Detection of target analytes by cross-spray ESI mass spectrometry

Abstract

The present invention relates to a method, diagnostic system, dopant, and use thereof for enhancing the detection of an analyte of interest by cross-spray electrospray ionization (ESI) mass spectrometry. The dopant is preferably a sulfone. The method includes two electrospray ionization sources for two analyte streams in gaseous or aerosol form. The second stream contains a dopant or derivatization reagent. The first and second analyte streams are mixed, and mass spectrometry is used to determine the presence or level of the analyte of interest in a sample with improved sensitivity.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to a method, a diagnostic system, a dopant, and the use thereof for enhancing the detection of a target analyte by cross-spray ESI mass spectrometry. [Background technology]

[0002] Background of the Invention Mass spectrometry (MS) is a widely used technique for the qualitative and quantitative analysis of chemicals ranging from small molecules to large molecules. Generally, it is a highly sensitive and specific method, enabling the analysis of complex biological samples, such as environmental or clinical samples. However, the sensitivity of the measurement remains a concern when analyzing certain analytes, particularly those from complex biological matrices like serum.

[0003] In many cases, MS is combined with chromatographic techniques, particularly gas and liquid chromatography, such as HPLC. Here, the target molecule (analyte) to be analyzed is separated by chromatography and subjected to individual mass spectrometry (Higashi et al. (2016) J. of Pharmaceutical and Biomedical Analysis 130 p.181-190).

[0004] To ensure reliable and highly sensitive mass spectrometry detection (avoiding matrix effects and interference, and improving sensitivity), the target analyte must be separated chromatographically as much as possible. Generally, this can be done isocratic or gradient systems, such as reversed-phase HPLC columns and gradients from aqueous to organic phases. The columns used in HPLC require a flow rate of 0.1–1.0 ml / min. Under these optimal flow conditions, very narrow chromatographic peaks with very small peak volumes are produced.

[0005] ESI (Electrospray Ionization) is a technique used in mass spectrometry to generate ions using electrospray, which generates aerosols or gases by applying a high voltage to a liquid.

[0006] However, when analyzing MS analysis methods, especially for substances present in small quantities or when only small amounts of material such as biopsy tissue are available, it is necessary to increase their sensitivity.

[0007] It is known that adding DMSO to the LC-MS eluent increases sensitivity to peptide signals. Adding a dopant to the liquid phase, such as DMSO, can alter chromatographic parameters such as retention time, which is disadvantageous if a different solvent needs to be introduced into the HPLC system to wash all piping from the previous solvent with the dopant, e.g., DMSO. Furthermore, if the dopant (e.g., DMSO) is in the eluent, the dopant will permanently enter the ion source and thus contaminate the ion source over time. The mixing behavior of the dopant and its respective eluent must be matched to eliminate precipitation or reaction effects. If post-column injection of any dopant is used, it will cause a widening of the chromatographic peak width. This is disadvantageous if very sharp peak shapes result from (modern) UHPLC separation and / or ultrashort gradient times.

[0008] When the reactants for derivatization are injected before the ion source, there is a high risk of contaminating the analyte ESI emitter, leading to peak width issues.

[0009] Therefore, there is an urgent need in this field for methods that not only reveal chemical structures without negatively impacting MS measurement workflows, but also enable highly sensitive detection of analytes from complex biological matrices. This is particularly important in random-access, high-throughput MS setups where several different analytes exhibiting different chemical properties must be measured in a short time.

[0010] The present invention relates to a method for measuring the level of a target analyte in a sample, enabling highly sensitive measurement of analyte molecules such as steroids, proteins, and other types of analytes in biological samples. The reagents are designed in a modular manner to allow for individual adaptations for specific needs arising from the measurement of particular analytes or for specific workflow adaptations.

[0011] The object of the present invention is to provide a method, a diagnostic system, a dopant, and its use for enhancing the detection of a target analyte by cross-spray ESI mass spectrometry. This objective is resolved by the subject matter of the independent claim. Further embodiments are provided according to the dependent claims. [Overview of the project]

[0012] In the following, the present invention relates to the following aspects.

[0013] In a first aspect, the present invention relates to a method for determining the presence or level of a target analyte in a sample, a) A step of supplying a first electrospray ionization source for a first stream of analytes containing the target analyte, b) A step of supplying a second electrospray ionization source for a second analyte stream containing a dopant or derivatization reagent, wherein each of the first analyte stream and the second analyte stream is contained in gaseous form or aerosol, c) A step of mixing a stream of a first analyte and a stream of a second analyte to form a mixture or a derivatized analyte of interest, wherein the mixture comprises the analyte of interest and a dopant, and d) A step of determining the presence or level of the target analyte in the sample using mass spectrometry. This includes methods.

[0014] In a second aspect, the invention relates to the use of the method of the first aspect of the invention for determining the presence or level of a target analyte in a sample.

[0015] In a third aspect, the invention relates to a diagnostic system for determining the presence or level of a target analyte in a sample, the diagnostic system comprising a first electrospray ionization source and a second electrospray ionization source, and a mass spectrometry unit for performing the method of the first aspect of the invention.

[0016] In a fourth aspect, the invention relates to a dopant for enhancing the sensitivity of mass spectrometric detection of a target analyte, having the formula I: R1-SO2-R2 (I) (wherein R1 and R2 are each independently selected from alkyl or aryl, alkyl contains 1 to 6 C atoms, and aryl contains from 1 to 6 C atoms) and relates to a dopant comprising the same.

[0017] In a fifth aspect, the invention relates to the use of the dopant of the fourth aspect of the invention in the method according to the first aspect of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] ]> [Figure 1] FIG. 1 shows a schematic diagram of a diagnostic system according to the invention. [Figure 2] FIG. 2 shows the TIC (total ion count) as a percentage of the retention time of a mixture containing a peptide as the target analyte and DMSO as the dopant at various concentrations (0% (without DMSO), 10%, 20% and 30% (v / v)). [Figure 3] FIG. 3 shows the TIC as a percentage of the retention time of a mixture containing tramacine as the target analyte and DMSO as the dopant at various concentrations (0% (without DMSO), 5% and 30% (v / v)). [Figure 4A]Figures 4A to 4D show the TIC (Time Indication) as a percentage of the retention time for mixtures containing estradiol as the analyte of interest, with or without ammonium fluoride as the dopant. [Figure 4B] Figures 4A to 4D show the TIC (Time Indication) as a percentage of the retention time for mixtures containing estradiol as the analyte of interest, with or without ammonium fluoride as the dopant. [Figure 4C] Figures 4A to 4D show the TIC (Time Indication) as a percentage of the retention time for mixtures containing estradiol as the analyte of interest, with or without ammonium fluoride as the dopant. [Figure 4D] Figures 4A to 4D show the TIC (Time Indication) as a percentage of the retention time for mixtures containing estradiol as the analyte of interest, with or without ammonium fluoride as the dopant. [Figure 5-1] Figure 5 shows the TIC as a percentage of the retention time of testosterone using a hydrazone derivatization reagent for coupling. [Figure 5-2] Figure 5 shows the TIC as a percentage of the retention time of testosterone using a hydrazone derivatization reagent for coupling. [Figure 6-1] Figure 6 shows the TIC as a percentage of the testosterone retention time using a derivatization reagent. [Figure 6-2] Figure 6 shows the TIC as a percentage of the testosterone retention time using a derivatization reagent. [Figure 7] Figure 7 shows the multiplicative changes of each peptide in a peptide mixture (purchased from Sigma Aldrich) against a blank H2O / CAN (CAN = acetonitrile) cross-flow eluent. [Figure 8] Figure 8 shows a schematic experimental setup. The peptide test mixture is mixed with the dopant in the reaction coil after the column and before the mass spectrometer. [Figure 9]Figure 9 shows the analyte-dependent changes in the three peptides of interest upon addition of different dopants, such as DMSO, sulfolane, diphenylsulfone, 1,4-dioxane, and propylene glycol sulfite. [Modes for carrying out the invention]

[0019] Detailed description of the invention Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific embodiments and examples described herein, and that these may vary. Furthermore, it should be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the invention, which is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art.

[0020] Several documents are cited herein. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.), whether above or below, is incorporated herein by reference in its entirety. In the event of any conflict between the definition or teaching of a reference thus incorporated herein and the definition or teaching cited herein, the text of this specification shall prevail.

[0021] The elements of the present invention are described below. While these elements are listed in conjunction with specific embodiments, it should be understood that they can be combined in any way and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutations and combinations of all elements described in this application should be considered disclosed by the description of this application unless the context indicates otherwise.

[0022] definition The word "comprise," as well as variations such as "comprises" and "comprising," means to include a specified integer or process, or a group of integers or processes, but not to exclude any other integer or process, or a group of integers or processes. The terms "including" and "comprising" can be used interchangeably.

[0023] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple subjects unless otherwise explicitly indicated.

[0024] Ratios, concentrations, quantities, and other numerical data may be expressed or presented herein in the form of “ranges.” Such range forms are used merely for convenience and conciseness, and therefore should be interpreted flexibly to include not only the numbers explicitly listed as limiting the range, but also all or part of the individual numbers encompassed within that range, as if explicitly listed. For example, the numerical range “4% to 20%” should be interpreted to include not only the explicitly listed value of 4% to 20%, but also the individual values ​​and part of the range shown. Thus, this numerical range includes individual values ​​such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20%, and sub-ranges such as 4 to 10%, 5 to 15%, 10 to 20%, etc. This same principle applies to ranges listing minimum or maximum values. Furthermore, such interpretation should apply regardless of the width or characteristics of the range.

[0025] When used in relation to a number, the term "approximately" means to encompass a range of numbers that have a lower limit 5% less than the given number and an upper limit 5% greater than the given number.

