Patent Description:
In mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) is an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation. It is commonly applied for an analysis of biomolecules and various organic molecules, which tend to be fragile and fragment when ionized by more conventional ionization methods. The MALDI methodology is commonly a three-step process. Firstly, a sample is mixed with a suitable matrix material and is applied to a metal plate. Secondly, a pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material. Finally, analyte molecules are ionized by being protonated or deprotonated in a hot plume of ablated gases. Subsequently, they can be accelerated into a mass spectrometer for analysis.

A variation of this technology is the surface assisted laser desorption ionization (SALDI) process. The surface assisted laser desorption ionization is a soft laser desorption technique used for mass spectrometry analysis. In its first embodiment Koichi Tanaka used a cobalt/glycerol liquid matrix and subsequent applications included a graphite/glycerol liquid matrix as well as a solid surface of porous silicon. The porous silicon represents the first matrix-free SALDI surface analysis allowing for facile detection of intact molecular ions. Since then, a multitude of different surfaces have been reported to work as SALDI substrates with varying degrees of success. Based on the elemental composition, the majority of the SALDI substrates reported in the literature can commonly be classified into three main types: carbon-based, semiconductor-based and metallic-based.

The SALDI process using inorganic matrices for the preparations is described in several works. In Law et al. <NUM>, <NUM>, <NUM>, DOI <NUM>/s00216-<NUM>-<NUM>-<NUM>, recent advances in SALDI-MS techniques and their chemical and bioanalytical applications are described. As based on Law et al. a matrix free device may fulfill the following conditions. Firstly, a laser desorption ionization performance should be much higher than a performance of a direct laser desorption. A sanded metal or silicon surface may serve as an experimental control. Secondly, a laser fluence required to achieve laser desorption ionization should be no more than a normal operation of MALDI using conventional organic matrices. Thirdly, being a soft ionization technique, molecular ions, or quasi-molecular ions of the analyte should dominate the mass spectra. Fourthly, if fragmentation occurs, a fragmentation pattern should be both, orderly and predictable. Fifthly, a wide range of classes of compounds should be analyzed by the technique.

General main problems of organic matrices are that the sample and the matrix components need to come together in liquid solution followed by drying and therefore co-crystallization. Resulting ion species of organic matrix assisted laser desorption are protonated species [M+H]+. To the contrary, inorganic matrices mostly give metallized species e.g. [M+Na]+ which is mostly driven by only a heat transfer from matrix to the analyte and therefore there is commonly no need for a co-crystallization.

Coatings comprising amorphous diamond-like carbon materials with application in SALDI mass spectrometry are described in several works,.

In <NPL>, a simple and rapid approach to obtaining target plates for the investigation of low-molecular-weight compounds by surface-assisted laser desorption/ionization (SALDI) mass spectrometry is proposed. It consists of the vacuum sputtering of a carbon layer with a thickness of about <NUM> onto a metal surface. The resulting coatings are characterized by homogeneity, hydrophobicity, and high mechanical strength, which eliminates a possibility of mass spectrometer contamination. A comparison of the SALDI mass spectra of test compounds recorded using conventional carbon materials and carbon nanocoatings demonstrates advantages of the last named materials, such as high spectral resolution and the absence of spectral interferences at low m/z values.

In <NPL>, a nanostructured diamond-like carbon coated digital versatile disc is described for the use as a matrix-free target for laser desorption/ionization mass spectrometry. A large number of vacancies, defects, relative sp<NUM>-carbon content, and nanogrooves of DLC films support the LDI phenomenon. The observed absorptivity of DLC is in the range of <NUM>-<NUM> (nitrogen laser, <NUM>). The universal applicability is demonstrated through different analytes like amino acids, carbohydrates, lipids, peptides, and other metabolites.

<CIT> describes a use of composites or compositions of diamond/non-diamond material, e.g. diamond/non-diamond carbon material for chemical or biological analysis. Further, the use of this material in separation adherence and detection of chemical or biological samples is described. Applications of either structured substrates or mixed phase particles of this material include but are not limited to processes which involve desorption-ionization of a sample, more specifically mass spectroscopy.

<CIT> describes a target for a laser desorption/ionization mass spectrometer, comprising a substrate that is at least partially coated with a carbon-containing layer comprising a material selected from the group consisting of diamond, amorphous carbon, DLC (diamond-like carbon), graphite, nanotubes, nanowires, fullerenes and mixtures thereof.

<CIT> describes an apparatus for providing an ionized analyte for mass analysis by photon desorption comprising at least one layer for contacting an analyte, and a substrate on which said layer is deposited. Upon irradiation of said apparatus, said analyte desorbs and ionizes for analysis by mass spectrometry. The layer or layers of said apparatus comprise a continuous film, a discontinuous film or any combinations thereof.

In <NPL>, nanostructured weathering steel for matrix-free laser desorption ionization mass spectrometry and imaging of metabolites, drugs and complex glycans is described. Specifically, weathering steel has been employed for the first time to prepare sample plates for matrix-free laser desorption ionization mass spectrometry (LDI-MS) of small molecules up to a mass range of around <NUM> Da. The effective UV absorption, heat conductivity and porosity of the nanostructured inner rust layer formed during passivation determine the excellent performance in LDI-MS for a broad range of different analyte classes. The inexpensive material was evaluated in a series of relevant analytical applications ranging from the matrix-free detection of serum metabolites, lactose quantification, lipid analysis in milk to the glycoprofiling of antibodies and imaging mass spectrometry of brain tissue samples.

Despite the advantages achieved by the above-mentioned devices and methods, several technical challenges remain.

Surfaces which comprise microstructured metal/semiconductors and/or carbon-based structures commonly exhibit an inherent problem of being not very resistant against mechanical stress. If the microstructures are broken, the SALDI effect is usually diminished. Therefore, a reuse of those structures after the analysis is commonly not very practicable. Further, a process for production of such surface modifications commonly involves high technical and personal interaction. A large-scale process for the production of disposables is commonly not very practicable.

The ionization process for constructing pseudo molecular ions during ionization commonly relies on a surrounding of impurities besides the analyte. If a large excess of e.g. Na+ or K+ is present, a formation of metallized species is usually highly possible during ionization. A clean surface is commonly required to enhance the H+ (proton) adduct. This is generally necessary because the protonated species fragment in the mass spectrometric analysis much better (formation of analyte breakdown products) and can therefore serve as measurable form of the analyte for a multiple reaction monitoring (MRM) analysis. To clean a surface some mechanical stress is always applied which makes it advantageous to use a surface, which can stand mechanical stress.

Although previously developed surface coatings based on diamond like carbon (DLC) or different carbon allotropes have shown a certain energy transfer from the surface to the analytes, this process is commonly not efficient enough to result in small detection limits for the purpose to ensure a broader analyte scope and to facilitate quantitative analysis. A further development and modification of the surface is therefore necessary to fulfill the requirements of current analytical issues.

It is therefore desirable to provide a target for use in a laser desorption mass spectrometer, a use of a target, a laser desorption mass spectrometer, a continuous laser desorption mass spectrometer system, a method for preparing at least one sample for analysis in a laser desorption mass spectrometer and a method for detecting at least one analyte in a sample with a laser desorption mass spectrometer which at least partially address the above-mentioned technical challenges. Specifically, a surface with good mechanical strength shall be provided, which exhibits a good SALDI ion species generation efficiency.

This problem is addressed by a target for use in a laser desorption mass spectrometer, a use of a target, a laser desorption mass spectrometer, a continuous laser desorption mass spectrometer system, a method for preparing at least one sample for analysis in a laser desorption mass spectrometer and a method for detecting at least one analyte in a sample with a laser desorption mass spectrometer with the features of the independent claims. Advantageous embodiments which might be realized in an isolated fashion or in any arbitrary combinations are listed in the dependent claims as well as throughout the specification.

In a first aspect of the present invention, a target for use in a laser desorption mass spectrometer is disclosed. The target has at least one surface. The surface is covered at least partially with at least one layer. The layer is a hydrogen comprising, silicon-incorporated amorphous carbon (a-C:H:Si) layer. The a-C:H:Si layer comprises:.

A sum of carbon, hydrogen and silicon may be up to <NUM>%, specifically <NUM>%. However, the a-C:H:Si layer may also comprise additional elements. Thus, the sum of carbon, hydrogen and silicon may be less than <NUM> %. Specifically, the sum of carbon, hydrogen and silicon may be at least <NUM> %, specifically at least <NUM>%, specifically at least <NUM> %, specifically at least <NUM> %, specifically at least <NUM> %, specifically at least <NUM> %, specifically at least <NUM> %, specifically at least <NUM> %, specifically at least <NUM> %, specifically at least <NUM> %, specifically at least <NUM> %.

The a-C:H:Si layer may specifically be a hydrogen comprising, heteroatom modified, silicon-incorporated amorphous carbon (a-C:H:Si:X) layer. The heteroatom X may be selected from the group consisting of oxygen, nitrogen, fluorine, boron and the a-C:H:Si:X layer may further comprise:.

A sum of oxygen, nitrogen, fluorine and boron may be at least <NUM> at. %, specifically at least <NUM> at. %, specifically at least <NUM> at.

A sum of carbon, hydrogen, silicon, oxygen, nitrogen, fluorine and boron specifically may be <NUM> at. However, the a-C:H:Si:X layer may also comprise additional elements. Thus, the sum of carbon, hydrogen, silicon, oxygen, nitrogen, fluorine and boron specifically may be less than <NUM> at. Thus, the sum of carbon, hydrogen, silicon, oxygen, nitrogen, fluorine and boron may be at least <NUM> at. %, specifically at least <NUM> at. %, specifically at least <NUM> at. %, specifically at least <NUM> at. %, specifically at least <NUM> at. %, specifically at least <NUM> at. %, specifically at least <NUM> at. %, specifically at least <NUM> at. %, specifically at least <NUM> at. %, specifically at least <NUM> at. %, specifically at least <NUM> at. Also other heteroatoms may be feasible. The heteroatom may specifically be selected from the group consisting of: a metalloid, specifically germanium, specifically antimony, specifically selenium, specifically, tellurium; a post-transition metal, specifically aluminum; a transition metal, specifically titanium, specifically vanadium, specifically niobium, specifically tantalum, specifically chromium, specifically molybdenum, specifically tungsten, specifically iron, specifically cobalt, specifically copper, specifically silver; a nonmetal, specifically phosphorus, specifically sulfur, specifically chlorine, specifically bromine, specifically iodine.

The expression "at. %" may specifically refer to an indication of a percentage of atoms in a chemical substance. The percentage of atoms may be calculated by dividing a number of all atoms of a kind of element by a number of all atoms within the chemical substance. Thereafter, the result may be multiplied with <NUM>.

