaded with analyte. The spectrum of the analyte experiment shows the [M+H] of leucine-enkephalin. + The signal at m / z 556.3 corresponding to the signal is shown.

[0272] Figures 9 and 10 showed that the ionization matrix can be pipetted directly onto the analyte-microparticle complex located on the substrate or added to the washed microparticle dispersion and subsequently loaded onto the substrate. Both routes are possible workflows. The corresponding blank experiments without ionization matrix showed no background and no analyte signal.

[0273] Figure 11a)-d) show the MS spectra of leucine-enkephalin coated microparticles with pre-crystallized ionization matrix (here 3-NBN). Crystallization of 3-NBN was performed by mixing 20 μL of 3-NBN (100 mg / mL in ACN + 0.1% formic acid) + 10 μL of HO. An aliquot of 10 μL of pre-crystallized 3-NBN matrix was transferred to the triangular filter loaded with analyte. The spectrum of the analyte experiment on the left shows the [M+H] of leucine-enkephalin. + The signal at m / z 556.3 corresponding to the signal is shown. A blank experiment without 3-NBN matrix on the right shows no corresponding signal for leucine-enkephalin and almost no background signal.

[0274] Figure 12 shows the screening of different substances as ionization matrices, including the ionization matrix of salsalate. Leucine-enkephalin was used as a model analyte at a concentration of 100 μg / mL. The ionization matrix and the analyte solution were mixed and measured directly by MS. The marking (X) represents the MS signal of the matrix and / or the analyte alone. This means that the ionization matrix of salsalate alone shows the analyte MS signal without interfering with the MS signal itself.

[0275] Figure 13 shows the screening of different nitrobenzene reaction products as ionization matrices. The nitrobenzene reaction products were previously prepared by condensation reaction of the corresponding acid chlorides of structures A)-E) with molecules 1)-10). As ionization matrices, all nitrobenzene reaction products were dissolved (100 mg / mL in ACN+0.1% formic acid). Leucine-enkephalin was used as a model analyte at a concentration of 100 μg / mL. The model analyte solution (1 μl) was mixed with each ionization matrix solution (2 yL), co-crystallized and measured by MS. No MS signals of the matrix and / or analyte were visible.

[0276] FIG. 14 shows an inlet for ion transport to a mass spectrometer. The inlet includes a cut sample inlet and a filter. The filter is placed in the sample inlet of a cone-shaped cut inlet device to prevent particles from entering the ion mobility spectrometry or mass spectrometry. The analyte-particle complex from step d) is retained in front of the filter and the cut inlet device. The filter forms a barrier. The filter can be a nylon mesh, a membrane or a metal grid. The filter can be replaceable. Other materials for the filter are possible, for example polyester mesh, poly(tetrafluoroethylene) filter membrane, polypropylene filter membrane or poly(ether ether ketone) filter membrane. The dimensions of the inlet as shown in FIG. 14 are examples and can be changed.

[0277] Figures 15a1)-d2) show the respective extracted ion mobilograms (Figures 15a1)-15d1); drift time range 0 ms-10 ms) as well as the corresponding full-scan mass spectra (Figures 15a2)-15d2); m / z range 200-900) applying the inlet for ion transport with and without the filtering material to the mass spectrometer.

[0278] Figure 15d1) and Figure 15d2) were recorded by analyzing the crystallization spot of 1 μL of leucine-enkephalin coated microparticle suspension (obtained from 100 μL of 1 μg / mL aqueous solution) with 2 μL of 3-NBN matrix (100 mg / mL in ACN + 0.1% formic acid) in combination with a filtration material (woven nylon filter, 5 μm mesh size, Repligen). Figure 15c1) and Figure 15c2) were recorded by analyzing the crystallization spot of 1 μL of leucine-enkephalin (1 μg / mL) coated microparticle suspension with 2 μL of 3-NBN matrix (100 mg / mL in ACN + 0.1% formic acid) without filtration material. For comparison, Figure 15b1) and Figure 15b2) were recorded by analyzing only the crystallization spot of 3-NBN matrix applying inlet without filtration material. Additionally, crystallization spots of leucine-enkephalin (1 μL of 1 μg / mL aqueous solution) were analyzed using 3-NBN matrix (2 μL, 100 mg / mL in ACN+0.1% formic acid) and recorded in Fig. 15a1) and Fig. 15b1). All spectra were recorded in IMS-ToF mode on a Synapt G2Si mass spectrometer (Waters) modified with an inlet for ion transport as shown in Fig. 14. The source temperature was set to 50 °C and all crystallization samples were measured for a total analysis time of 30 s. Nitrogen was used as the IMS drift gas. The IMS pulse height and wave velocity settings were 30 V, 800 m / s, respectively. The extracted ion mobilogram showed leucine-enkephalin [M+H] at m / z 556.3; +The signal was obtained by extracting the signal. To determine the S / N ratio, the signal range was set between 4.2 ms and 4.8 ms, and the noise range was set between 1.0 ms and 3.8 ms. The number of counts observed from the leucine-enkephalin-coated microparticles in Figure 15c1) was clearly increased compared to the leucine-enkephalin solution itself (Figure 15a1). The blank 3-NBN matrix itself in Figure 1b1) was significantly higher than the leucine-enkephalin [M+H] + The results show no detectable signal but a certain noise level. Applying a filtering material between the sample and the inlet significantly reduces the background signal in the mass spectrum of Fig. 15d2) compared to Fig. 15c2), while significantly increasing the S / N ratio of the extracted ion mobilogram of Fig. 15d1) compared to Fig. 15c1).