[0026] In the context of this invention, the terms “compound,” “derivative reagent,” “label,” or “derivative agent” are used interchangeably and refer to chemical substances having a specific chemical structure. The compound may contain one or more reactive groups. Each reactive group may perform a different function, and two or more reactive groups may perform the same function. The reactive groups include, but are not limited to, reactive units, charged units, and neutral loss units. The derivatizing reagent may undergo an atmospheric pressure chemical reaction with the analyte of interest.

[0027] The terms “Mass Spectrometry” (“Mass Spec” or “MS”), “mass spectrometric determination,” or “mass spectrometric analysis” relate to analytical techniques used to identify compounds by their mass. MS is a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio or “m / z.” MS techniques generally involve (1) ionizing a compound to form a charged compound; and (2) detecting the molecular weight of the charged compound and calculating its mass-to-charge ratio. Compounds can be ionized by any suitable means and then detected. A “mass spectrometer” generally includes an ionizer and an ion detector. Generally, one or more molecules of interest are ionized, and these ions are then introduced into a mass spectrometer, in which, due to a combination of magnetic and electric fields, the ions follow a path in space depending on their mass (“m”) and charge (“z”). The term "ionization" refers to the process of generating analyte ions having a net charge equal to one or more units. Negative ions have a net negative charge of one or more units, and positive ions have a net positive charge of one or more units. MS methods can be performed in either an "anion mode," where anions are generated and detected, or a "cation mode," where cations are generated and detected.

[0028] Tandem mass spectrometry, or MS / MS, involves multiple steps of selected mass spectrometry in which analyte fragmentation occurs between steps. In a tandem mass spectrometer, ions are generated in an ion source and separated by mass-to-charge ratio in the first step of mass spectrometry (MS1). Ions with a specific mass-to-charge ratio (precursor or parent ion) are selected, and fragment ions (daughter ions) are generated by collision-induced dissociation, ionic-molecular reactions, or photodissociation. The resulting ions are then separated and detected in the second step of mass spectrometry (MS2).

[0029] Mass spectrometers separate and detect ions of slightly different masses, thus easily distinguishing different isotopes of a given element. Therefore, mass spectrometry is an important method for the accurate mass determination and characterization of analytes, including, but not limited to, low molecular weight analytes, peptides, polypeptides, or proteins. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits, and functional interactions, and the whole-body analysis of proteins in proteomics. De novo sequencing of peptides or proteins by mass spectrometry can usually be performed without prior knowledge of the amino acid sequence.

[0030] Most sample workflows in MS further include sample preparation and / or concentration steps, for example, separating the target analyte(s) from the matrix using gas or liquid chromatography. Typically, mass spectrometry involves the following three steps: 1. Samples containing the target analyte are ionized, usually by complex formation with cations, and often by protonation to cations. Ionization sources include, but are not limited to, electrospray ionization (ESI), nano electrospray ionization (nano-ESI), and atmospheric pressure chemical ionization (APCI). 2. The ions are classified and separated according to their mass and charge. High-field asymmetric waveform ion mobility spectroscopy (FAIMS) may be used as the ion filter. 3. The separated ions are detected, for example, in multiple reaction mode (MRM), and the results are displayed on a chart.

[0031] The term "electrospray ionization" or "ESI" refers to a method in which a solution is passed through a short capillary tube, and a high positive or negative potential is applied to the end of the capillary tube. The solution that reaches the end of the tube is vaporized (atomized) into a jet or spray of very small droplets in solvent vapor. This mist of droplets passes through an evaporation chamber, which is slightly heated to evaporate the solvent and prevent condensation. The smaller the droplets, the higher the electrical surface charge density becomes until ions and neutral molecules are released due to the natural repulsion between like charges.

[0032] The terms "nanoelectrospray ionization" or "nanoESI" typically refer to methods using flow rates of less than 1 μL / min in either static or dynamic mode. Preferably, nanoESI uses flow rates of 50–500 nl / min, for example, 500 nl / min. 500 nl / min is equal to 0.5 μL / min.

[0033] The term "atmospheric pressure chemical ionization" or "APCI" refers to a mass spectrometry method similar to ESI. However, APCI generates ions through ionic molecular reactions occurring within an atmospheric pressure plasma. The plasma is maintained by an electrical discharge between a spray capillary and a counter electrode. The ions are then extracted into a mass spectrometer, typically using a set of differentially pumped gap stages. To improve solvent removal, a counterflow of dry and preheated Ni gas may be used. Gas-phase ionization in APCI can be more effective than ESI for analyzing less polar entities.

[0034] High-field asymmetric waveform ion mobility spectroscopy (FAIMS) is an atmospheric pressure ion mobility technique that separates gas-phase ions based on their behavior under strong and weak electric fields.

[0035] "Multiple reaction mode" or "MRM" is a detection mode for MS instruments in which precursor ions and one or more fragment ions are selectively detected. Mass spectrometry determination may be combined with additional analytical methods, including chromatographic methods such as gas chromatography (GC), liquid chromatography (LC), particularly HPLC, and / or separation techniques based on ion mobility.

[0036] In the context of this disclosure, the terms “analyte,” “analyte molecule,” or “analyte of interest” are used interchangeably to refer to chemical species analyzed by mass spectrometry, particularly nano-ESI mass spectrometry. Chemical species suitable for analysis via mass spectrometry, i.e., analytes, can be any type of molecule present in a living organism, including but not limited to nucleic acids (e.g., DNA, mRNA, miRNA, rRNA, etc.), amino acids, peptides, proteins (e.g., cell surface receptors, cytosolic proteins, etc.), metabolites or hormones (e.g., testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (e.g., vitamin D), molecules characteristic of specific modifications of other molecules (e.g., sugar moieties or phosphoryl residues on proteins, methyl residues on genomic DNA), or substances internalized by a living organism (e.g., therapeutic agents, drugs of abuse, toxins, etc.), or metabolites of such substances. Such analytes may serve as biomarkers. In the context of this invention, the term “biomarker” refers to a substance within a biological system used as an indicator of the biological state of that system.

[0037] In this context, the term "dopant" can refer to a chemical substance that interacts with the target analyte by undergoing atmospheric pressure chemical interactions with it.

[0038] The terms “permanent charge” or “permanently charged” are used in the context of this disclosure when the charge of a unit, for example, positive or negative charge, is not easily reversible by, for example, washing, dilution, filtration, etc. Permanent charge may be, for example, the result of covalent bonding. Reversible charge (non-permanent charge) may be, for example, the result of electrostatic interaction, in contrast to permanent charge.

[0039] The terms “permanent net charge” or “net charge” are used in the context of this disclosure, where permanent net charge is the total permanent charge possessed by an ion or molecule. Permanent net charge can be calculated as follows: number of protons - number of electrons = permanent net charge. Permanent net charge can be viewed as a covalent combination of atoms that form a charged mojety within the molecule through bond rearrangement (e.g., quaternary nitrogen, tetramethylammonium), and net charge can also be present through the addition or subtraction of atoms, e.g., hydrogen, [M + H] + or [M - H] - This can result in a pseudomolecular ion consisting of (2) permanent positive charges and (2) permanent negative charges. For example, if a compound has two (2) permanent positive charges and one permanent negative charge, the permanent net charge is +1. * (+1) + (-1) = (+1).

[0040] The phrase "a compound can covalently bond to an analyte" means that the compound is suitable for bonding to the analyte. The bond between the compound and the analyte is covalent.

[0041] The term "mass," e.g., m1, m2, m3, m4, or mx (x>4), refers to atomic mass, specifically unified atomic mass. The unit of unified atomic mass is u. In the biomedical field, daltons [Da] may be used instead of unified atomic mass [u]. Daltons are not SI units. Daltons are equivalent to unified atomic mass in that there is no conversion factor between these units. A "mass spectrum" is a two-dimensional representation of signal intensity (vertical coordinate) versus m / z (horizontal coordinate). The position of the peak, usually called the signal, reflects the m / z of the ions produced from the compound, analyte, or combination thereof (complex) in the ion source. The intensity of this peak correlates with the abundance of that ion. Often, though not always, the highest m / z peak is found in intact ionized molecules, molecular ions, M +It arises from the detection of molecular ions. The molecular ion peak is usually accompanied by several lower or higher m / z peaks caused by fragmentation of the compound, analyte, or complex, resulting in fragment ions. Therefore, each peak in the mass spectrum can be referred to as a fragment ion peak or daughter ion peak. m / z is dimensionless by definition.

[0042] The term "fragmentation" may mean that a compound, analyte, and / or complex dissociates by passing through the ionization chamber of a mass spectrometer, forming ions, such as at least one daughter ion. Fragments cause a pattern characteristic of the mass spectrum. The term "fragmentation" may also refer to the dissociation of a single molecule into two or more distinct molecules. As used herein, the term fragmentation refers to a particular fragmentation event in which the breakdown point of the parent molecule in which the fragmentation event occurs is clearly defined, and the two or more daughter molecules resulting from the fragmentation event are well characterized. Methods for determining the breakdown points of the parent molecule and the resulting two or more daughter molecules are well known to those skilled in the art. The resulting daughter molecules may be stable or may dissociate during subsequent fragmentation events. For example, if the parent molecule undergoing fragmentation contains an N-benzylpyridinium unit, a person skilled in the art can determine, based on the overall structure of the molecule, whether the pyridinium unit fragments and releases the benzyl entity, or whether it is completely released from the parent molecule; that is, the resulting daughter molecule is either a benzyl molecule or a parent molecule lacking benzyl.Fragmentation can occur via collision-induced dissociation (CID), electron-capture dissociation (ECD), electron-transfer dissociation (ETD), negative electron-transfer dissociation (NETD), electron-detachment dissociation (EDD), photodissociation, particularly infrared multiphoton dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD), surface-induced dissociation (SID), higher-energy C-trap dissociation (HCD), charge-remote fragmentation.