The term "mass spectrometer" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary analytical device which is configured for determining or measuring a mass-to-charge ratio of ions. Measurement results may specifically be presented as a mass spectrum, e.g. a plot of intensity as a function of the mass-to-charge ratio.

The term "laser desorption mass spectrometer" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary mass spectrometer based on an ionization technique using a laser. Specifically, the term may refer to a mass spectrometer which uses a medium and a laser for desorbing and ionizing a sample or parts of a sample from the medium. Specifically, the medium may absorb energy from a laser and may then transfer the energy to the sample or parts thereof. The ionization technique may also be referred to as soft ionization technique. The laser desorption mass spectrometer may specifically be configured as a surface-assisted laser desorption/ionization (SALDI) technique. The SALDI technique may comprise at least three different stages. At a first stage, a sample may be applied on a target. At a second stage, laser pulses of a laser may be applied to the target and the target may absorb laser energy and transfer the laser energy to molecules of the sample. At a third stage, desorption and ionization may occur and a potential difference may accelerate produced ions into a mass analyzer. The laser desorption mass spectrometer may specifically comprise the at least one target, at least one laser and at least one mass analyzing unit. Further details on these components will be given in further detail below. The term "laser desorption mass spectrometric detection" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a process of analyzing a sample by using a laser desorption mass spectrometer. The detection may specifically refer to an identifying of an analyte of a sample. The detection may be a qualitative and/or a quantitative detection. Further, specifically, the laser desorption mass spectrometric detection may be a laser desorption imaging mass spectrometric detection. The laser desorption imaging mass spectrometric detection may include a visualization of a spatial distribution of molecules by their molecular masses. After collecting a mass spectrum at one spot, the target may be moved to reach another region, and so on, until a region of the target is scanned. By choosing a peak in resulting spectra that corresponds to an analyte of interest, its distribution across the target may be mapped.

The term "target" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary article, device or element which is exposed or exposable to a beam, specifically a laser beam. As an example, the target may be configured as a solid target having a predefined shape, such as a flat target disc or wafer having flat target surface and e.g. having a round, oval or polygonal shape. Specifically, the target may be exposed or may be exposable to a laser beam of a laser of a mass spectrometer, specifically of a laser desorption mass spectrometer. The target may specifically be a reusable target. The term "reusable target" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary target which may be configured for being used more than one time. As will be outlined in further detail below, a preparing of last one sample for analysis in a laser desorption mass spectrometer may include an applying at least one sample to the target. After conducting at least one measurement, the target, specifically a surface of the target, may be cleaned, e.g. the sample may be removed. Thereafter, a further sample may be applied to the target and a further measurement may be conducted.

The target may specifically have a thickness of <NUM> to <NUM>, preferably <NUM> to <NUM>. Further, the target may have a thickness of less than <NUM>, preferably of less than <NUM>.

The target may specifically comprise at least one substrate. The term "substrate" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary flat element, such as a flat element having a lateral extension exceeding its thickness by at least a factor of <NUM>, at least a factor of <NUM>, at least a factor of <NUM>, or even at least a factor of <NUM> or more. The substrate may have an arbitrary shape. Specifically, the substrate may have a round, oval or polygonal shape, such as a rectangular or round shape. Further, as will be described in further detail below, the substrate may have a strip-shape. However, also other shapes may be feasible.

The substrate may be made of at least one electrically conductive material or may comprise at least one layer of the at least one electrically conductive material. The electrically conductive material may specifically have a sheet resistance which is smaller than or equal to <NUM>Ω/sq, preferably smaller than or equal to <NUM>Ω/sq. Thus, exemplarily, the substrate may be made of the least one electrically conductive material and the a-C:H:Si layer may be deposited on the surface of the substrate.

Further, exemplarily, the substrate may be made of at least one electrically insulating material or of the at least one electrically conductive material and at least one layer of at least one electrically conductive material may be deposited on the substrate. Thereby, the a-C:H:Si layer may be deposited on a surface of the layer of the at least one electrically conductive material. Thus, the a-C:H:Si layer may form an outermost layer of the target. The layer of the at least one electrically conductive material may form an intermediate layer of the target. Further, the target may comprise a layer structure having the at least one a-C:H:Si layer and the at least one layer of the at least one electrically conductive material. Specifically, the layer structure may comprise a plurality of the layers of the at least one electrically conductive material. The plurality of the layers of the at least one electrically conductive material may form intermediate layers of the target. Further, the layer structure may comprise one or more layers of at least one electrically insulating material. The layer of the at least one electrically conductive material may also be referred to as electrically conductive contact layer. The surface of the substrate or the surface of the layer of the at least one electrically conductive material on which the a-C:H:Si layer may be deposited may enable a good bonding of the a-C:H:Si layer to the surface of the substrate or to the surface of the layer of the at least one electrically conductive material. Exemplarily, the substrate may be made of glass and the electrically conductive material may be indium tin oxide (ITO). Thus, the substrate being made of glass may comprise at least one ITO layer and the a-C:H:Si layer may be deposited on a surface of the ITO layer. Further, a pretreatment, specifically a plasma treatment, of the surface of the substrate and/or of the intermediate layer may be conducted before the a-C:H:Si layer is deposited, specifically in order to increase an adhesion of the a-C:H:Si layer on the substrate.

Specifically, the substrate may at least partially be made of at least one material or may comprise at least one material which is selected from the group consisting of: glass; steel, specifically stainless steel; aluminum; silicon; germanium titanium; copper; cobalt; chromium; molybdenum; nickel; tungsten; tantalum; graphite; a polymeric material, specifically polyethylene, specifically polypropylene, specifically polycarbonate, specifically polystyrene, specifically polyacrylate. Further, the polymeric material may be a conductive polymeric material, specifically polyaniline, specifically poly(<NUM>,<NUM>-ethylenedioxythiophene) polystyrene sulfonate, specifically polypyrrole, specifically polythiophene. Also other materials may be feasible such as alloys which comprise at least one of the metals as outlined above and at least one further element.

The term "surface" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an entirety of areas which delimit an arbitrary body from the outside. Thus, the body may have a plurality of surfaces. The term "layer" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an amount of material which is deposited on a surface of an arbitrary element. The layer may specifically be a coating. The layer may cover the object completely or may only cover a part or parts of the object. The layer may specifically have a lateral extension exceeding its thickness by at least a factor of <NUM>, at least a factor of <NUM>, at least a factor of <NUM>, or even at least a factor of <NUM> or more. Specifically, the a-C:H:Si layer may have a thickness of: <NUM> to <NUM>, preferably <NUM> to <NUM>. However, also other dimensions may be feasible.

The term "being at least partially covered" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a property of an arbitrary element of being fully or partially covered with something. Specifically, a surface of the arbitrary element may be fully or partially covered with something. In case the surface is partially covered with something, the covered surface may also be referred to as surface section. As further used herein, the term "surface section" may refer to a part, specifically to a distinct part, of a surface. Exemplarily, the term surface section may refer to at least <NUM> %, at least <NUM> %, at least <NUM><NUM>%, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> %, at least <NUM> % of the surface. However, other embodiments may be feasible. The layer may specifically form a continuous layer covering a surface section or even a whole surface of the target, specifically of the substrate of the target.

The term "hydrogen comprising, silicon-incorporated amorphous carbon (a-C:H:Si) layer" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an amorphous carbon layer, which comprises hydrogen and silicon. The hydrogen and/or the silicon, specifically, may be embedded or dispersed in the carbon layer without, however, being bound to the carbon by covalent chemical binding. The a-C:H:Si layer may specifically be an amorphous silicon-incorporated diamond-like carbon layer. The amorphous silicon-incorporated diamond-like carbon may have structural, mechanical, electrical, optical, chemical, and/or acoustic properties similar to those of diamond. Specifically, the amorphous silicon-incorporated diamond-like carbon may be a metastable form of amorphous carbon comprising sp<NUM> hybridized carbon atoms. More specifically, in the amorphous silicon-incorporated diamond-like carbon, the carbon may exist in three hybridizations, sp<NUM>, sp<NUM>, and sp<NUM>. The physical properties of the amorphous silicon-incorporated diamond-like carbon as described above may derive from its mixture of carbon bonds. Specifically, the sp<NUM> hybridization, as in diamond, may have a strong σ bond, which may lead to a high mechanical hardness and chemical inertness. The sp<NUM> hybridization, as in graphite, may have a strong intralayer σ bond and a weak van der Waals bond between its layers. The physical properties may specifically depend on a ratio of sp<NUM> to sp<NUM> bonds.

The term "hydrogen comprising, silicon-incorporated, heteroatom modified, amorphous carbon (a-C:H:Si:X) layer" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an amorphous carbon layer, which comprises hydrogen and silicon and which further, besides hydrogen and silicon, also comprises one or more kinds of heteroatoms. The hydrogen, the silicon, as well as the heteroatoms, specifically, may be embedded or dispersed in the carbon layer without, however, being bound to the carbon by covalent chemical binding. The a-C:H:Si:X layer may specifically be an amorphous heteroatom modified silicon-incorporated diamond-like carbon layer. The term "heteroatom" may refer to any atom that differs from carbon or hydrogen. As outlined above, the heteroatom is selected from the group consisting of: oxygen, nitrogen, fluorine, boron. However, also other heteroatoms may be feasible. The heteroatom may specifically be selected from the group consisting of: a metalloid, specifically germanium, specifically antimony, specifically selenium, specifically, tellurium; a post-transition metal, specifically aluminum; a transition metal, specifically titanium, specifically vanadium, specifically niobium, specifically tantalum, specifically chromium, specifically molybdenum, specifically tungsten, specifically iron, specifically cobalt, specifically copper, specifically silver; a nonmetal, specifically phosphorus, specifically sulfur, specifically chlorine, specifically bromine, specifically iodine.

A substrate having a surface which is covered at least partially with at least one layer comprising hydrogen comprising, silicon-incorporated amorphous carbon (a-C:H:Si) is commercially available from e.g. CeWOTec GmbH (Chemnitzer Werkstoff- und Oberflächentechnik, Chemnitz, Germany). CeWOTec GmbH provides a "technical data sheet for our XLC-PURA coating" including the information according to the following Table <NUM>. The technical data sheet originates from January <NUM>, <NUM> and the information is provided in the German language. The following table comprises a translation of the text into the English language.

As further outlined above, an elemental composition of the a-C:H:Si:X layer can be specified in <NUM> at. % to <NUM> at. % of carbon; <NUM> at. % to <NUM> at. % of hydrogen; <NUM> at. % to <NUM> at. % of silicon; up to <NUM> at. % of oxygen; up to <NUM> at. % of nitrogen; up to <NUM> at. % of boron; and up to <NUM> at. % of fluorine. As outlined above, a sum of oxygen, nitrogen, fluorine and boron is at least <NUM> at. Thus, the a-C:H:Si:X layer may comprise at least <NUM> at. % of one of the heteroatoms or some or all of the heteroatoms. Specifically, the sum of oxygen, nitrogen, fluorine and boron may be at least <NUM> at. %, preferably at least <NUM> at.