[0279] This patent application claims priority to European Patent Application No. 21197383.9, the contents of which are incorporated herein by reference. [Explanation of symbols]

[0280] 110 Analytes 112 Samples 114 Ionization Matrix 116 Container 118 Fine particles 120 Surface of fine particles 122 Analyte-Particulate Complex 124 Matrix: Analyte-Particulate Sample 126 Ion Mobility Spectroscopy and / or Mass Spectrometry 128 Base material surface

Claims

1. A method for determining at least one analyte (110) in a sample (112), said method comprising the following steps: a) providing a substrate having said at least one analyte (110), at least one microparticle (118), at least one ionization matrix (114), and a substrate surface (128); b) incubating said analyte (110) with said microparticles (118) having at least one microparticle surface (120), whereby said analyte is adsorbed to said microparticle surface (120) and an analyte-microparticle complex (122) is formed; c) contacting the analyte-particle complex (122) with the ionization matrix (114) to form a matrix:analyte-particle sample (124); d) providing said matrix:analyte-particulate sample (124) on said substrate surface (128); e) ionizing at least the analyte (110), wherein the ionization is mechanical ionization; f) determining said analytes (110) by ion mobility spectrometry and / or mass spectrometry (126); A method comprising:

2. said mechanical ionization is induced by a mechanical force, preferably comprising or consisting of a shear force; and / or the mechanical ionization is induced by a mechanical stimulus, preferably the mechanical stimulus being triboluminescence; and / or the mechanical ionization is not induced by evaporation or sublimation; The method of claim 1.

3. 2. The method according to claim 1, wherein the ionization in step e) is matrix-assisted ionization (MAI), preferably solid dual-support matrix-assisted ionization.

4. the microparticles (118) are magnetic particles or immunobeads, When the particles (118) are magnetic particles, the particles (118) are coated with a glass coating or a polymer coating. The method of claim 1.

5. 2. The method of claim 1, wherein the ionization matrix (114) is selected from the group consisting of salsalate, 3-nitrobenzonitrile, 2,2'-azobis(2-methylpropane), 2-nitrobenzonitrile, 5-methyl-2-nitrobenzonitrile, coumarin, methyl-2-methyl-3-nitrobenzoate, methyl-5-nitro-2-furoate, 2-bromo-2-nitropropane-1,3-diol), 3-nitrobenzaldehyde, 6-nitro-o-anisinonitrile, phthalic anhydride, or mixtures thereof.

6. Use of the method according to any one of claims 1 to 5 for determining said at least one analyte.

7. A sample element suitable for determining at least one analyte and for carrying out the method according to any one of claims 1 to 5, comprising a substrate surface (128), an ionization matrix (114) arranged on said substrate surface (128) for use in matrix-assisted ionization; - an analyte-particle complex (122) disposed on said substrate surface (128); Including, said ionization matrix (114) is selected from the group consisting of salsalate, 3-nitrobenzonitrile, 2,2'-azobis(2-methylpropane), 2-nitrobenzonitrile, 5-methyl-2-nitrobenzonitrile, coumarin, methyl-2-methyl-3-nitrobenzoate, methyl-5-nitro-2-furoate, 2-bromo-2-nitropropane-1,3-diol, 3-nitrobenzaldehyde, 6-nitro-o-anisinonitrile, phthalic anhydride, or mixtures thereof; - the ionization matrix (114) and / or the analyte-particle complex (122) are crystallized; - the particles (118) of the analyte-particle complex (122) are magnetic, A sample element, in which the analyte-particle complex (122) and the ionization matrix (114) are in contact with each other.

8. Use of the sample element of claim 7 for determining at least one analyte.

9. 6. An inlet suitable for carrying out the method according to any one of claims 1 to 5 and suitable for transporting ions to a mass spectrometer or ion mobility spectrometer (126) or to a detector of a mass spectrometer or ion mobility spectrometer with a truncated sample inlet and filter.

10. Use of the inlet according to claim 9 for determining at least one analyte.

11. A composition for vacuum ionization or inlet ionization comprising an ionization matrix (114), said ionization matrix (114) comprising or consisting of salsalate.

12. Use of the composition according to claim 11 for determining at least one analyte.

13. (A) an ionization matrix (114); (B) a solvent or further solvent; (C) fine particles (118); (D) optionally, at least one internal standard; A kit suitable for carrying out the method according to any one of claims 1 to 5, comprising:

14. Use of the kit according to claim 13 in the method according to any one of claims 1 to 5.

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