[0043] The term "m1 / z1 < m2 / z2" means that the mass-to-charge ratio (m1 / z1) of a compound is less than the mass-to-charge ratio (m2 / z2) of at least one or exactly one daughter ion of the compound.

[0044] The term "limit of detection" or "LOD" is the lowest concentration of an analyte at which a bioanalytical method can reliably distinguish the analyte from background noise.

[0045] The term "signal-to-noise ratio" or S / N represents the uncertainty of intensity measurements and provides a quantitative measure of the quality of a signal by quantifying the ratio of the intensity of the signal to the noise.

[0046] The analyte may be present in the sample of interest, for example, in a biological or clinical sample. The terms “sample” and “target sample” are used interchangeably herein and refer to a tissue, organ, or part or portion of an individual, typically smaller than such tissue, organ, or individual intended to represent the whole. For analysis purposes, the sample provides information about the tissue state, or the health or disease state of the organ or individual. Examples of samples include, but are not limited to, liquid samples such as blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva, and lymph, or solid samples such as dried blood spots and tissue extracts. Further examples of samples are cell cultures or tissue cultures.

[0047] A "covalent bond," "covalently linked," or "covalently bonded" is a chemical bond in which at least one electron pair is shared between atoms or molecules, for example, between a compound and an analyte.

[0048] The terms "compound" and "label" or "derivative reagent" may be used interchangeably.

[0049] The numerical value of the charge, for example 1, 2, 3, 4, 5, or 6, for example z1, z2, z3, z4, or zx (x>4), is the absolute value of the charge. For example, a net charge z1=2 could mean that the net charge z1 is +2 or the net charge is -2. Preferably, in this case the charge is a positive number, for example 2=+2.

[0050] In this context, “level” or “level value” encompasses absolute quantities, relative quantities or concentrations, as well as any values ​​or parameters that correlate with or can be derived from them.

[0051] As used herein, the terms “determine” and “determine” the level of the analyte of interest refer to the quantification of the analyte of interest, for example, determining or measuring the level of the analyte of interest in a pre-treated sample. The level of the analyte of interest is determined by nano-ESI mass spectrometry.

[0052] In this context, “pre-treated sample” refers to a sample prepared for mass spectrometry, particularly nano-ESI mass spectrometry. Specifically, a pre-treated sample is a sample provided and / or prepared before steps (a) and / or (b) of the present method are carried out. Before analyzing the analyte by mass spectrometry, the sample may be pre-treated in a manner specific to the sample and / or analyte. In the context of this disclosure, the terms “pre-treatment” or “pre-treated” refer to any means necessary to enable the subsequent analysis of the desired analyte by mass spectrometry, particularly nano-ESI mass spectrometry. Typical pre-treatment means include, but are not limited to, elution of solid samples (e.g., elution of dried blood spots), addition of hemolytic reagents (HR) to whole blood samples, and addition of enzymatic reagents to urine samples. Similarly, the addition of an internal standard (ISTD) is considered sample pre-treatment. In particular, sample pre-treatment does not include concentration steps, for example, by using magnetic or paramagnetic beads.

[0053] The term "hemolytic reagent" (HR) refers to a reagent that lyses cells present in a sample, and in the context of this invention, it refers to a hemolytic reagent, in particular a reagent that lyses cells present in a blood sample, including, but not limited to, red blood cells present in a whole blood sample. A well-known hemolytic reagent is water (H2O). Further examples of hemolytic reagents include, but are not limited to, deionized water, highly osmotic liquids (e.g., 8M urea), ionic liquids, and different surfactants.

[0054] Typically, an “internal standard” (ISTD) is a known amount of a substance that exhibits characteristics similar to the analyte of interest when applied to the mass spectrometry detection workflow (i.e., including any pretreatment, concentration, and actual detection steps). The ISTD exhibits characteristics similar to the analyte of interest but can be clearly distinguished from the analyte of interest. By way of example, during chromatographic separation such as gas or liquid chromatography, the ISTD has approximately the same retention time as the analyte of interest from the sample. Thus, both the analyte and the ISTD enter the mass spectrometer simultaneously. The ISTD, however, exhibits a different molecular weight from the analyte of interest from the sample. Thereby, using different mass / charge (m / z) ratios, the ions from the ISTD and the ions from the analyte can be distinguished by mass spectrometry. Both are subjected to fragmentation to obtain daughter ions. These daughter ions can be distinguished by their m / z ratios relative to each other and their respective parent ions. As a result, separate determination and quantification of the signals from the ISTD and the analyte can be performed. Since the ISTD is added in a known amount, the signal intensity of the analyte from the sample can be attributed to a specific quantitative amount of the analyte. Thus, the addition of the ISTD enables a relative comparison of the amount of the detected analyte and enables clear identification and quantification of the analyte(s) of interest present in the sample when the analyte(s) reach the mass spectrometer. Typically, but not necessarily, the ISTD is an isotopically labeled variant of the analyte of interest (e.g., 2 H, 13 C, or 15 N, etc., including such labels).

[0055] In addition to sample preparation, the sample may also be subjected to one or more concentration steps. In the context of this disclosure, the terms “first concentration process” or “first concentration workflow” refer to a concentration process that occurs following sample preparation and provides a sample containing a concentrated analyte compared to the initial sample. The first concentration workflow may include chemical precipitation (e.g., using acetonitrile) or the use of a solid phase. Preferred solid phases include, but are not limited to, solid-phase extraction (SPE) cartridges and beads. The beads may be non-magnetic, magnetic, or paramagnetic. The beads may be coated differently to be specific to the analyte of interest. The coating may vary depending on the intended application, i.e., the intended capture molecule. Which coating is suitable for which analyte is well known to those skilled in the art. The beads may be made from a variety of different materials. The beads may have a variety of sizes and may have a surface with or without pores.

[0056] In the context of this disclosure, the terms “second enrichment process” or “second enrichment workflow” refer to an enrichment process performed after sample pretreatment and the first enrichment process, which provides a sample containing a concentrated analyte relative to the initial sample and the sample after the first enrichment process. In the context of this disclosure, a sample may originate from an “individual” or “subject.” Typically, a subject is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0057] As used herein, the term “serum” refers to the clear liquid portion of blood that can be separated from coagulated blood. As used herein, the term “plasma” refers to the clear liquid portion of blood that contains blood cells. Unlike plasma, serum is the liquid portion of normal, uncoagulated blood that contains red blood cells, white blood cells, and platelets. The difference between serum and plasma is the blood clot. As used herein, the term “whole blood” includes all components of blood, such as white blood cells and red blood cells, platelets, and plasma.

[0058] The term "in vitro method" is used to indicate that the method is performed outside of an organism, preferably on body fluids, isolated tissues, organs, or cells.

[0059] As used herein, the terms “automatically” or “automated” are broad terms, and should be given their ordinary and customary meanings to those skilled in the art, and should not be limited to any special or customized meanings. Specifically, the terms may refer to processes that are carried out entirely by at least one computer and / or computer network and / or machine, without any manual action and / or user interaction.

[0060] The term "chromatography" refers to the process by which a chemical mixture, carried by a liquid or gas, is separated into its components as a result of the different distributions of chemical components as it flows around or over a stationary liquid or solid phase.

[0061] The term "liquid chromatography" or "LC" refers to the process of selectively delaying one or more components of a fluid solution as a fluid uniformly permeates through a column or capillary pathway of finely divided substances. The delay is due to the distribution of the mixture components between one or more stationary phases and the bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). Methods where the stationary phase has higher polarity than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal-phase liquid chromatography (NPLC), while methods where the stationary phase has lower polarity than the mobile phase (e.g., a water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) are called reversed-phase liquid chromatography (RPLC).

[0062] High-performance liquid chromatography (HPLC) is a method of liquid chromatography in which the degree of separation is increased by passing a mobile phase, typically a densely packed column, through a stationary phase under pressure. Typically, the column is packed with a stationary phase consisting of irregular or spherical particles, a porous monolithic layer, or a porous membrane. Historically, HPLC has been divided into two distinct subclasses based on the polarity of the mobile and stationary phases.

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

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

[0065] The term "reactive unit" refers to a unit that can react with another molecule, that is, a unit that can form a covalent bond with another molecule, such as the analyte of interest. Typically, such covalent bonds are formed by chemical groups present in the other molecule. Therefore, during a chemical reaction, the reactive unit of a compound forms a covalent bond with a suitable chemical group present in the analyte molecule. Because this chemical group present in the analyte molecule performs the function of reacting with the reactive unit of the compound, the chemical group present in the analyte molecule is also called the "functional group" of the analyte. The formation of a covalent bond occurs in each chemical reaction in which a new covalent bond is formed between the atom of the reactive group and the functional group of the analyte. It is well known to those skilled in the art that an atom is lost during this chemical reaction when a covalent bond is formed between the reactive group and the functional group of the analyte.

[0066] The phrase "analyte flow is contained in gaseous or aerosol form" in this context may mean that an aerosol consists of, for example, microdroplets larger than 1 nm, while in a gaseous state or gaseous form, one or more analytes are free or surrounded only by a solvate shell, forming minute compartments smaller than 1 nm. The term "size" refers to particle diameter.

[0067] In the context of this disclosure, the terms “complex” and “derivativeized analyte of interest” are used interchangeably and refer to the product produced by the reaction between a compound and an analyte molecule. This reaction creates a covalent bond between the compound and the analyte. Therefore, the term “complex” refers to the covalently bonded reaction product formed by the reaction between a compound and an analyte molecule. In the context of the present invention, the method of the first aspect of the present invention may also be referred to as a method having a cross-flow spray configuration. In the context of the present invention, the diagnostic system of the third aspect of the present invention may also be referred to as a system having a cross-flow spray configuration.