As outlined above, the a-C:H:Si layer comprises up to <NUM> at. % of oxygen. Specifically, a percentage of oxygen may vary within the a-C:H:Si layer. The closer a region of the a-C:H:Si layer is to the surface of the target, the lower may be the percentage of oxygen. Thus, within the a-C:H:Si layer, the percentage of oxygen may continuously or discontinuously decrease in a direction perpendicular to the surface of the target, i.e. the surface which is exposed to the laser radiation. In a bulk region of the a-C:H:Si layer the percentage of oxygen may be less than <NUM> at. %, specifically less than <NUM> at. %, more specifically less than <NUM> at.

As outlined above, the a-C:H:Si layer is an amorphous layer. The term "amorphous layer" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary layer being made of at least one material which lacks a long-range order. Thus, atoms of the material may form an irregular pattern and may only have a short-range order. However, the material may also have an internal structure made of interconnected structural blocks. The interconnected structural blocks may correspond to crystalline phase of the material. Specifically, the a-C:H:Si layer may be deposited on the surface of the target by a plasma-supported surface coating process. Specifically, the plasma-supported surface coating process may be a plasma assisted chemical vapor deposition process (PA-CVD). The PA-CVD may be performed by a process temperature of <NUM> to <NUM>. The term "plasma assisted chemical vapor deposition" may generally refer to a deposition method wherein a substrate is exposed to one or more volatile precursors which react and/or decompose on a surface of the substrate to produce a desired deposit. However, also other deposition methods may be feasible.

Specifically, the a-C:H:Si layer may form an outermost layer of the target which may specifically face an outer environment of the target. Further, the a-C:H:Si layer may form a continuous layer on the surface of the target. Further, a structural shape of a surface of the a-C:H:Si layer may resemble a shape of the surface of the substrate. Specifically, on a smooth substrate, the a-C:H:Si layer may also form a continuous and smooth surface with only low numbers of defect positions.

The a-C:H:Si layer may have a microhardness of <NUM> GPa to <NUM> GPa, preferably of <NUM> GPa to <NUM> GPa, most preferably of <NUM> GPa to <NUM> GPa. However, also other values may be feasible. The term "microhardness", also be referred to as "indention hardness", as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a hardness of a material exposed to low applied loads. During testing of the microhardness, a diamond indenter of specific geometry may be impressed into a surface of a test specimen using a known applied force, which may also be referred to as load, such as of <NUM> N. Thereby, indentations of about <NUM> may be produced. Specifically, for the purpose of determining the microhardness of the material, a Vickers hardness test or a Knoop hardness test may be applied. However, also other methods may be feasible.

Specifically, the a-C:H:Si layer may have a friction coefficient of <NUM> to <NUM>, preferably of <NUM> to <NUM>, most preferably of <NUM> to <NUM>. However, also other values may be feasible. The term "friction coefficient" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a dimensionless value which describes a ratio of a force of friction between two bodies and a force pressing them together. Specifically, the term may refer to a static friction and may specifically be determined by dry sliding against steel or carbide at a temperature of less than <NUM>.

In a further aspect of the present invention, a use of a target as described above or as will further be described below in more detail for a laser desorption mass spectrometric detection of at least one analyte in a sample is disclosed.

The term "analyte" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary chemical or biological substance or species, such as a molecule or a chemical compound, to be detected and/or measured. Specifically, a presence, an absence, a concentration and/or an amount of the analyte in a sample may be detected or measured. Specifically, the analyte may be a biological molecule or macromolecule. The analyte may be selected from the group consisting of: a steroid; specifically a ketosteroid, specifically a secosteroid; a therapeutically active substance; a detergent; a glycoside; a peptide; a protein; a dye; an ion; a nucleic acid; an amino acid; a metabolite; a hormone; a fatty acid; a lipid; a carbohydrate. Further, the analyte may be a molecule characteristic of a certain modification of another molecule or a substance that has been internalized by an organism or a metabolite of such a substance or a combination thereof. However, also different kinds of analytes may be feasible. The steroid may be selected from the group consisting of: progesterone, testosterone, estradiol, androstenedione, cortisol, cortisone, <NUM>-deoxycortisol. However, also other steroids may be feasible. The therapeutically active substance may be selected from the group consisting of: digitoxin, mycophenolic acid, theophylline, lidocaine, digoxin, voriconazole, <NUM>-hydoxyalprazolam. However, also other therapeutically active substances may be feasible. The analyte may comprise permanently positive charged molecules or permanently negative charged molecules. Further, the analyte may have an isotope pattern. The analyte may have a molar mass from <NUM> Da to <NUM> Da, preferably from <NUM> Da to <NUM> Da, most preferably from <NUM> Da to <NUM> Da. Specifically, Lithium may be desorbed having a molar mass of <NUM> Da.

The term "sample" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary sample such as a biological sample, also called test sample, a quality control sample, an internal standard sample. The sample may comprise one or more analytes of interest. The sample may specifically be a liquid sample, in particular a liquid sample comprising at least one biological material. Further, the analyte may be provided in a sample, specifically in a tissue sample or in a processed serum sample. The tissue sample may specifically have a slice thickness of less than <NUM>. For example, the sample may be selected from the group consisting of: a physiological fluid, including blood, serum, plasma, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cells or the like. The sample may be used directly as obtained from the respective source or may be subject of a pretreatment and/or sample preparation workflow. For example, the sample may be pretreated by adding an internal standard and/or by being diluted with another solution and/or by being mixed with reagents or the like. The quality control sample may be a sample that mimics the test sample, and that comprises known values of one or more quality control substances. The quality control substance may be identical to the analyte of interest or may be an analyte which generates by reaction or derivatization an analyte identical to the analyte of interest and/or may be an analyte of which the concentration is known and/or may be a substance which mimics the analyte of interest or that can be otherwise correlated to a certain analyte of interest. The internal standard sample may be a sample comprising at least one internal standard substance with a known concentration.

In a further aspect of the present invention, a laser desorption mass spectrometer is disclosed. The laser desorption mass spectrometer comprises:.

The term "laser" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary device which is configured for emitting light through a process of optical amplification based on a stimulated emission of electromagnetic radiation. Specifically, the laser may be a pulsed laser. An energy of a pulse may be in a range of less than <NUM>µJ, specifically in a range of less than <NUM>µJ. Further, the laser may have a laser repetition rate in the range of <NUM> to <NUM>, specifically of <NUM> to <NUM>. For example, the laser may be configured for generating a laser beam in the UV spectral range. Specifically, a laser wavelength may be in the range between <NUM> to <NUM>. More specifically, the laser may be a neodymium-doped yttrium aluminum garnet laser (Nd:YAG laser) having a wavelength of <NUM>.

As outlined above, the laser is configured for providing energy to the target such that at least one ion of at least one analyte is generated. Thus, the target may be irradiated with at least one laser beam. The term "irradiating" as used herein, is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a process of exposing an object, specifically a surface of an object, to laser light. In particular, a first portion of the object, specifically a first portion of the surface of the object, may be irradiated with at least one laser beam and a second portion of the object, specifically a second portion of the surface of the object, may preferably not be irradiated with the at least one laser beam. Specifically, the target may absorb laser energy and transfer the laser energy to molecules of the sample and desorption and ionization may occur.

As outlined above, the laser desorption mass spectrometer may further comprise at least one mass analyzing unit. The mass analyzing unit may be configured for detecting or determining at least one mass-to-charge ratio of the at least one ion emitted from the target. The term "mass analyzing unit" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an apparatus configured for detecting incoming ions. The mass analyzing unit may be configured for detecting charged particles. The mass analyzing unit may be or may comprise at least one electron multiplier. The mass analyzing unit may be configured for determining at least one mass spectrum of the detected ions. As used herein, the term "mass spectrum" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a two-dimensional representation of signal intensity vs the mass-to-charge ratio m/z, wherein the signal intensity corresponds to abundance of the respective ion. The mass-to-charge ratio may refer to a reciprocal of a specific charge. The mass spectrum may be a pixelated image. For determining resulting intensities of pixels of the mass spectrum, signals detected with the mass analyzing unit within a certain m/z range may be integrated. The mass analyzing unit may comprise at least one evaluation device. The analyte in the sample may be identified by the at least one evaluation device. Specifically, the evaluation device may be configured for correlating known masses to the identified masses or through a characteristic fragmentation pattern.

Further, the laser desorption mass spectrometer may comprise at least one vacuum pump. The term "vacuum pump" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary device which is configured for drawing gas molecules from a sealed volume in order to leave behind a partial vacuum and/or for generating an underpressure, e.g. a pressure below normal pressure. The vacuum pump may be configured for generating a relative vacuum within a capacity such as a sealed volume. The underpressure may be generated by allowing or forcing gas to flow from the sealed volume to the ambient atmosphere and/or to another room or chamber. Specifically, the vacuum pump may be configured to provide a pressure gradient between the sealed volume and the ambient atmosphere, wherein the value of the pressure within the sealed volume may be smaller than the value of the pressure of the ambient atmosphere. The vacuum pump may be configured for generating an underpressure below <NUM> mbar, such as to a pressure of below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar or even less. Specifically, the vacuum pump may be configured for generating an underpressure of <NUM> mbar to <NUM> mbar, specifically of <NUM> mbar to <NUM> mbar.

The laser desorption mass spectrometer may comprise at least one chamber. The vacuum pump may be configured for generating the underpressure within the chamber. The target may be received within the chamber. Further, the mass analyzing unit may be received within the chamber. Moreover, the laser desorption mass spectrometer may comprise at least one field generator. The field generator may be configured for generating an electric and/or magnetic field. The electric and/or magnetic field may be configured for drawing away generated ions from the target.

Further, the laser desorption mass spectrometer may comprise at least one ion-mobility spectrometry device having a least one ion-mobility spectrometry cell. The term "ion-mobility spectrometry device" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary analytical technique which is configured to separate and identify ionized molecules in a gas phase based on their mobility in a carrier buffer gas. The ion-mobility spectrometry device may specifically be coupled to the laser desorption mass spectrometer, specifically in order to achieve a multi-dimensional separation. The ion-mobility spectrometry device may be configured for detecting at least one drift time of the ion through the ion-mobility spectrometry cell.

In a further aspect of the present invention, a continuous laser desorption mass spectrometer system comprising at least one laser desorption mass spectrometer as described above or as will further be described below in more detail is disclosed. The target is provided as a material strip or as a stack of platelets.

The term "continuous laser desorption mass spectrometer system" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary desorption mass spectrometer system which is configured for measuring a plurality of samples successively, preferably without cessation between the measurements. The continuous laser desorption mass spectrometer system may also be referred to just as desorption mass spectrometer system.

As outlined above, the target is provided as a material strip or as a stack of platelets. In case the target is provided as a material strip, the target may specifically be provided in a coiled manner. The material strip may be configured to be unwound before one or more samples are applied to the material strip, specifically to a surface of the material strip. The material strip may be configured to pass through the continuous laser desorption mass spectrometer system. Specifically, the material strip may be configured to pass through different stations of the continuous laser desorption mass spectrometer system. The different stations may comprise the laser desorption mass spectrometer and may further comprise one or more liquid handling systems and one or more vacuum zones. Further details on the liquid handling system and the vacuum zone may be provided below in more detail. The material strip may specifically be made of steel or aluminum. However, also other materials may be feasible. The material strip may have a width of <NUM> to <NUM>, preferably of <NUM> to <NUM>. Further, the material strip may have a thickness of <NUM> to <NUM>, preferably of <NUM> to <NUM>. Further, the material strip may have a length of <NUM> to <NUM>. However, also other dimensions may be feasible. Specifically, the length of the material strip may not be limited. Specifically, the material strip may be manufactured by performing a coating of the substrate during a winding process. Thus, exemplarily, the a-C:H:Si layer may be formed on the surface of the substrate during the winding process. This manufacturing process may exemplarily also be referred to as roll to roll PA-CVD coating process.

Further, as outlined above, the target is provided as a stack of platelets. The platelets may specifically have a rectangular shape such as a square shape. However, also other shapes may be feasible such as a round shape. The platelets may specifically have a thickness of <NUM> to <NUM>, preferably of <NUM> to <NUM>. Further, the platelets may have a width in the range of <NUM> to <NUM>, preferably of <NUM> to <NUM>, most preferably of <NUM> to <NUM>. Further, the platelets may have a length in the range of <NUM> to <NUM>, preferably of <NUM> to <NUM>, most preferably of <NUM> to <NUM>.

The platelets may be provided as being stacked on top of each other. Specifically, the continuous laser desorption mass spectrometer system may comprise at least one platelet holder configured for receiving the stack of platelets. The platelet holder may be configured for releasing the platelets successively. Specifically, the continuous laser desorption mass spectrometer system may comprise at least one conveyor belt. The platelet holder may be configured for releasing the platelets successively on the conveyor belt. The conveyor belt may be configured for successively passing the platelets through the continuous laser desorption mass spectrometer system. Specifically, the conveyor belt may be configured for successively passing the platelets through the different stations of the continuous laser desorption mass spectrometer system.

Moreover, the continuous laser desorption mass spectrometer system may comprise the at least one liquid handling system. The liquid handling system may be configured for applying at least one sample having at least one analyte on the target, specifically on the material strip or on one of the platelets. The term "liquid handling system" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary device which is configured for applying liquid, specifically a defined or desired amount of liquid to another object. The amount of liquid may be adjustable. The liquid handling system may specifically comprise one or more pipetting units. The pipetting unit may comprise at least one chamber being configured for holding or receiving at least one liquid. The pipetting unit may be configured for creating a partial vacuum above the chamber and for selectively releasing the partial vacuum to draw up and dispense the liquid. Further, additionally or alternatively, the liquid handling system may comprise at least one acoustic droplet ejection unit. The acoustic droplet ejection unit may be configured for using a pulse of ultrasound to move volumes of fluids without any physical contact. However, also other embodiments may be feasible.

Further, the continuous laser desorption mass spectrometer system may comprise at least one vacuum system. The continuous laser desorption mass spectrometer system may be configured for passing the material strip or the platelets through the vacuum system. The term "vacuum system" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to an arbitrary device which is configured to create a vacuum, e.g. a region having a gaseous pressure which is below normal pressure, in a defined space such as a chamber. For this purpose, the vacuum system may comprise at least one vacuum pump. For further details on the vacuum pump, reference can be made to the description of the vacuum pump above. Specifically, the vacuum system may comprise at least one vacuum zone, preferably at least two vacuum zones. The term "vacuum zone" may refer to a defined space such as a chamber having a gaseous pressure which is below normal pressure. The at least two vacuum zones may be arranged successively. The material strip or the stack of platelets may be configured for passing the vacuum system, specifically one or more of the vacuum zones before passing the laser desorption mass spectrometer. The vacuum zone may be configured for providing an underpressure below <NUM> mbar, such as to a pressure of below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar, below <NUM> mbar or even less. Specifically, the at least two vacuum zones may be configured for providing underpressures which differ from one each other. Also other parameters may be feasible. The vacuum system, specifically the vacuum zones, may be configured for drying the sample on the target.

In a further aspect of the present invention, a kit is disclosed.

The term "kit" as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to a set of several items such as chemicals which are utilized for conducting a desired method, specifically for a sample preparation method and/or a sample analysis method. Specifically, the items may be provided in a housing, e.g. a packaging.

The kit comprises: (i) at least one target as described above or as will further be described below in more detail; and (ii) at least one internal standard.

As used herein, the term "internal standard" may refer to an analyte which is present with a defined concentration in a sample. Thus, specifically, the concentration of the internal standard may be known; it may be, however, also envisaged that the concentration of the standard is unknown, but is the same for at least the sample of interest and at least one calibration sample; in such case, specifically, the concentration of the internal standard may be the same for all samples analyzed. The internal standard may specifically be structurally similar or identical to the analyte. In particular, the internal standard may be an isotope-labelled molecule, specifically an isotope-labelled version of the analyte, e.g. a <NUM>H (deuterated), <NUM>N, and/or <NUM>C-labelled derivative. The internal standard sample may be a sample comprising at least one internal standard substance with a known, e.g. pre-determined, concentration.

The kit may optionally comprise further elements. The further elements may be selected from the group consisting of: an auxiliary reagent, specifically a derivatization reagent; a bead suspension for a purification step.

In a further aspect of the present invention, a method for preparing at least one sample for analysis in a laser desorption mass spectrometer is disclosed.

The method comprises the following steps which specifically may be performed in the given order. It shall be noted, however, that a different order is also possible. Further, it is also possible to perform one or more of the method steps once or repeatedly. Further, it is possible to perform two or more of the method steps simultaneously or in a timely overlapping fashion. The method may comprise further method steps which are not listed.

In step ii. the sample may be applied by a liquid handling system, preferably a pipetting unit, for applying the sample to the target. For further details on the liquid handling system reference may be made to the description above.

Further, the method may comprise the following step:
iii. drying the sample.

The step iii. may specifically be conducted after step ii. The step iii. may comprise a naturally drying of the sample on the target, e.g. a drying in the open air. Further, additionally or alternatively, the step iii. may comprise a drying of the sample via at least one vacuum system. For further details on the vacuum system, reference may be made to the description above.

Moreover, the method may additionally comprise at least one cleaning step. During the cleaning step, the a-C:H:Si layer may be cleaned. The cleaning step may be conducted before step ii. is conducted.

Specifically, the cleaning step may refer to an initial cleaning after a production process or manufacturing process of the target. Further, specifically, the cleaning step may be conducted after the a-C:H:Si layer is deposited on the surface of the target such as by a plasma supported surface coating process. Thus, the cleaning step may comprise a removal of impurities caused by the deposition process of the a-C:H:Si layer. Further, the cleaning step may comprise a removal of contamination such as with dust and dirt caused during storing of the target. The initial cleaning after the production process may increase a visibility of protonated pseudo molecular ions. However, the initial cleaning after the production process may only be optional. The target having the a-C:H:Si layer may also be suitable for the laser desorption mass spectrometric detection without conducting the initial cleaning after the production process.

Further, the cleaning step may refer to a cleaning after a previous application of the target. Thus, the cleaning step may comprise a removal of a sample which was previously applied to the target. Further, the cleaning step may comprise a removal of contamination such as with dust and dirt caused during storing of the target. Thus, the target may also be referred to as reusable target. For further details on the reusable target, reference may be made to the description above.

The cleaning step may specifically comprise a rinsing or sonication of the a-C:H:Si layer with at least one solvent. Specifically, the solvent may be deionized water. Further, the solvent may be an organic solvent, specifically an organic solvent mixture, specifically an organic solvent mixture with deionized water. The organic solvent may selected from the group consisting of: tetrahydrofuran, acetonitrile, methanol, ethanol. However, also other organic solvents may be feasible such as other hydrocarbons or alcohols.

In a further aspect of the present invention, a method for detecting at least one analyte in a sample with a laser desorption mass spectrometer is disclosed.

The methods and devices according to the present invention provide a large number of advantages over known methods and devices.

The presented invention presents an utilization of a plasma assisted surface modification of amorphous carbon (a-C:H:Si:X, wherein X = O, N, F and/or B). An elemental composition of the plasma assisted surface modification of amorphous carbon may be specified in ranges of <NUM> at. % to <NUM> at. % of carbon, <NUM> at. % to <NUM> at. % of hydrogen, <NUM> at. % to <NUM> at. % of silicon, up to <NUM> at. % of oxygen, up to <NUM> at. % of nitrogen, up to <NUM> at. % of boron and up to <NUM> at. % of fluorine. This may build up a surface with optimum mechanical strength and which may exhibit a very good SALDI ion species generation efficiency compared to non-functionalized substrates or different surface plasma process modifications. The substrate which may specifically be made of stainless steel or glass may specifically be modified via a plasma supported surface coating process. The gray steel may become a brownish/gold like color and may also become slightly iridescent. The target may specifically have a microhardness of the surface of <NUM> GPa to <NUM> GPa. The target may specifically have a friction coefficient of <NUM> to <NUM>. To use the target the only sample-processing step may be pipetting and air-drying to gain a sufficient MS signal after laser irradiation. To clean the plate initially after the production process may enable or improve a visibility of protonated pseudo molecular ions.

Summarizing and without excluding further possible embodiments, the following embodiments may be envisaged:
In an embodiment, a target for use in a laser desorption mass spectrometer is dislosed, wherein the target has at least one surface, wherein the surface is covered at least partially with at least one layer, wherein the layer is a hydrogen comprising, silicon-incorporated amorphous carbon (a-C:H:Si) layer, wherein the a-C:H:Si layer comprises:.

In an embodiment, the a-C:H:Si layer is a hydrogen comprising, heteroatom modified, silicon-incorporated amorphous carbon (a-C:H:Si:X) layer, wherein the heteroatom X is selected from the group consisting of oxygen, nitrogen, fluorine, boron, wherein the a-C:H:Si:X layer further comprises:.

wherein a sum of oxygen, nitrogen, fluorine and boron is at least <NUM> at.