[0068] Embodiment In a first aspect, the present invention relates to a method for determining the presence or level of a target analyte in a sample, a) A step of supplying a first electrospray ionization source for a first stream of analytes containing the target analyte, b) A step of supplying a second electrospray ionization source for a second analyte stream containing a dopant or derivatization reagent, wherein each of the first analyte stream and the second analyte stream is contained in gaseous form or aerosol, c) A step of mixing a stream of a first analyte and a stream of a second analyte to form a mixture or a derivatized analyte of interest, wherein the mixture comprises the analyte of interest and a dopant, and d) A step of determining the presence or level of the target analyte in the sample using mass spectrometry. This includes methods.

[0069] In an embodiment of the first aspect of the present invention, the amount, concentration, or level of an analyte, particularly the relative amount of the analyte in a pre-treated sample, can be determined. This method is highly accurate and, when repeatedly determining the amount of analyte, gives a coefficient of variation (CV) of 20% or less, particularly 10% or less, more specifically 2% or less, for example, 1% to 2%.

[0070] In the first embodiment of the present invention, the sample may be a pre-treated sample.

[0071] In the first embodiment of the present invention, the method is an in vitro method.

[0072] In an embodiment of the first aspect of the present invention, the sample is obtained from a patient sample selected from the group consisting of serum, plasma, and whole blood samples from an individual.

[0073] In the first embodiment of the present invention, the sample is a human sample, preferably a hemolytic whole blood sample, and more particularly a hemolytic human whole blood sample.

[0074] In embodiments of the first aspect of the present invention, the sample is a hemolyzed whole blood sample, particularly a hemolyzed human whole blood sample, derived from, for example, blood, the subject to be tested for the amount of analyte of interest. Hemolysis is carried out, in particular, by dilution with water (H2O), such as deionized water or distilled water, in a sample:water ratio of about 1:2 to about 1:20, particularly about 1:5 to about 1:10, and particularly about 1:9 (v / v). The sample may be hemolyzed for a time of less than about 30 minutes, less than about 10 minutes, less than about 5 minutes, or even less than 2 minutes. In certain embodiments, the sample is hemolyzed for a time of about 10 to about 60 seconds.

[0075] In certain embodiments, hemolysis is performed by mixing the sample with water, particularly by vortexing the sample with water. Specifically, the sample and water are mixed for about 1 to about 60 seconds, particularly for about 5 to about 30 seconds, particularly for about 10 seconds, and particularly vortexed.

[0076] During hemolysis, the sample can be maintained at a temperature of 20°C to 30°C, especially 22°C to 25°C, and especially room temperature.

[0077] In certain embodiments, hemolysis of the sample is performed by mixing the sample with water in a 1:9 ratio by vortexing at room temperature for 10 seconds.

[0078] According to step (a), a first electrospray ionization source is provided. The first electrospray ionization source is for a first analyte stream. The first analyte stream contains the analyte of interest.

[0079] In an embodiment of the first aspect of the present invention, the first electrospray ionization source is designed so that ions are generated by an ESI support tip by forming a Taylor cone that can lead the generated ions to the inlet of a mass spectrometer. The ion source may be sealed as well as connected to ambient air. The material may be stainless steel, glass, or a nonpolar material, which generally does not disturb the electric field.

[0080] In an embodiment of the first aspect of the present invention, the first analyte stream is contained in gaseous form or aerosol.

[0081] According to step (b), a second electrospray ionization source is provided. The second electrospray ionization source is for a second analyte stream. The second analyte stream contains a dopant or derivatization reagent.

[0082] In an embodiment of the first aspect of the present invention, the flow of the second analyte is contained in gaseous form or aerosol.

[0083] According to step (c), a stream of the first analyte and a stream of the second analyte are mixed to form a mixture or the derivatized analyte of the choice. The mixture contains the analyte of the choice and a dopant. The cross-mixing step preferably includes crossing the stream of the first analyte and the stream of the second analyte for signal enhancement.

[0084] In an embodiment of the first aspect of the present invention, the mixture is formed in step (c). The mixture comprises or consists of at least one analyte of interest and a dopant.

[0085] In an embodiment of the first aspect of the present invention, the desired derivatized analyte or at least the desired derivatized analyte is formed in step (c). The desired derivatized analyte is obtained by reacting at least one desired analyte with a derivatizing reagent.

[0086] In an embodiment of the first aspect of the present invention, an internal standard is provided. The internal standard (preferably an isotope-labeled analyte) is dissolved in a suitable solvent and added to the sample at a specified concentration.

[0087] In an embodiment of the first aspect of the present invention, the method further includes step c1) after step c). c1) The step of introducing the target mixture or derivatized analyte into a unit for mass spectrometry.

[0088] In an embodiment of the first aspect of the present invention, the method is carried out to enhance the signal, preferably by increasing the number of analytes ionized, in order to increase the sensitivity of mass spectrometry detection.

[0089] In an embodiment of the first aspect of the present invention, the method is performed to enhance the mass spectrometry signal of the target analyte.

[0090] In the first embodiment of the present invention, in step b), a derivatizing reagent for a chemical reaction with the target analyte is added.

[0091] In the first embodiment of the present invention, a dopant for ion enhancement is added in step b).

[0092] In an embodiment of the first aspect of the present invention, the first analyte stream is preferably chromatographically purified before it passes through the first electrospray ionization source.

[0093] In an embodiment of the first aspect of the present invention, the first analyte stream is mixed with the second analyte stream after exiting the first and second electrospray ionization sources.

[0094] In an embodiment of the first aspect of the present invention, the flow of the first analyte and / or the flow of the second analyte have a flow rate in the range of 1 to 1000 μL / min, preferably in the range of 50 to 500 μL / min.

[0095] In an embodiment of the first aspect of the present invention, the first electrospray ionization source and / or the second electrospray ionization source is a borosilicate glass capillary and / or a metal-based capillary.

[0096] In an embodiment of the first aspect of the present invention, the first electrospray ionization source and / or the second electrospray ionization source may be ESI or nanoESI.

[0097] In an embodiment of the first aspect of the present invention, the first electrospray ionization source and the second electrospray ionization source are arranged at an angle of at least 10° to 180° according to the plan view of the two electrospray ionization sources, and at an angle of at least 10° to 180° according to the front view of the electrospray ionization source. Preferably, the first electrospray ionization source and the second electrospray ionization source are arranged at an angle of at least 70° to 100°, e.g., 90°, according to the plan view of the two electrospray ionization sources, and / or at an angle of at least 70° to 100°, e.g., 90°, according to the front view of the electrospray ionization source.

[0098] In an embodiment of the first aspect of the present invention, the flow of the first analyte and the flow of the second analyte are mixed without being obstructed by a rotatable baffle, preferably.

[0099] In the first embodiment of the present invention, the dopant is DMSO or NH4F.

[0100] In an embodiment of the first aspect of the present invention, the second flow includes a dopant or derivatizing reagent in a concentration in the range of 5 to 35% (v / v).

[0101] In the first embodiment of the present invention, the dopant is DMSO having a concentration in the range of 5 to 35% (v / v).

[0102] In an embodiment of a first aspect of the present invention, prior to step (a), a stream of a first analyte containing the analyte of interest undergoes a chromatography step comprising at least one method selected from the group consisting of chromatography, high-performance liquid chromatography (HPLC), liquid chromatography-high-performance liquid chromatography (LC-HPLC), gel permeation chromatography (GPC), and flash chromatography. The chromatography is, for example, size exclusion chromatography.

[0103] In the first embodiment of the present invention, the method is automated.

[0104] According to step (b), the target analyte in the sample is derivatized.

[0105] In an embodiment of the first aspect of the present invention, step (b) is carried out with a derivatizing reagent.

[0106] In an embodiment of the first aspect of the present invention, step (b) is performed within a time range of up to 5 minutes, preferably up to 3 minutes, and more preferably up to 2 minutes.

[0107] In an embodiment of the first aspect of the present invention, the compound is capable of covalently bonding to the analyte, or covalently bonding to the analyte.

[0108] In an embodiment of the first aspect of the present invention, the analyte of interest is derivatized in step (b) with a derivatizing reagent capable of forming a covalent bond to the analyte of interest, and in particular, after step (b), the derivatizing reagent is covalently bonded to the analyte of interest to form a derivatized analyte of interest. A complex of the analyte and the compound is formed.

[0109] In the first embodiment of the present invention, the derivatization reagent is a simple permanent positive charge or a simple permanent negative charge.

[0110] In the first embodiment of the present invention, the derivatization reagent is a double permanent positive charge or a double permanent negative charge.

[0111] In an embodiment of the first aspect of the present invention, the derivatization reagent comprises, for example, three or more permanent positive charges such as 3, 4, 5, 6, or 7, or three or more permanent negative charges such as 3, 4, 5, 6, or 7.

[0112] In the first embodiment of the present invention, the derivatization reagent does not contain a permanent charge.

[0113] In an embodiment of the first aspect of the present invention, the derivatization reagent has a net charge z1, particularly before fragmentation. After fragmentation, the compound can be split or cleaved into at least one daughter ion. The daughter ion has a net charge z2, which is smaller than the net charge z1 (z2 < z1). The complex containing or constituting the analyte and the compound has a net charge z3, particularly before fragmentation. After fragmentation, the complex can be split or cleaved into at least one daughter ion having a net charge z4 (z4 < z3) that is smaller than the net charge z3. At least one daughter ion can, in this context, mean that one or more daughter ions are formed after fragmentation. One daughter ion and other daughter ions are distinguishable from each other at least by their mass, charge or structure.

[0114] In an embodiment of the first aspect of the present invention, the derivatization reagent contains a permanent charge, particularly a permanent net positive charge, and the compound can covalently bond to the analyte of interest. The derivatization reagent has a mass m1 and a net charge z1. After fragmentation by mass spectrometry measurement, the derivatization reagent can form at least one daughter ion having a mass m2 < m1 and a net charge z2 < z1. m1 / z1 < m2 / z2.