In an embodiment, the a-C:H:Si layer is deposited on the surface of the target by a plasma supported surface coating process.

In an embodiment, the a-C:H:Si layer has a micro hardness of <NUM> GPa to <NUM> GPa, preferably of <NUM> GPa to <NUM> GPa, most preferably of <NUM> GPa to <NUM> GPa.

In an embodiment, the a-C:H:Si layer has a friction coefficient of <NUM> to <NUM>, preferably of <NUM> to <NUM>, most preferably of <NUM> to <NUM>.

In an embodiment, the target comprises at least one substrate.

In an embodiment, the substrate is made of at least one material which is selected from the group consisting of: glass; steel, specifically stainless steel; aluminum; silicon; germanium; titanium; copper; cobalt; chromium; molybdenum, nickel; tungsten; tantalum; graphite; a polymeric material, specifically polyethylene, specifically polypropylene, specifically polycarbonate, specifically polystyrene, specifically polyacrylate, specifically polyaniline, specifically poly(<NUM>,<NUM>-ethylenedioxythiophene) polystyrene sulfonate, specifically polypyrrole, specifically polythiophene.

In an embodiment, the substrate is made of at least one electrically conductive material or wherein the substrate is made of at least one electrically insulating material and at least one layer of at least one electrically conductive material is deposited on the substrate. Embodiment <NUM>: The target according to any one of the preceding embodiments, wherein the target is a reusable target.

In an embodiment, the a-C:H:Si layer has a thickness of: <NUM> to <NUM>, preferably <NUM> to <NUM>.

In an embodiment, a use of a target according to the present invention for a laser desorption mass spectrometric detection of at least one analyte in a sample is disclosed.

In an embodiment, the analyte is selected from the group consisting of: a steroid; specifically a ketosteroid, specifically a secosteroid; a therapeutically active substance; a detergent; a glycoside; a peptide; a protein; a dye; an ion; a nucleic acid; an amino acid; a metabolite; a hormone; a fatty acid; a lipid; a carbohydrate.

In an embodiment, the analyte has a molar mass from <NUM> Da to <NUM> Da, preferably from <NUM> Da to <NUM> Da.

In an embodiment, the analyte comprises permanently positive charged molecules or permanently negative charged molecules.

In an embodiment, the analyte has an isotope pattern.

In an embodiment, the analyte is provided in a sample, wherein the sample is selected from the group consisting of: a physiological fluid, including blood, serum, plasma, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid, tissue or cells.

In an embodiment, the laser desorption mass spectrometric detection is a laser desorption imaging mass spectrometric detection.

In an embodiment, a laser desorption mass spectrometer is disclosed comprising:.

In an embodiment, the laser desorption mass spectrometer further comprises:
d) at least one vacuum pump.

In an embodiment, the vacuum pump is configured for generating an underpressure of <NUM> mbar to <NUM> mbar, specifically of <NUM> mbar to <NUM> mbar.

In an embodiment, the laser wavelength is in the range between <NUM> to <NUM>.

In an embodiment, the laser desorption mass spectrometer further comprises at least one ion-mobility spectrometry device having a least one ion-mobility spectrometry cell, wherein the ion-mobility spectrometry device is configured for detecting at least one drift time of the ion through the ion-mobility spectrometry cell.

In an embodiment, a continuous laser desorption mass spectrometer system is disclosed, comprising at least one laser desorption mass spectrometer according to the present invention, wherein the target is provided as a material strip or as a stack of platelets.

In an embodiment, the continuous laser desorption mass spectrometer system further comprises at least one liquid handling system, preferably at least one pipetting unit, wherein the liquid handling system is configured for applying at least one sample having at least one analyte on the target.

In an embodiment, the continuous laser desorption mass spectrometer system further comprises at least one vacuum system, wherein the continuous laser desorption mass spectrometer system is configured for passing the material strip or the platelets through the vacuum system.

In an embodiment, the vacuum system comprises at least one vacuum zone, preferably at least two vacuum zones.

In an embodiment, a kit is disclosed, comprising (i) at least one target according to any one of the present invention; and (ii) at least one internal standard.

In an embodiment, a method for preparing at least one sample for analysis in a laser desorption mass spectrometer is disclosed, wherein the method comprises:.

In an embodiment, the method further comprises the following step:
iii. drying the sample.

In an embodiment, step ii. comprises a liquid handling system, preferably a pipetting unit, for applying the sample to the target.

In an embodiment, the method additionally comprises at least one cleaning step, wherein during the cleaning step, the a-C:H:Si layer is cleaned, wherein the cleaning step is conducted before step ii. is conducted.

In an embodiment, the cleaning step comprises a rinsing or sonication of the a-C:H:Si layer with at least one solvent.

In an embodiment, the solvent is deionized water.

In an embodiment, the solvent is an organic solvent, specifically an organic solvent mixture, specifically an organic solvent mixture with deionized water.

In an embodiment, the organic solvent is selected from the group consisting of: tetrahydrofuran, acetonitrile, methanol, ethanol.

In an embodiment, a method for detecting at least one analyte in a sample with a laser desorption mass spectrometer is disclosed, wherein the method comprises:.

The scope of the invention is not restricted by the preferred embodiments, but is defined by the appended claims.

<FIG> show exemplary embodiments of a target <NUM> according to the present invention.

As illustrated in <FIG>, the target <NUM> has at least one surface <NUM>. The surface <NUM> is covered at least partially with at least one layer <NUM>. The layer <NUM> is a hydrogen comprising, silicon-incorporated amorphous carbon (a-C:H:Si) <NUM>. Specifically, the a-C:H:Si layer <NUM> may have a thickness t of: <NUM> to <NUM>. The target <NUM> may specifically comprise at least one substrate <NUM>. The substrate <NUM> may exemplarily be made of steel. The surface <NUM> may be a surface of the substrate <NUM>.

<FIG> shows a further embodiment of the target <NUM>. The substrate may comprise at least one layer <NUM> of at least one electrically conductive material. The layer <NUM> of the at least one electrically conductive material may also be referred to as electrically conductive contact layer <NUM>. Thereby, the a-C:H:Si layer <NUM> may be deposited on a surface <NUM> of the layer <NUM> of the at least one electrically conductive material. The layer <NUM> of the at least one electrically conductive material may form an intermediate layer and the a-C:H:Si layer <NUM> may be an outermost layer.

<FIG> shows an exemplary embodiment of a laser desorption mass spectrometer <NUM> according to the present invention.

The laser desorption mass spectrometer <NUM> comprises at least one target <NUM>. The target <NUM> may correspond at least partially to the target <NUM> as depicted in <FIG>. Thus, reference to the description of <FIG> above is made. Further, the laser desorption mass spectrometer <NUM> comprises at least one laser <NUM>. The laser <NUM> is configured for providing energy to the target <NUM> such that at least one ion of at least one analyte is generated. Further, the laser desorption mass spectrometer <NUM> may comprise at least one mass analyzing unit <NUM>. The mass analyzing unit <NUM> may be configured for detecting at least one mass-to-charge ratio of the at least one ion emitted from the target <NUM>.

The laser desorption mass spectrometer <NUM> may comprise at least one chamber <NUM>. Further, the laser desorption mass spectrometer <NUM> may comprise at least one vacuum pump <NUM>. The vacuum pump <NUM> may be configured for generating an underpressure within the chamber <NUM>. The underpressure may be generated by allowing or forcing gas to flow from the chamber to the ambient atmosphere which is schematically depicted with arrow <NUM>.

The vacuum pump <NUM> may specifically be configured for generating an underpressure below <NUM> mbar.

The laser <NUM> may specifically be a pulsed laser <NUM>. The laser <NUM> may be configured for generating a laser beam in the UV spectral range. The laser <NUM> may be arranged relative to the target <NUM> such that the laser beam, which is schematically depicted with arrow <NUM>, hits the target <NUM> at an angle of <NUM>° to <NUM>°, preferably of <NUM>° to <NUM>°. Specifically, the target <NUM> may absorb laser energy and transfer the laser energy to molecules of the sample and desorption and ionization may occur.

Moreover, the laser desorption mass spectrometer <NUM> may comprise at least one mass separating module <NUM>. A setup of the mass separating module <NUM> may be dependent on an applied mass spectrometry technique.

The mass analyzing unit <NUM> having read-out electronics <NUM> may be received within the chamber <NUM>. Further, the mass analyzing unit <NUM> may be arranged at a certain distance to the target <NUM>. The mass analyzing unit <NUM> is configured for detecting or determining at least one mass-to-charge ratio of the at least one ion emitted from the target <NUM>.

<FIG> show two different exemplary embodiments of a continuous laser desorption mass spectrometer system <NUM> according to the present invention.

The continuous laser desorption mass spectrometer system <NUM> according to <FIG> comprises at least one laser desorption mass spectrometer <NUM>. The laser desorption mass spectrometer <NUM> corresponds at least partially to the laser desorption mass spectrometer <NUM> as depicted in <FIG>. Thus, reference to the description of <FIG> above is made.

<FIG> shows the continuous laser desorption mass spectrometer system <NUM> wherein the target <NUM> is provided as a material strip <NUM>. The material strip <NUM> may specifically be made of steel or aluminum. The material strip <NUM> may be rolled up to a role <NUM>. The material strip <NUM> may be configured to be unwound before one or more samples are applied to the material strip <NUM>, specifically to a surface <NUM> of the material strip <NUM>. The material strip <NUM> may be configured to pass through the continuous laser desorption mass spectrometer system <NUM>. Specifically, the material strip <NUM> may be configured to pass through different stations <NUM> of the continuous laser desorption mass spectrometer system <NUM>. The different stations <NUM> may comprise the laser desorption mass spectrometer <NUM> and may further comprise one or more liquid handling systems <NUM> and/or a vacuum system <NUM>.

The liquid handling system <NUM> may be configured for applying at least one sample <NUM> having at least one analyte on the target <NUM>, specifically on the material strip <NUM>. The liquid handling system <NUM> may specifically comprise one or more pipetting units <NUM>. Specifically, the liquid handling system <NUM> may be configured for applying a plurality of samples <NUM> successively on different regions <NUM> of the material strip <NUM>. The different regions <NUM> may be spaced apart from one another.

The vacuum system <NUM> may comprise one or more vacuum zones <NUM>. The vacuum zones <NUM> may be arranged successively. The material strip <NUM> may be configured for passing the vacuum system <NUM>, specifically one or more of the vacuum zones <NUM> before passing the laser desorption mass spectrometer <NUM>. Specifically, the material strip <NUM> may be configured for passing at least one first vacuum zone <NUM> and at least one second vacuum zone <NUM> before passing the laser desorption mass spectrometer <NUM>. The first vacuum zone <NUM> may be configured for providing a first underpressure and the second vacuum zone <NUM> may be configured for providing a second underpressure. The first underpressure may be higher than the second underpressure or vice versa. The first underpressure and the second underpressure may be below <NUM> mbar. The vacuum system <NUM>, specifically the vacuum zones <NUM>, may be configured for drying the sample on the target <NUM>. Further, the vacuum system <NUM> may comprise at least one third vacuum zone <NUM> and at least one fourth vacuum zone <NUM>. Specifically, the material strip <NUM> may be configured for passing the at least one third vacuum zone <NUM> and the at least one fourth vacuum zone <NUM> after passing the laser desorption mass spectrometer <NUM>. The third vacuum zone <NUM> and the fourth vacuum zone <NUM> may be configured for ensuring a continuous outward transfer and for keeping the vacuum in the laser region as low as technically possible at the same time, specifically for ensuring a reliable measurement.

The laser desorption mass spectrometer <NUM> may exemplarily comprise a quadrupole with subsequent ion trapping, isobaric separation via ion mobility, fragmentation in a collision cell and may be followed by quadrupole or time-of-flight (ToF) mass analysis. Other techniques of ion manipulations, like magnetic sector or ion trap, and different combinations of the corresponding units are also possible.

<FIG> shows the continuous laser desorption mass spectrometer system <NUM> wherein the target <NUM> is provided as a stack <NUM> of platelets <NUM>. The platelets <NUM> may specifically have a rectangular shape such as a square shape. The platelets <NUM> may be made of steel, glass or aluminum. However, also other materials may be feasible. The platelets <NUM> may be provided as being stacked on top of each other. Specifically, the platelets <NUM> may be stored in a platelet holder <NUM>. The platelet holder <NUM> may be configured for releasing the platelets <NUM> successively. Specifically, the continuous laser desorption mass spectrometer system <NUM> may comprise at least one conveyor belt (not shown). The platelet holder <NUM> may be configured for releasing the platelets <NUM> successively on the conveyor belt. The conveyor belt may be configured for successively passing the platelets <NUM> through the different stations <NUM> of the continuous laser desorption mass spectrometer system <NUM>. Specifically, the conveyor belt may be configured for successively passing the platelets <NUM> through the liquid handling system <NUM>. After individual collection of the platelets <NUM> out of the stack <NUM> the platelets <NUM> may be loaded with the sample <NUM> comprising an analyte solution by using the pipetting unit <NUM>. The sample <NUM> may be pipetted on a surface <NUM> of the platelets <NUM>. Further, the conveyor belt may be configured for successively passing the platelets <NUM> through the vacuum system <NUM> and the laser desorption mass spectrometer <NUM>. For further details on the liquid handling system <NUM>, the vacuum system <NUM> and the laser desorption mass spectrometer <NUM>, reference may be made of the description of <FIG> above.

The following examples serve to illustrate the invention. They must not be interpreted as limiting with regard to the scope of protection.

For the following examples samples comprising analytes were prepared. Specifically, the analytes were selected from steroids and therapeutically relevant substances. For some of the following examples, a mixture of seven naturally occurring steroids was prepared. The naturally occurring steroids (S) were selected from the group consisting of: Progesterone (Pr), Testosterone (Te), Estradiol (Es), Androstenedione (S7), Cortisol (S9), Cortisone (S10), <NUM>-Deoxycortisol (S19). A concentration of each of the naturally occurring steroids was <NUM>µg/mL and the solvent was a mixture of deionized water and acetonitrile (H<NUM>O/MeCN = <NUM>/<NUM>). Further, a mixture of seven therapeutically substances (T) was prepared. The therapeutically substances were selected from the group consisting of: Digitoxin (T7), Mycophenolic acid (T16), Theophylline (T29), Lidocain (T37), Digoxin (T41), Voriconazole (T62), <NUM>-Hydroxyalprazolam (4OHAlp). A concentration of each of the therapeutically substances was <NUM>µg/mL and the solvent was a mixture of deionized water and acetonitrile (H<NUM>O/MeCN = <NUM>/<NUM>). 4OHAlp was ordered from Enzo Life Sciences Inc. The deionized water was obtained from a Milli-Q® water purification system from Merck KGaA, acetonitrile and methanol were ordered from Biosolve BV, tetrahydrofurane was ordered from Merck KGaA. The remaining chemicals were ordered from Sigma-Aldrich Inc.

The measurements were performed on a Maldi-Synapt-G2Si mass spectrometer (Waters Inc. ), which is also capable of quadrupole mass filtering and ion mobility separation. The laser repetition rate was set to <NUM> for the imaging experiments and to <NUM> for all other measurements, with a Nd:YAG-laser wavelength of <NUM>. Analyte spots were each measured by a linear profile and a plate movement of <NUM> during the laser irradiation. The laser intensity could be varied in a relative scale of up to <NUM>, resembling a maximum output energy of <NUM>µJ. Individual voltage settings were set to the following gradient parameters: 'Sample plate' <NUM> V, 'Extraction' <NUM> V, 'Hexapole' <NUM> V and 'Aperture <NUM>' <NUM> V, as well as a hexapole RF amplitude of <NUM> V.

The mass spectra as described below in more detail show a relative abundance ra in % in dependence of the mass-to-charge ratio m/z.

As a first example a screening of a laser desorption ionization performance of different surface coatings was conducted.

<FIG> show mass spectra of the steroid mix measured on different targets having different surface coatings at a laser intensity of <NUM> in comparison, normalized to a highest peak intensity.

<FIG> show mass spectra of the steroid mix measured on different targets having different surface coatings at a laser intensity of <NUM> in comparison, normalized to a highest peak intensity having individual enlargements in the range between m/z <NUM> to m/z <NUM>.

Different types of targets were tested. The substrate of the targets were respectively made of steel. The mass spectra as depicted in <FIG> and <FIG> were obtained by utilizing an a-C:H:Si:X layer. Thereby, the targets were respectively coated via plasma assisted chemical vapor deposition. The mass spectra as depicted in <FIG> and <FIG> were obtained by utilizing a diamond like carbon layer (DLC, C≈<NUM> at. %, H≈<NUM> at. % and Si≈<NUM> at. Thereby, the targets were respectively coated via plasma assisted chemical vapor deposition. The mass spectra as depicted in <FIG> and <FIG> were obtained by utilizing an electrostatic dissipative diamond-like carbon layer, comprising a-C:H:Si:N (C≈<NUM> at. %, H≈<NUM> at. %, Si≈<NUM> at. % and N≈<NUM> at. Thereby, the targets were respectively coated via plasma assisted chemical vapor deposition.

The mass spectra as depicted in <FIG> and <FIG> were obtained by utilizing a titanium carbon nitride (TiCN) layer. Thereby, the targets were respectively coated via physical vapor deposition (PVD). The mass spectra as depicted in <FIG> and <FIG> were obtained by utilizing a titanium carbide / titanium nitride (TiC/TiN) layer. Thereby, the targets were respectively coated via high temperature chemical vapor deposition. The mass spectra as depicted in <FIG> and <FIG> were obtained by utilizing a titanium aluminum nitride (TiAlN) layer. Thereby, the targets were respectively coated via physical vapor deposition. The mass spectra as depicted in <FIG> and <FIG> were obtained by utilizing a titanium nitride (TiN) layer. Thereby, the targets were respectively coated via physical vapor deposition.

Direct spotting of <NUM>µL of the steroid-mix and measuring using the as described voltage settings with a laser energy of <NUM>, resulted in the mass spectra shown in <FIG> as well as in <FIG> show individual enlargements in the range between m/z <NUM> to m/z <NUM>. As shown in <FIG> and <FIG>, the a-C:H:Si:X layer hereby evinced superior capabilities in the desorption and ionization of the tested steroid analytes, specifically without revealing noticeable own background signals. The steroids Progesterone, Testosterone, Androstenedione, Cortisol, Cortisone and <NUM>-Deoxycortisol all appeared as sodium adducts [M+Na]+ in the positive ion mode from the untreated surface of the a-C:H:Si:X layer. Similar to that, as shown in <FIG> as well as in <FIG>, TiC/TiN and TiAlN also demonstrated certain capabilities in SALDI-MS analysis, resulting mainly in the formation of sodiated adducts [M+Na]+, but with less intensity, compared to the heteroatom-modified hydrogen-comprising carbon. As shown in <FIG> and <FIG>, the a-C:H:Si:N layer even resulted in the detection of some [M+H]+ and [M+K]+ steroid adducts, but also in the formation of background signals itself. Summarizing, it can be conducted that a high percentage of silicon in the a-C:H:Si:X layer was decisive for a significantly better performance in comparsion to the a-C:H:Si:N layer. Contrary, as shown in <FIG> and <FIG>, the DLC layer just showed traces of desorption and ionization of [S7+Na]+ and [Te+Na]+, but other steroid analytes could not be recognized. Thus, a diamond-like carbon layer having a small percentage of silicon may be applicable but may not evince superior capabilities. Further, as shown in <FIG> and <FIG> as well as in <FIG> and <FIG>, TiCN and TiN resulted in no laser desorption ionization of the analytes at all.

As a second example an analyte scope regarding SALDI-MS on a-C:H:Si:X layers was evaluated.

In order to assure a broad applicability of the herein described a-C:H:Si:X layers in the desorption and ionization of analytes, obtained by ultraviolet laser irradiation, a broad range of low molecular weight compounds was tested. These analytes were selected to represent important therapeutic or metabolic substances, as well as a broad scope of different functionalities and molecular weights in both, the positive and negative ionization mode. Additionally, a formation of protonated adducts in the positive ionization, instead of the formation of alkali adducts, was investigated. This would be an important achievement, due to the incompatibility of tandem mass spectrometry experiments of several alkali adducts, especially with steroid analytes.

To favor the formation of protonated adducts over the alkali species, a purification of the heteroatom-modified hydrogen-comprising carbon coating was conducted as well as the optimization of the laser energy during SALDI measurement. The purification of the surface coating aimed the reduction of alkali ions absorbed on the surface and could be achieved by simply rinsing with a sufficient amount of solvents, for example tetrahydrofuran, deionized water and acetonitrile. The surface-enhanced laser desorption/ionization measurement of <NUM>µL of the steroid mix previously dried on the herein described purified heteroatom-modified hydrogen-comprising carbon surface coating resulted at a laser intensity of <NUM> in the preferential formation of protonated adducts [M+H]+ of the steroids Pr, Te, S7, S9, S10, S19 as well as the water loss of estradiol [Es+H-H<NUM>O]+ in the positive ion mode, as can be seen in <FIG> show a full scan mass spectrum of the steroid mix measured on a purified heteroatom-modified hydrogen-comprising carbon surface at a laser energy of <NUM> units in the positive ion mode (<FIG>) with a respective enlargement in the mass range between m/z <NUM> to m/z <NUM> (<FIG>). Even some multiple steroid adducts, for example m/z <NUM> [Te+Pr+H]+ could be observed. Beside the protonated species, some minor sodiated and potassiated steroid adducts also occurred.