[0115] In an embodiment of the first aspect of the present invention, the compound is in the following group:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0116] In an embodiment of the first aspect of the present invention, the derivatization reagent comprises a reaction unit K that can react with a carbonyl group, phenol group, amine, hydroxyl group, or diene group of the analyte of interest.

[0117] In the first embodiment of the present invention, K is selected from the group consisting of hydrazides, hydrazines, hydroxylamines, Br, F-aromatics, 4-substituted 1,2,4-triazolin-3,5-diones (TAD), active esters, sulfonyl chlorides, and reactive carbonyls.

[0118] In the first embodiment of the present invention, the derivatizing reagent includes a counterion for forming a salt, and the counterion is preferably from the following group:Cl - , Br - F - formate, trifluoroacetate, PF6 - Selected from sulfonates, phosphates, and acetates.

[0119] In the first embodiment of the present invention, step b) is carried out at a temperature of at least 20°C or higher.

[0120] In the first embodiment of the present invention, step b) is carried out at a temperature of at least 30°C, for example, 35°C.

[0121] In an embodiment of the first aspect of the present invention, step b) is carried out at a temperature of at least 40°C, for example, 45°C.

[0122] In an embodiment of the first aspect of the present invention, step b) is carried out at a temperature of at least 50°C, for example, 55°C.

[0123] In the first embodiment of the present invention, step b) is performed at a temperature of at least 60°C, for example, 65°C.

[0124] In the first embodiment of the present invention, step b) is performed at a temperature of at least 70°C, for example, 75°C.

[0125] In the first embodiment of the present invention, step b) is performed at a temperature of at least 80°C, for example, 85°C.

[0126] In the embodiment of the first aspect of the present invention, step b) includes the addition of further substances or further substances. These further substances or further substances are, for example, additives. The further substances or further substances are, for example, for protonation and / or catalysis. In particular, the further substances or further substances for catalysis are Lewis bases or a plurality of Lewis bases.

[0127] In an embodiment of the first aspect of the present invention, the further substance for protonation or further substance is selected from the group consisting of protonated organic acids, such as formic acid.

[0128] In the first embodiment of the present invention, further substances for catalytic action or further substances are selected from the group consisting of Lewis bases, for example, phenylenediamine.

[0129] In the first embodiment of the present invention, the derivatization reagent is of formula A or B: [ka] (In the formula, X is a reactive unit that can form a covalent bond with the analyte of interest, L1 and L2 are independent of each other, and can be substituted or not substituted. Linkers, especially branched or linear linkers, Y is a neutral loss unit, Z is a charged unit that includes at least one permanently charged portion, in particular one permanently charged portion. This includes any salt.

[0130] In the first embodiment of the present invention, the derivatization reagent of formula A is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] Alternatively, a selection is made from a group consisting of combinations of these.

[0131] In the first embodiment of the present invention, the derivatization reagent of formula B is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

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[0132] In an embodiment of the first aspect of the present invention, the derivatization reagent is selected from the group consisting of dansilloride, carbamic acid, N-[2-[[[2-(diethylamino)ethyl]amino]carbonyl]-6-quinolinyl]-, 2,5-dioxo-1-pyrrolidinyl ester (RapiFluor-MS), 4-substituted 1,2,4-triazolin-3,5-dione (Cockson-type reagent), 4-phenyl-1,2,4-triazolin-3,5-dione derivative (Amplifex Diene), 1-propaneaminium, 3-(aminooxy)-N,N,N-trimethyl compound containing a suitable counterion (Amplifex Keto), acetyldrazide trimethylammonium chloride (Girard T), 1-(carboxymethyl)pyridinium chloride hydrazide (Girard P), and pyridylamine.

[0133] In an embodiment of the first aspect of the present invention, at least one possible chemical structure of the derivatizing reagent is [ka] [ka] [ka] [ka] [ka] That is the case.

[0134] In an embodiment of the first aspect of the present invention, the method is expressed by formula PI: [ka] (In the formula, one of the substituents B1, B2, B3, B4, B5 is a coupling group Q that can form a covalent bond with the analyte, The other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, allyroyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, allyroyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotopes, or derivatives thereof. Y1 and Y2 are independently selected from hydrogen, methyl, ethyl, methoxy, substituted aromatic, unsubstituted aromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heteroaromatic, unsubstituted heteroaromatic, or amine, or Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, or unsubstituted heteroaromatic. It contains the compound.

[0135] In an embodiment of a first aspect of the present invention, A3 is ammonium, and B1 or B5 is Q, where Q does not contain at least one atom selected from O, N, S, and Br. The phrase "Q does not contain at least one atom selected from O, N, S, and Br" means, in this specification and in the context of this disclosure, that Q does not contain O or N or S or Br or any combination thereof. In particular, Q does not contain O, N, S, and Br. In particular, if A3 is ammonium and B1 is Q, then Q does not contain O, N, S, and Br. Or, if A3 is ammonium and B5 is Q, then Q does not contain O, N, S, and Br.

[0136] In the first embodiment of the present invention, the derivatization reagent of formula PI is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] Alternatively, a selection is made from a group consisting of combinations of these.

[0137] In an embodiment of the first aspect of the present invention, the method is expressed by formula DI: [ka] (In the formula, one of the substituents B1, B2, and B4 is a coupling group Q that can form a covalent bond with the analyte, The other substituents A1, A2, A3, A4, A5, B1, B2, B4 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, allyroyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, allyroyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotopes, or derivatives thereof. B3 is selected from alkyl, acetyl, vinyl, substituted aromatic, unsubstituted aromatic, substituted benzyl, unsubstituted benzyl, substituted cycloalkyl, unsubstituted cycloalkyl, isotopes and their derivatives. Y1 and Y2 are independently selected from hydrogen, methyl, ethyl, methoxy, substituted aromatic, unsubstituted aromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heteroaromatic, unsubstituted heteroaromatic, or amine, or Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, or unsubstituted heteroaromatic. It contains a derivatization reagent.

[0138] In the first embodiment of the present invention, the derivatization reagent of formula DI is [ka] [ka] Alternatively, a selection is made from a group consisting of combinations of these.

[0139] In an embodiment of the first aspect of the present invention, the method is expressed by formula CI: [ka] (In the formula, one of the substituents B1, B2, B3, B4, B5 is a coupling group Q that can form a covalent bond with the analyte, The other substituents A1, A2, B1, B2, B3, B4, and B5 are each independently selected from hydrogen, halogen, alkyl, modified alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, allyroyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, allyroyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, sulfur, isotopes, or derivatives thereof. A3 comprises ammonium, pyridinium, phosphonium, or derivatives thereof. If A3 is ammonium and B1 or B5 is coupling group Q, coupling group Q contains four carbon atoms separated from the carbon atom of the CA1A2A3 substituent by single or double bonds, and coupling group Q contains five carbon atoms separated from the carbon atom of the CA1A2A3 substituent by single or double bonds. It contains a derivatization reagent.

[0140] In the first embodiment of the present invention, the derivatization reagent of formula CI is [ka] [ka]

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[0141] In an embodiment of the first aspect of the present invention, in step (b), the ratio of the target analyte to the compound is within the range of 1:1 to 1:6,000,000. In particular, the ratio of the target analyte to the compound is within the range of 1:50,000 to 1:100,000, or 1:5,000 to 1:10,000, or 1:1 to 1:100, or 1:100 to 1:1,000, or 1:1,000,000 to 1:2,000,000. This ratio depends on the type of reaction, the compound (derivatization reagent), the reaction kinetics such as the reaction rate, and / or the temperature. The compound may be provided in excess compared to the analyte.

[0142] In an embodiment of the first aspect of the present invention, the target analyte is selected from the group consisting of nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, seco-steroids, molecules characterized by specific modifications of another molecule, substances internalized by a living body, metabolites of such substances, and combinations thereof.

[0143] In the first embodiment of the present invention, the analyte molecule contains a functional group selected from the group consisting of carbonyl groups, diene groups, hydroxyl groups, amine groups, imine groups, ketone groups, aldehyde groups, thiol groups, diol groups, phenol groups, epoxy groups, disulfide groups, nucleic acid base groups, carboxylic acid groups, terminal cysteine ​​groups, terminal serine groups, and azide groups, each of which can form a covalent bond with the reactive unit K of the compound. Furthermore, it is also considered within the scope of the present invention that the functional groups present on the analyte molecule are first converted into other groups that can be easily utilized by reaction with the reactive unit K of the compound.

[0144] In the first embodiment of the present invention, the analyte molecule contains a carbonyl group as a functional group selected from the group consisting of carboxylic acid groups, aldehyde groups, keto groups, masked aldehydes, masked keto groups, ester groups, amide groups, and anhydride groups. Aldoses (aldehydes and ketos) exist as acetals and hemiacetals, which are a type of masked form of the parent aldehyde / keto.

[0145] In the first embodiment of the present invention, the carbonyl group is an amide group, and those skilled in the art are well aware that while the amide group itself is a stable group, it can be hydrolyzed to a carboxylic acid group and an amino group. Hydrolysis of the amide group can be achieved by an acid / base catalyzed reaction or by an enzymatic process, both of which are well known to those skilled in the art. In the first embodiment of the present invention, where the carbonyl group is a masked aldehyde group or a masked keto group, the respective group is either a hemiacetal group or an acetal group, particularly a cyclic hemiacetal group or an acetal group. In the first embodiment of the present invention, the acetal group is converted to an aldehyde group or a keto group before reacting with the compound.