Another important aspect regarding the usability of the herein described heteroatom-modified hydrogen-comprising carbon coating is specifically the laser desorption and ionization of analytes in the negative ionization mode. This was also tested after drying <NUM>µL of the steroid mix on the purified heteroatom-modified hydrogen-comprising carbon surface and starting the measurement by irradiation with a laser intensity of <NUM> units. The corresponding full scan mass spectrum as well as an enlargement of the relevant range is given in <FIG> show a full scan mass spectrum of the steroid mix measured on the purified heteroatom-modified hydrogen-comprising carbon surface at a laser energy of <NUM> units in the negative ion mode (<FIG>) with a respective enlargement in the mass range between m/z <NUM> to m/z <NUM> (<FIG>). Here, the most abundant signal in the negative ionization mode belongs to estradiol [Es-H]-, but also most of the other steroids could be detected. This result proves the ability of the heteroatom-modified hydrogen-comprising carbon surface for the analysis of steroids by laser desorption and ionization.

A further screening regarding the scope of analytes was performed using therapeutic relevant substances that represent a variety of organic functional groups (for example acid, amide, amine, glycoside, halide, hydroxyl, aromatic or heteroaromatic moieties). Therefore, <NUM>µL of the therapeutic mix was dried on the purified heteroatom-modified hydrogen-comprising carbon surface and was measured by laser irradiation with an intensity of <NUM> units. A resulting full scan mass spectrum, as well as the corresponding enlargements, are given in <FIG> show a full scan mass spectrum of a therapeutic mix measured on the purified heteroatom-modified hydrogen-comprising carbon surface at a laser energy of <NUM> units in the positive ion mode (<FIG>) with the respective enlargements in the mass ranges between m/z <NUM> to m/z <NUM> (<FIG>), m/z <NUM> to m/z <NUM> (<FIG>) and m/z <NUM> to m/z <NUM> (<FIG>). All seven therapeutic relevant substances Alp, T7, T10, T16, T29, T41 and T62 were detected in the positive ionization mode, either as protonated adducts [M+H]+ or as the alkaline adducts [M+Na/K]+.

Also, this therapeutic analyte mix was examined for the laser desorption ionization on the heteroatom-modified hydrogen-comprising carbon surface utilizing the negative ionization mode. Therefore, <NUM>µL was previously dried on the purified heteroatom-modified hydrogen-comprising carbon surface and was irradiated with a laser energy of <NUM> units. The resulting full scan mass spectrum, as well as the corresponding enlargements, are given in <FIG> show a full scan mass spectrum of a therapeutic mix measured on the purified heteroatom-modified hydrogen-comprising carbon surface at a laser energy of <NUM> units in the negative ion mode (<FIG>) with the respective enlargements in the mass ranges between m/z <NUM> to m/z <NUM> (<FIG>), m/z <NUM> to m/z <NUM> (<FIG>) and m/z <NUM> to m/z <NUM> (<FIG>). Besides T62, which was detected in traces, all other analytes showed effective laser desorption ionization on the heteroatom-modified hydrogen-comprising carbon surface. Resulting ions were generally the deprotonated analyte species [M-H]-. These results show that the heteroatom-modified hydrogen-comprising carbon surface is capable of the laser desorption ionization of diverse therapeutically relevant analytes in the small molecular weight range, both in the positive and negative ionization mode.

Another important application of the a-C:H:Si:X layer is the combination of the laser desorption and ionization with ion mobility spectrometry (IMS), allowing the ion generation using the herein described a-C:H:Si:X layer with the ion separation capability of IMS. This was also measured using the therapeutic mix in the positive ionization mode as before, but operating additionally in the IMS-measurement mode of the Synapt G2-Si mass spectrometer. As drift gas, nitrogen is used, at a wave velocity of <NUM>/s and a wave height of <NUM> V. <FIG> respectively show a full scan mass spectrum of the therapeutic mix measured on the purified a-C:H:Si:X surface at a laser energy of <NUM> in the positive ion mode plotted against the drift time td in ms after IMS separation in a 3D illustration (<FIG>) and in a 2D illustration (<FIG>). In the following Table <NUM>, data from the ion mobility spectrum are provided in tabular form. As can be seen in <FIG>, a separation of all seven analytes was successfully achieved and the plot of the m/z ratio against the drift time td resulted in additional valuable data. This proves the broad applicability of the a-C:H:Si:X surface, even when a separation of the analyte mixture is needed.

Other experiments, illustrating the applicability of the heteroatom-modified hydrogen-comprising carbon surface also in the presence of analytes, which cover a broader mass range, were performed. Therefore, two substances were chosen, which comprised each polyethylene glycol (PEG) chains with different chain lengths and their corresponding size distributions. One first analyte solution covered the detergent Triton X-<NUM> (<NUM>µg/mL in H<NUM>O/MeCN = <NUM>/<NUM>) and a second analyte solution covered PEG1970 (Mp=<NUM>, PDI=<NUM>, <NUM>µg/mL in H<NUM>O/MeCN = <NUM>/<NUM>). Each solution was spotted (<NUM>µL) separately on the heteroatom-modified hydrogen-comprising carbon surface and dried. Subsequent SALDI measurements with a laser intensity of <NUM> units resulted in the mass spectra as displayed in <FIG> show enlargements in the mass ranges of full scan mass spectra of the analytes Triton X-<NUM> (<FIG>) and PEG1970 (<FIG>), measured by laser desorption ionization on a purified heteroatom-modified hydrogen-comprising carbon surface at a laser energy of <NUM> units in the positive ion mode. Triton X-<NUM> hereby showed the characteristic protonated adduct peaks in the mass range mainly between m/z <NUM> and m/z <NUM>. The PEG1970 sample presented mainly the sodiated adducts [M+Na]+ with also its minor potassiated adduct species [M+K]+. The size distribution of the PEG1970 sample was localized predominantly in the range between m/z <NUM> to m/z <NUM>. This proved the suitability of the heteroatom-modified hydrogen-comprising carbon surface for SALDI analysis over a broad range of small to mid molecular weight analytes.

An important class of analytes in the low to mid molecular weight region generally are peptides. To test the suitability of the laser desorption ionization measurement on the heteroatom-modified hydrogen-comprising carbon surface, the therapeutically important cyclic peptide Cyclosporin A (CsA, <NUM>µL, <NUM>µg/mL in MeCN/H<NUM>O = <NUM>/<NUM>) was selected. The respective enlargement of the resulting mass spectrum is displayed in <FIG> shows an enlargement in the mass range between (m/z <NUM> to m/z <NUM>) of a full scan mass spectrum of Cyclosporin A, measured by laser desorption ionization on a purified heteroatom-modified hydrogen-comprising carbon surface at a laser energy of <NUM> in the positive ion mode. Cyclosporin A resulted mainly in the detection of the sodiated adduct [CsA+Na]+, with also some potassiated adduct [M+K]+ and even few protonated analyte adducts [M+H]+. With CsA, an analytically important peptide was successfully detected by SALDI-MS on the heteroatom-modified hydrogen-comprising carbon surface.

Beside the successful laser desorption ionization of analytes from the heteroatom-modified hydrogen-comprising carbon surface, a further experiment was performed to demonstrate the capability of the desorption of permanently positive charged molecules which do not require further ionization. For this, a solution of testosterone, which was derivatized with Girard T reagent, was prepared (TeGT, <NUM>µg/mL in H<NUM>O/MeCN = <NUM>/<NUM>) and <NUM>µL was spotted on the heteroatom-modified hydrogen-comprising carbon surface and was dried. Measuring this cationic analyte with a laser irradiation (intensity <NUM> units) resulted in the full scan mass spectrum, as is shown in <FIG>. In <FIG>, a full scan mass spectrum of the permanently positive charged analyte Testosterone-Girard T, measured on a purified heteroatom-modified hydrogen-comprising carbon surface at a laser energy of <NUM> units in the positive ion mode is shown. The major signal could be assigned to the analyte [TeGT]+ itself, with only minor fragments, such as [TeGT-NMe<NUM>]+. This provided the confirmation, that the laser energy transfer by the heteroatom-modified hydrogen-comprising carbon surface is relatively soft, without extensive fragmentation of the analyte during desorption.

Also, this experiment showed the compatibility regarding a derivatization of analytes, which is often used for example in the analysis of steroids.

Moreover, a permanent negative analyte was tested. The solution of sodium dodecyl sulfate (SDS, <NUM>µg/mL in H<NUM>O), was applied on the heteroatom-modified hydrogen-comprising carbon surface (<NUM>µL) and dried. Subsequently, a SALDI measurement was performed by irradiation with an intensity of <NUM> units. The product ion scan of m/z <NUM> (CE = <NUM> eV) was recorded to show the corresponding fragmentation of dodecyl sulfate, as shown in <FIG> shows a product ion scan of the permanently negative charged analyte dodecyl sulfate (DS), measured on a purified heteroatom-modified hydrogen-comprising carbon surface at a laser energy of <NUM> units in the negative ion mode. The dodecyl sulfate anion [DS]- was observed in quite high intensity. As main fragment, [HSO<NUM>]- occurred, indicating the elimination of the sulfate end group of dodecyl sulfate. The desorption of a negatively charged analyte on the heteroatom-modified hydrogen-comprising carbon surface was therefore successfully shown.

A further observation regarding the SALDI performance of the heteroatom-modified hydrogen-comprising carbon surface aimed to display the desorption and ionization of analytes with an accurate isotope pattern. As model substrate, the histological important stain Eosin Y (EY) was selected, due to the presence of four bromine atoms inside the structure. <NUM>µL of the analyte solution (EY, <NUM>/mL in MeOH/H<NUM>O = <NUM>/<NUM>) was spotted on the heteroatom-modified hydrogen-comprising carbon surface, dried, and subsequently measured by SALDI mass spectrometry (laser intensity <NUM> units) in the negative ion mode. The observed full scan showed a main formation of the deprotonated molecule [EY-H]-. An enlargement of the range between m/z <NUM> to m/z <NUM>, as shown in <FIG> confirmed the predicted shape of the isotope pattern of the species [C<NUM>H<NUM>Br<NUM>O<NUM>]-. <FIG> show a corresponding enlargement of the isotope pattern of [EY-H]- in the range between m/z <NUM> to m/z <NUM> (<FIG>) and the calculated ion pattern of [EY-H]-(<FIG>). This experiment underlines the broad applicability of the herein described heteroatom-modified hydrogen-comprising carbon surface, because it is an important consideration for quantitative analysis, where isotope-labeled internal standards are commonly used.