[0146] In the first embodiment of the present invention, the carbonyl group is a keto group. In the first embodiment of the present invention, the keto group can be transferred to an intermediate imine group before reacting with the reactive units of the compound. In the first embodiment of the present invention, the analyte molecule containing one or more keto groups is a ketosteroid. In a particular embodiment of the first aspect of the present invention, the ketosteroid is selected from the group consisting of testosterone, epitestosterone, dihydrotestosterone (DHT), deoxymethyltestosterone (DMT), tetrahydrogestrinone (THG), aldosterone, estrone, 4-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone, 16-ketoestradiol, 16-alpha-hydroxyestrone, 2-hydroxyestrone-3-methyl ether, prednisone, prednisolone, pregnenolone, progesterone, dehydroepiandrosterone (DHEA), 17-hydroxypregnenolone, 17-hydroxyprogesterone, androsterone, epiandrosterone, Δ4-androstenedione, 11-deoxycortisol, corticosterone, 21-deoxycortisol, 11-deoxycorticosterone, allopregnanolone, and aldosterone.

[0147] In the first embodiment of the present invention, the carbonyl group is a carboxyl group. In the first embodiment of the present invention, the carboxyl group either reacts directly with the compound or is converted to an activated ester group before reacting with the compound. In the first embodiment of the present invention, the analyte molecule containing one or more carboxyl groups is selected from the group consisting of Δ8-tetrahydrocannabinolic acid, benzoylecgonine, salicylic acid, 2-hydroxybenzoic acid, gabapentin, pregabalin, valproic acid, vancomycin, methotrexate, mycophenolic acid, montelukast, repaglinide, furosemide, telmisartan, gemfibrozil, diclofenac, ibuprofen, indomethacin, zomepirac, isoxepac, and penicillin. In the first embodiment of the present invention, the analyte molecule containing one or more carboxyl groups is an amino acid selected from the group consisting of arginine, lysine, aspartic acid, glutamic acid, glutamine, asparagine, histidine, serine, threonine, tyrosine, cysteine, tryptophan, alanine, isoleucine, leucine, methionine, phenylalanine, valine, proline, and glycine.

[0148] In the first embodiment of the present invention, the carbonyl group is an aldehyde group. In the first embodiment of the present invention, the aldehyde group can be transferred to an intermediate imine group before reacting with the reactive units of the compound. In the first embodiment of the present invention, the analyte molecule containing one or more aldehyde groups is selected from the group consisting of pyridoxal, N-acetyl-D-glucosamine, alkafutazine, streptomycin, and josamycin.

[0149] In the first embodiment of the present invention, the carbonyl group is a carbonyl ester group. In the first embodiment of the present invention, the analyte molecule containing one or more ester groups is selected from the group consisting of cocaine, heroin, ritalin, aceclofenac, acetylcholine, amcinonide, amyloxate, amylocaine, anirelizine, arnidipine artesunate, and pethidine.

[0150] In the first embodiment of the present invention, the carbonyl group is an anhydride. In the first embodiment of the present invention, the analyte molecule containing one or more anhydrides is selected from the group consisting of cantharidin, succinic anhydride, trimellitic anhydride, and maleic anhydride.

[0151] In the first embodiment of the present invention, the analyte molecule comprises one or more diene groups, particularly conjugated diene groups, as functional groups. In the first embodiment of the present invention, the analyte molecule comprising one or more diene groups is a secosteroid. In the embodiment, the secosteroid is selected from the group consisting of cholecalciferol (vitamin D3), ergocalciferol (vitamin D2), calcifediol, calcitriol, tachysterol, lumisterol, and tacalcitol. In particular, the secosteroid is vitamin D, particularly vitamin D2 or D3, or derivatives thereof. In certain embodiments, the secosteroid is selected from the group consisting of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 (calcifediol), 3-epi-25-hydroxyvitamin D2, 3-epi-25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3 (calcitriol), 24,25-dihydroxyvitamin D2, and 24,25-dihydroxyvitamin D3. In embodiments of the first aspect of the present invention, the analyte molecule containing one or more diene groups is selected from the group consisting of vitamin A, tretinoin, isotretinoin, alitretinoin, natamycin, sirolimus, amphotericin B, nystatin, everolimus, temsirolimus, and fidaxomicin.

[0152] In embodiments of the first aspect of the present invention, the analyte molecule contains one or more hydroxyl groups as functional groups. In embodiments of the first aspect of the present invention, the analyte molecule contains a single hydroxyl group or two hydroxyl groups. In embodiments where multiple hydroxyl groups are present, the two hydroxyl groups (1,2-diol) may be adjacent to each other or may be separated by one, two, or three carbon atoms (1,3-diol, 1,4-diol, and 1,5-diol, respectively). In specific embodiments of the first aspect, the analyte molecule contains a 1,2-diol group. In embodiments where only one hydroxyl group is present, the analyte is selected from the group consisting of primary alcohols, secondary alcohols, and tertiary alcohols. In embodiments of the first aspect of the present invention, the analyte molecule comprises one or more hydroxyl groups, and the analyte is selected from the group consisting of benzyl alcohol, menthol, L-carnitine, pyridoxine, metronidazole, isosorbide mononitrate, guaifenesin, clavulanic acid, miglitol, zalcitabine, isoprenaline, acyclovir, methocarbamol, tramadol, venlafaxine, atropine, clofedanol, alpha-hydroxyalprazolam, alpha-hydroxytriazolam, lorazepam, oxazepam, temazepam, ethyl glucuronide, ethyl morphine, morphine, morphine-3-glucuronide, buprenorphine, codeine, dihydrocodeine, p-hydroxypropoxyfen, O-desmethyltramadol, desmethadol, dihydroquinidine, and quinidine.In an embodiment of the first aspect of the present invention, the analyte molecule comprises two or more hydroxyl groups, and the analyte is vitamin C, glucosamine, mannitol, tetrahydrovepterin, cytarabine, azacitidine, ribavirin, phloxuridine, gemcitabine, streptozotocin, adenosine, vidarabine, cladribine, estriol, trifluridine, clofarabine, nadolol, zanamivir, lactulose, adenosine monophosphate, idoxuridine, regadenosone, lincomycin, clindamycin, canaglyph The following are selected from the group consisting of rosin, tobramycin, netylmycin, kanamycin, ticagrelor, epirubicin, doxorubicin, arbekacin, streptomycin, ouabain, amikacin, neomycin, flamycetin, palymosetin, erythromycin, clarithromycin, azithromycin, vindesine, digitoxin, digoxin, metrizamide, acetyldigitoxin, deslanoside, fludarabine, clofarabine, gemcitabine, cytarabine, capecitabine, vidarabine, and plicamycin.

[0153] In the first embodiment of the present invention, the analyte molecule contains one or more thiol groups (including, but not limited to, alkylthiol and arylthiol groups) as functional groups. In the first embodiment of the present invention, the analyte molecule containing one or more thiol groups is selected from the group consisting of thiomandelic acid, DL-captopril, DL-thiorphan, N-acetylcysteine, D-penicillamine, glutathione, L-cysteine, zofenoprilat, thiopronine, dimercaprol, and succimer.

[0154] In the first embodiment of the present invention, the analyte molecule contains one or more disulfide groups as functional groups. In the first embodiment of the present invention, the analyte molecule containing one or more disulfide groups is selected from the group consisting of glutathione disulfide, dipyrithione, selenium sulfide, disulfiram, lipoic acid, L-cystine, fursultiamine, octreotide, desmopressin, vapreotide, terlipressin, linaclotide, and peginesatide. Selenium sulfide may be selenium disulfide, SeS2, or selenium hexasulfide, Se2S6.

[0155] In the first embodiment of the present invention, the analyte molecule comprises one or more epoxide groups as functional groups. In the first embodiment of the present invention, the analyte molecule comprising one or more epoxide groups is selected from the group consisting of carbamazepine-10,11-epoxide, carfilzomib, furosemide epoxide, fosfomycin, sevelamer hydrochloride, cerurenin, scopolamine, tiotropium, tiotropium bromide, methylscopolamine bromide, eplerenone, mupirocin, natamycin, and troleandmycin.

[0156] In an embodiment of the first aspect of the present invention, the analyte molecule contains one or more phenol groups as functional groups. In a specific embodiment of the first aspect of the present invention, the analyte molecule containing one or more phenol groups is a steroid or a steroid-like compound. In an embodiment of the first aspect of the present invention, the analyte molecule containing one or more phenol groups is sp 2The steroid or steroid-like compound has a hybridizing A ring and an OH group at the 3-position of the A ring. In a particular embodiment of the first aspect of the present invention, the steroid or steroid-like analyte molecule is selected from the group consisting of estrogen, estrogen-like compounds, estrone (E1), estradiol (E2), 17a-estradiol, 17b-estradiol, estriol (E3), 16-epiestriol, 17-epiestriol, and 16,17-epiestriol and / or their metabolites. In the embodiment, the metabolites are estriol, 16-epiestriol (16-epiE3), 17-epiestriol (17-epiE3), 16,17-epiestriol (16,17-epiE3), 16-ketoestradiol (16-ketoE2), 16a-hydroxyestrone (16a-OHEl), 2-methoxyestrone (2-MeOEl), 4-methoxyestrone (4-MeOEl), 2-hydroxyestrone-3-methyl ether (3-MeOEl), 2-methoxyestradiol (2-MeOE2), 4-methoxyestradiol (4-MeOE2), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2-hydroxyestradiol A selection of compounds from the group consisting of ol (2-OHE2), estrone (El), estrone sulfate (Els), 17a-estradiol (E2a), 17b-estradiol (E2B), estradiol sulfate (E2S), equine (EQ), 17a-dihydroequine (EQa), 17b-dihydroequine (EQb), equrenin (EN), 17-dihydroequrenin (ENa), 17α-dihydroequrenin, 17β-dihydroequrenin (ENb), Δ8,9-dihydroestrone (dEl), Δ8,9-dihydroestrone sulfate (dEls), Δ9-tetrahydrocannabinol, and mycophenolic acid, wherein β or b may be used interchangeably, and α and a may be used interchangeably.

[0157] In the embodiment of the first aspect of the present invention, the analyte molecule contains an amine group as a functional group. In the embodiment of the first aspect of the present invention, the amine group is an alkylamine group or an arylamine group. In the embodiment of the first aspect of the present invention, the analyte molecule containing one or more amine groups is selected from the group consisting of proteins and peptides. In the embodiment of the first aspect of the present invention, the analyte molecule containing an amine group is 3,4-methylenedioxyamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, 3,4-methylenedioxymethamphetamine, amphetamine, methamphetamine, N-methyl-1,3-benzodioxolylbutanamine, 7-aminoclonazepam, 7-aminoflunitrazepam, 3,4-dimethylmethcathinone, 3-fluorometh Cathinone, 4-Methoxymethcathinone, 4-Methylethcathinone, 4-Methylmethcathinone, Amphepramon, Butyron, Ethcathinone, Elephehedron, Methcathinone, Methylone, Methylenedioxypyrovalerone, Benzoylecgonine, Dehydronorketamine, Ketamine, Norketamine, Methadone, Normethadone, 6-Acetylmorphine, Diacetylmorphine, Morphine, Norhydrocodone, Oxycodone, Oxy The following are selected from the group consisting of morphone, phencyclidine, norpropoxifen, amitriptyline, clomipramine, dotiepin, doxepin, imipramine, nortriptyline, trimipramine, fentanyl, glycylxylidide, lidocaine, monoethylglycylxylidide, N-acetylprocainamide, procainamide, pregabalin, 2-methylamino-1-(3,4-methylenedioxyphenyl)butane, N-methyl-1,3-benzodioxolylbutanamine, 2-amino-1-(3,4-methylenedioxyphenyl)butane, 1,3-benzodioxolylbutanamine, normeperidine, O-destramadol, desmethamadol, tramadol, lamotrigine, theophylline, amikacin, gentamicin, tobramycin, vancomycin, methotrexate, gabapentin cystocyane, and 5-methylcystocyane.

[0158] In the first embodiment of the present invention, the analyte molecule is a carbohydrate, or a substance having a carbohydrate portion, such as a glycoprotein or nucleoside. In the first embodiment of the present invention, the analyte molecule is selected from monosaccharides, particularly from the group consisting of ribose, deoxyribose, arabinose, ribulose, glucose, mannose, galactose, fucose, fructose, N-acetylglucosamine, N-acetylgalactosamine, neuraminic acid, N-acetylneuraminic acid, etc. In the embodiment, the analyte molecule is selected from the group consisting of oligosaccharides, particularly from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and polysaccharides. In the first embodiment of the present invention, the disaccharides are selected from the group consisting of sucrose, maltose, and lactose. In the first embodiment of the present invention, the analyte molecule is a substance containing the above-mentioned monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharide portions.

[0159] In the first embodiment of the present invention, the analyte molecule includes an azide group as a functional group selected from groups consisting of alkyl or aryl azides. In the first embodiment of the present invention, the analyte molecule containing one or more azide groups is selected from the group consisting of zidovudine and azidocillin.

[0160] Such analyte molecules may be present in biological or clinical samples such as body fluids, e.g., blood, serum, plasma, urine, saliva, cerebrospinal fluid, tissue or cell extracts. In embodiments of the first aspect of the present invention, the analyte molecules are present in biological or clinical samples selected from the group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid, and dried blood spots. In some embodiments of the first aspect of the present invention, the analyte molecules may be present in purified or partially purified samples, e.g., samples that are purified or partially purified protein mixtures or extracts.

[0161] In an embodiment of the first aspect of the present invention, the reactive unit K is selected from the group consisting of carbonyl reactive units, diene reactive units, hydroxyl reactive units, amino reactive units, imine reactive units, thiol reactive units, diol reactive units, phenol reactive units, epoxide reactive units, disulfide reactive units, and azide reactive units.

[0162] In the first embodiment of the present invention, the reactive unit K is a carbonyl reactive unit that can react with any type of molecule having a carbonyl group. In the first embodiment of the present invention, the carbonyl reactive unit is selected from the group consisting of carboxyl reactive units, keto reactive units, aldehyde reactive units, anhydride reactive units, carbonyl ester reactive units, and imide reactive units. In the first embodiment of the present invention, the carbonyl reactive unit may have either an adjacent O or N atom NH2-N / O, or a supernucleophilic N atom enhanced by the α effect via a dithiol molecule.

[0163] In the first embodiment of the present invention, the carbonyl reactive unit is (i) Hydrazine units, e.g., H2N-NH- or H2N-NR1- units (wherein R1 is an aryl or C1-C4 alkyl, especially a C1 or C2 alkyl, and is optionally substituted), (ii) Hydrazide units, especially carbohydrazides or sulfohydrazides, especially H2N-NH-C(O)- or H2N-NR2-C(O)- units (wherein R2 is an aryl or C1-C4 alkyl, especially a C1 or C2 alkyl, and is optionally substituted), (iii) Hydroxylamino units, e.g., H2N-O- units, (iv) Dithiol units, especially 1,2-dithiol or 1,3-dithiol units It is selected from the group consisting of the following.

[0164] In the first embodiment of the present invention, the carbonyl-reactive unit is a carboxyl-reactive unit, and the carboxyl-reactive unit reacts with a carboxyl group on the analyte molecule. In the first embodiment of the present invention, the carboxyl-reactive unit is selected from the group consisting of diazo units, alkyl halides, amines, and hydrazine units.

[0165] In the first embodiment of the present invention, the analyte molecule contains a ketone or aldehyde group, and Q is a carbonyl reactive unit. (i) Hydrazine unit, (ii) Hydrazide unit, (iii) Hydroxylamino unit, and (iv) Dithiol units Selected from the group.

[0166] In the first embodiment of the present invention, the reactive unit K is a diene-reactive unit that can react with an analyte containing a diene group. In the first embodiment of the present invention, the diene-reactive unit is selected from the group consisting of Cookson-type reagents that can act as dienophiles, for example, 1,2,4-triazolin-3,5-dione.

[0167] In embodiments of the first aspect of the present invention, reactive unit K is a hydroxyl-reactive unit that can react with an analyte containing a hydroxyl group. In embodiments of the first aspect of the present invention, the hydroxyl-reactive unit is selected from the group consisting of sulfonyl chloride, activated carboxylic acid esters (NHS or imidazolides), and fluoroaromatic / heteroaromatic compounds that can undergo nucleophilic substitution of fluorine (T. Higashi, "J Steroid Biochem Mol Biol." (September 2016), Vol. 162, pp. 57-69). In embodiments of the first aspect of the present invention, reactive unit K is a diol-reactive unit that reacts with a diol group on the analyte molecule. In embodiments of the first aspect of the present invention where the reactive unit is a 1,2-diol-reactive unit, the 1,2-diol-reactive unit includes a boronic acid. In further embodiments, the diol may be oxidized to a ketone or aldehyde, which can then react with a ketone / aldehyde-reactive unit(s) K.

[0168] In the first embodiment of the present invention, the amino-reactive unit reacts with an amino group on the analyte molecule. In the first embodiment of the present invention, the amino-reactive unit is selected from the group consisting of active ester groups such as N-hydroxysuccinimide (NHS) ester or sulfo-NHS ester, pentafluorophenyl ester, carbonylimidazole ester, squalate ester, hydroxybenzotriazole (HOBt) ester, 1-hydroxy-7-azabenzotriazole (HOAt) ester, and sulfonyl chloride unit.

[0169] In the first embodiment of the present invention, the thiol-reactive unit reacts with a thiol group on the analyte molecule. In the first embodiment of the present invention, the thiol-reactive unit is selected from the group consisting of haloacetyl groups, which is selected from the group consisting of unsaturated imide units such as Br / I-CH2-C(=O)- units, acrylamide / ester units, maleimide, methylsulfonylphenyloxadiazole, and chloride sulfonyl units.

[0170] In the first embodiment of the present invention, the phenol-reactive unit reacts with the phenol group on the analyte molecule. In the first embodiment of the present invention, the phenol-reactive unit is selected from the group consisting of active ester units such as N-hydroxysuccinimide (NHS) ester or sulfo-NHS ester, pentafluorophenyl ester, carbonylimidazole ester, squalate ester, hydroxybenzotriazole (HOBt) ester, 1-hydroxy-7-azabenzotriazole (HOAt) ester, and sulfonyl chloride units. The phenol group present on the analyte molecule can be reacted with a highly reactive electrophile such as triazolinedione (TAD, etc.) via a reaction (H. Ban et al., J. Am. Chem. Soc., 2010, 132(5), pp 1523-1525), by diazotization, or by orthonitration and subsequent reduction to an amine, and then reacted with an amine-reactive reagent. In the first embodiment of the present invention, the phenol-reactive unit is fluoro-1-pyridinium.

[0171] In the first embodiment of the present invention, the reactive unit K is an epoxide reactive unit which can react with an analyte containing an epoxide group. In the first embodiment of the present invention, the epoxide reactive unit is selected from the group consisting of amino, thiol, and supernucleophilic N atoms, which are enhanced by the α effect via an adjacent O or N atom NH2-N / O molecule. In the first embodiment of the present invention, the epoxide reactive unit is Hydrazine units, e.g., H2N-NH- or H2N-NR 1 -Unit (in the formula, R 1 This includes aryls, aryls containing one or more heteroatoms, or C 1~4 Alkyl, particularly C1 or C2 alkyl, optionally halo, hydroxyl, and / or C 1~3 (substituted with alkoxy), Hydrazide units, especially carbohydrazide or sulfohydrazide units, especially H2N-NH-C(O)- or H2N-NR 2 -C(O)-unit (in the formula, R 2This includes aryls, aryls containing one or more heteroatoms, or C 1-4 Alkyl, particularly C1 or C2 alkyl, and optionally, for example, halo, hydroxyl, and / or C 1-3 (substituted with alkoxy), Hydroxylamino units, for example, H2N-O- units Selected from the group.