As a third example a quantification of analytes regarding SALDI-MS on a heteroatom-modified hydrogen-comprising carbon surface was evaluated.

While the qualitative performance and usability of the heteroatom-modified hydrogen-comprising carbon surface regarding SALDI-MS is shown above in detail, a quantitative capability would additionally be highly valuable. For this analysis, two serial dilutions were performed with targets having heteroatom-modified hydrogen-comprising carbon surfaces, in the positive and negative ion mode respectively, and the results were compared to a blank steel plate and a weathered steel plate.

At first, a serial dilution of progesterone comprising <NUM>C<NUM>Pr as internal standard was selected. The concentration of Pr in detail was <NUM>µg/mL, <NUM>µg/mL, <NUM> ng/mL and <NUM> ng/mL, while <NUM>C<NUM>Pr was added to all samples in a concentration of <NUM>µg/mL. Each analyte solution was spotted separately with <NUM>µL sample volume on a purified heteroatom-modified hydrogen-comprising carbon surface and dried. The following SALDI-MS measurements, irradiating each spot with a laser intensity of <NUM> units, result in the detection of [M+H]+ and [M+Na]+ adducts for Pr as well as <NUM>C<NUM>-Pr. The combined intensities [Pr+H]+ and [<NUM>C<NUM>Pr+H]+ of each analyte spot were evaluated and the combined intensity ratios Pr/<NUM>C<NUM>Pr, CIR Pr/<NUM>C<NUM>Pr, were calculated and plotted against the corresponding concentration c, as can be seen in <FIG> shows a plot of a combined intensity ratio Pr/<NUM>C<NUM>Pr CIR Pr/<NUM>C<NUM>Pr against a concentration c of Pr in the range between <NUM>µg/ml to <NUM> ng/mL, with the linear regression line and, additionally, an enlargement of the range between <NUM>µg/mL to <NUM> ng/mL. Over the whole range, a linear dependence was observed. Even at <NUM> ng/mL, progesterone was detected successfully, and considering the low sample volume, this resembled a total of <NUM> fmol on a single spot. The heteroatom-modified hydrogen-comprising carbon surface is therefore applicable for the quantification of progesterone with an internal standard by SALDI mass spectrometry in the positive ion mode.

The quantitative analysis using the heteroatom-modified hydrogen-comprising carbon surface was furthermore tested in the negative ion mode with a serial dilution of estradiol. The concentration of Es in detail was <NUM>µg/mL, <NUM>µg/mL, <NUM> ng/mL and <NUM> ng/mL, while <NUM>C<NUM>Es was added as internal standard to all samples in a concentration of <NUM>µg/mL. Each analyte solution was spotted separately with <NUM>µL sample volume on a purified heteroatom-modified hydrogen-comprising carbon surface and was dried. The following SALDI-MS measurements, irradiating each spot with a laser intensity of <NUM> units in the negative ion mode, resulted in the detection of [M-H]- for Es as well as <NUM>C<NUM>Es. The combined intensities [Es-H]- and [<NUM>C<NUM>Es-H]- of each analyte spot were evaluated and the combined intensity ratios Es/<NUM>C<NUM>Es CIR Es/<NUM>C<NUM>Es were calculated and plotted against the corresponding concentration, as can be seen in <FIG> shows a plot of a combined intensity ratio Es/<NUM>C<NUM>Es CIR Es/<NUM>C<NUM>Es against a concentration c of Es in the range between <NUM>µg/ml to <NUM> ng/mL, with the linear regression line and, additionally, an enlargement of the range between <NUM>µg/mL to <NUM> ng/mL. Up to the concentration of <NUM> ng/mL, a linear dependence was observed. Considering the low sample volume, this resembled a total of <NUM> fmol on a single spot. The heteroatom-modified hydrogen-comprising carbon surface is therefore applicable for the quantification of estradiol with an internal standard by SALDI mass spectrometry in the negative ion mode.

To verify that the desorption and ionization of analytes is affected by the heteroatom-modified hydrogen-comprising carbon surface, the serial dilution of estradiol was measured again on a bare steel plate and also on a rusted steel plate. The latter one was additionally chosen, because similar weathering effects of stainless steel were mentioned in Reichardt et al. , Analyst <NUM>, <NUM>, <NUM>, DOI <NUM>/c4an00216d to appear in some SALDI performance. The weathering of a steel plate was performed in an aqueous NaCl bath over one day and cleaned sufficiently with deionized water, tetahydrofuran and acetonitrile. On both, the bare purified steel plate and the rusted steel plate, the dilution series of estradiol - as prepared before - were spotted with <NUM>µL sample volume and dried. The following SALDI-MS measurements, irradiating each spot with a laser intensity of <NUM> units in the negative ion mode, resulted in mostly no detection of [M-H]- of Es as well as <NUM>C<NUM>Es. The combined number of counts [Es-H]- of each analyte spot were evaluated and plotted against the corresponding concentration in comparison to the results with the heteroatom-modified hydrogen-comprising carbon surface, as can be seen in <FIG> shows a plot of a combined number of counts N of [Es-H]- against a concentration c of Es in the range between <NUM>µg/ml to <NUM> ng/mL on the heteroatom-modified hydrogen-comprising carbon surface (<NUM>, rectangles) in comparison to a bare steel plate (<NUM>, circles) and a rusted steel plate (<NUM>, triangles). Only the bare steel plate resulted on a single spot (<NUM>µg/mL) in some detection of estradiol, but this seemed to be more an outlier, due to no further detection of analytes at all other concentrations. Also the rusted steel plate led to no detection of analytes - not even at the highest concentration at <NUM>µg/mL estradiol. On the contrary, the heteroatom-modified hydrogen-comprising carbon surface resulted in a significant desorption and ionization of the estradiol serial dilution samples. These experiments proved that the desorption and ionization of analytes are initiated by the heteroatom-modified hydrogen-comprising carbon surface itself during laser irradiation, whereas a simple steel plate as well as a rusted steel plate do not have the ability to desorb and ionize analytes effectively, especially in a quantitative manner.

As a fourth example a reusability of the heteroatom-modified hydrogen-comprising carbon surface was evaluated.

Another beneficial feature of a SALDI surface coating would be its reusability without reducing significantly the functional efficiency. Because there are no visible signs of a deterioration of the heteroatom-modified hydrogen-comprising carbon surface after a measurement with a laser irradiation of up to <NUM> units, an additional experiment was implemented to estimate whether a reusability of the heteroatom-modified hydrogen-comprising carbon surface is possible. For this, a new target having a heteroatom-modified hydrogen-comprising carbon surface was cleaned, spotted in duplicate with an analyte solution, consisting of Pr and <NUM>C<NUM>Pr, both in a concentration of <NUM>µg/mL, dried and measured by SALDI-MS with a laser intensity of <NUM> units. Cleaning again the target having the heteroatom-modified hydrogen-comprising carbon surface, this procedure was iterated for additional four times. The normalized combined intensity ratio Pr/<NUM>C<NUM>Pr NCIR Pr/<NUM>C<NUM>Pr was calculated for both, [M+H]+ and [M+Na]+, and plotted against a number of times Nt the position was used. An example of the relevant part of the first full scan mass spectrum is given in <FIG>, as well as the plot of the combined intensity ratio in <FIG> shows a respective part (m/z <NUM> to m/z <NUM>) of a full scan mass spectrum of [Pr+Na]+ and [<NUM>C<NUM>Pr+Na]+ resulting from a first SALDI-MS measurement on a heteroatom-modified hydrogen-comprising carbon surface with a laser intensity of <NUM> units and <FIG> shows a normalized combined intensity ratio Pr/<NUM>C<NUM>Pr NCIR Pr/<NUM>C<NUM>Pr of [M+H]+ (<NUM>, triangles) and [M+Na]+ with N=<NUM> (<NUM>, circles) plotted against the number of times Nt the position was used. It can be concluded that there is no significant loss of the laser desorption ionization performance of a heteroatom-modified hydrogen-comprising carbon surface even after <NUM> uses of the same spot and at increased laser intensity.

As a fifth example a performance in the presence of realistic matrices was evaluated.

Relating to the broad analyte scope, quantification and reusability opportunities of the heteroatom-modified hydrogen-comprising carbon surface at SALDI mass spectrometry, a last consideration was taken to elucidate the performance in the presence of realistic matrices, such as tissue, serum, whole cell samples. A successful application of the heteroatom-modified hydrogen-comprising carbon surface in the analysis of diverse biological or diagnostic samples, would resemble a huge leap in the state of the art of SALDI mass spectrometry.

The first experiment aimed to demonstrate a desorption and ionization of a selected analyte in the presence of a processed formalin fixed paraffin embedded (FFPE) tonsillar tissue sample. The paraffin of the FFPE tissue sample was softened with cautious heating to <NUM> and washed away with small amounts of tetrahydrofuran. The tonsillar tissue sample was macerated with some drops of deionized water and transferred to the heteroatom-modified hydrogen-comprising carbon surface of the target. On a defined spot of the tissue sample, <NUM>µL of a <NUM>µg/mL estradiol sample solution was added on top of the tonsillar tissue sample. The following SALDI measurement in the negative ion mode was performed with a laser intensity of <NUM> units - with one mass spectrum recording the background of the processed FFPE tonsillar sample and another mass spectrum recording the spot, where estradiol was spiked on top. Both mass spectra can be compared in <FIG> show full scan SALDI mass spectra of a processed FFPE tonsillar tissue sample on a heteroatom-modified hydrogen-comprising carbon surface with the background of the tissue (<FIG>) and the tissue spiked with estradiol (<FIG>) operating in the negative ion mode with a laser intensity of <NUM> units. A background of the tissue sample itself and residual paraffin was clearly visible in the corresponding mass spectrum. Despite the background signals, the sample spot with previous addition of estradiol resulted clearly in the detection of [Es-H]-. This is a quite important result, because it clarifies that the SALDI performance of the heteroatom-modified hydrogen-comprising carbon surface is not just limited to sample solutions alone, but is also useful in the analysis of tissue samples with a challenging matrix.

Claim 1:
A target (<NUM>) for use in a laser desorption mass spectrometer, wherein the target (<NUM>) has at least one surface (<NUM>), wherein the surface (<NUM>) is covered at least partially with at least one layer (<NUM>), characterised in that the layer (<NUM>) is a hydrogen comprising, silicon-incorporated amorphous carbon (a-C:H:Si) layer (<NUM>), wherein the a-C:H:Si layer (<NUM>) comprises:
• <NUM> at.% to <NUM> at.% of carbon;
• <NUM> at.% to <NUM> at.% of hydrogen; and
• <NUM> at.% to <NUM> at.% of silicon.