[0172] In an embodiment of the first aspect of the present invention, the reactive unit K is a disulfide-reactive unit that can react with an analyte containing a disulfide group. In an embodiment of the first aspect of the present invention, the disulfide-reactive unit is selected from the group consisting of thiols. In a further embodiment, the disulfide group can be reduced to its respective thiol group and then reacted with the thiol-reactive unit Q.

[0173] In the first embodiment of the present invention, the reactive unit K is either a thiol reactive group or an amino reactive group such as an active ester group, such as an N-hydroxysuccinimide (NHS) ester or sulfo-NHS ester, a hydroxybenzotriazole (HOBt) ester, or a 1-hydroxy-7-akabenzotriazole (HOAt) ester group.

[0174] In an embodiment of the first aspect of the present invention, the reactive unit K is selected from 4-substituted 1,2,4-triazolin-3,5-dione (TAD), 4-phenyl-1,2,4-triazolin-3,5-dione (PTAD), or fluorosubstituted pyridinium.

[0175] In the first embodiment of the present invention, the reactive unit K is an azide-reactive unit that reacts with an azide group on the analyte molecule. In the first embodiment of the present invention, the azide-reactive unit reacts with the azide group by azide-alkyne cycloaddition. In the first embodiment of the present invention, the azide-reactive unit is selected from the group consisting of alkynes (alkyl or aryl), linear alkynes, or cyclic alkynes. The reaction between the azide and the alkyne can proceed with or without the use of a catalyst. In a further embodiment of the first embodiment of the present invention, the azide group may be reduced to its respective amino group and then react with the amino-reactive unit K.

[0176] In the first embodiment of the present invention, the functional group of the analyte is selected from the options listed in the left column of Table 1. The reactive group Q of the corresponding functional group of the analyte is selected from the groups listed in the right column of Table 1. [Table 1]

[0177] In the first embodiment of the present invention, the analyte of interest is a peptide selected from the group that preferably exhibits a free -NH2 or -COOH group.

[0178] In an embodiment of the first aspect of the present invention, steps a), then b), then c), and then d) are carried out.

[0179] Surprisingly, instead of adding a dopant permanently to the eluent stream, a method of cross-spray configuration of two ESI analytes (i.e., the process of intermixing the first analyte stream and the second analyte stream to form the desired mixture or derivatized analyte) was found to be used. The two ESI analyte streams merge in the space in front of the ion source and exhibit either a chemical reaction or ion enhancement mode of action. Furthermore, the ability of the cross-spray configuration can be used to bring about chemical reactions within the space in front of the ESI source, as well as the effects of DMSO or other dopants in cross-flow on peptides and small polysaccharides or estradiol.

[0180] In a second aspect, the present invention relates to the use of the method of the first aspect of the present invention for determining the presence or level of an analyte of interest in a sample. All embodiments described in the first aspect of the present invention are applicable to the second aspect of the present invention, and vice versa.

[0181] In a third aspect, the present invention relates to a diagnostic system for determining the presence or level of an analyte of interest in a sample, comprising a first electrospray ionization source and a second electrospray ionization source, and a mass spectrometry unit for carrying out the method of the first aspect of the present invention. All embodiments described in relation to the first aspect and / or the second aspect of the present invention are applicable to the third aspect of the present invention, and vice versa.

[0182] In a third embodiment of the present invention, the diagnostic system is a clinical diagnostic system.

[0183] In a third embodiment of the present invention, the first and / or second electrospray ionization source may be, for example, Advion's chip-based electrospray ionization technology. The second electrospray ionization source for applying each dopant, for example, a second ESI sprayer, may be an ESI spray capillary or a second chip-based system, for example, Advion's. This combines the advantages of liquid chromatography, mass spectrometry, chip-based injection, fraction collection, and direct surface analysis into a single integrated ion source platform. Other known nanoESI sources are also possible. NanoESI sources are known to those skilled in the art and will therefore not be described in detail.

[0184] In a third embodiment of the present invention, the mass spectrometry unit may be, for example, a triple quadrupole mass spectrometer or a linear ion trap mass spectrometer. Mass spectrometers are known to those skilled in the art and will therefore not be described in detail.

[0185] A "clinical diagnostic system" is a laboratory automation device dedicated to the analysis of samples for in vitro diagnosis. A clinical diagnostic system may have different configurations as needed and / or according to a desired laboratory workflow. Further configurations can be obtained by combining multiple instruments and / or modules. A "module" is a working cell, typically smaller in size than the entire clinical diagnostic system, with a dedicated function. This function may be an analytical function, a pre-analysis or post-analysis function, or an auxiliary function to any of the pre-analysis, analytical, or post-analysis functions. In particular, a module may be configured to work in conjunction with one or more other modules to perform dedicated tasks in a sample processing workflow, for example, by performing one or more pre-analysis and / or analytical and / or post-analysis steps. Specifically, a clinical diagnostic system may include one or more analytical instruments designed to perform respective workflows optimized for specific types of analysis, such as clinical chemistry, immunochemistry, coagulation, hematology, liquid chromatography separation, and mass spectrometry. Therefore, a clinical diagnostic system may comprise either a single analyzer or a combination of such analyzers, each having its own workflow, and pre-analysis and / or post-analysis modules may be coupled to individual analyzers or shared by multiple analyzers. Alternatively, pre-analysis and / or post-analysis functions may be performed by units integrated into the analyzer. A clinical diagnostic system may comprise functional units such as liquid handling units for pipetting and / or pumping and / or mixing of samples and / or reagents and / or system fluids, and similarly, functional units for sorting, storing, transporting, identifying, separating, and detecting.

[0186] A clinical diagnostic system may include a sample preparation station for the automated preparation of samples containing the analyte of interest, a liquid chromatography (LC) separation station optionally having multiple LC channels, and / or a sample preparation / LC interface for inputting the prepared sample into any one of the LC channels. In particular, the clinical diagnostic system does not include a separation station, such as an LC-HPLC unit or an HPLC unit.

[0187] The clinical diagnostic system may further include a controller programmed to assign samples to a predefined sample preparation workflow, each containing a predefined sequence of sample preparation steps and requiring a predefined time for completion depending on the analyte of interest. The clinical diagnostic system may further include an LC / MS interface for connecting a mass spectrometer (MS) and an LC separation station to the mass spectrometer.

[0188] A "sample preparation station" is a pre-analysis module coupled to one or more analyzers or units within an analyzer, designed to perform a series of sample processing steps aimed at removing or at least reducing interfering matrix components in a sample and / or concentrating the analyte of interest in the sample. Such processing steps may include one or more of the following operations performed sequentially, in parallel, or with time staggering on a sample or multiple samples: pipetting of fluids (aspiration and / or delivery), pumping of fluids, mixing with reagents, incubation at a specific temperature, heating or cooling, centrifugation, separation, filtering, sieving, drying, washing, resuspension, preparation, transfer, and storage.

[0189] A "liquid chromatography (LC) separation station" is an analyzer, module, or unit within an analyzer designed to chromatographically separate a prepared sample in order to separate the analyte of interest from, for example, matrix components, such as remaining matrix components after sample preparation that may still interfere with subsequent detection, such as mass spectrometry detection, and / or to separate the analytes of interest from each other in order to enable their individual detection. According to one embodiment, the LC separation station is an intermediate analyzer, module, or unit within an analyzer designed to prepare a sample for mass spectrometry and / or transfer the prepared sample to a mass spectrometer. In particular, the LC separation station is a multi-channel LC station containing multiple LC channels. Preferably, the clinical diagnostic system does not include a liquid chromatography (LC) separation station.

[0190] A clinical diagnostic system, such as a sample preparation station, may also include a buffer unit for receiving multiple samples before a new sample preparation initiation sequence is started, and the samples may be individually and randomly accessible, with their individual preparations being initiated according to the sample preparation initiation sequence.

[0191] Clinical diagnostic systems make LC coupled to mass spectrometry more convenient, reliable, and therefore suitable for clinical diagnosis. In particular, enabling online coupling to mass spectrometry while allowing for high throughput, e.g., up to 100 samples / hour or more, through random-access sample preparation and LC separation. Furthermore, the process can be fully automated to improve yield and reduce the required skill level.

[0192] In a third embodiment of the present invention, the diagnostic system does not include a baffle, preferably a rotatable baffle, which is located between the outlet of a first electrospray ionizing capillary / ESI sprayer and the outlet of a second electrospray ionizing capillary / ESI sprayer. Commercial baffle systems in mass spectrometers, e.g., Waters or Micromass, are used to separate two or more ion spray tips that continuously apply ESI spray. Continuously flowing the spray avoids phase loss and is advantageous for intended use, where two separated ESI sprays, which are separated baffles, are applied to recalibrate the mass spectrometer. When a dopant spray setting is used, it is not important whether the dopant appling ESI-sprayer functions continuously.

[0193] In a fourth aspect, the present invention relates to a dopant for enhancing the sensitivity of mass spectrometry detection of an analyte of interest, comprising formula I: R1-SO2-R2 (I) (In the formula, R1 and R2 are each independently selected from alkyl or aryl, where alkyl contains 1 to 6 carbon atoms and aryl contains 1 to 6 carbon atoms.) The present invention relates to dopants, including the following: Preferably, the total number of carbon atoms (n), which is the sum of the carbon atoms of R1 and R2 (R1 and R2 may be aryl and alkyl), is at most n=12. More preferably, the sum of the carbon atoms of R1 and R2 (R1 and R2 may be aryl and / or alkyl), is at most n=11, 10, 9, 8, 7, 6, or 5.

[0194] All embodiments described in relation to the first and / or second and / or third aspects of the present invention are applicable to the fourth aspect of the present invention, and vice versa. Preferably, the dopant of the fourth aspect of the present invention is used in the manner of the first aspect of the present invention.

[0195] A dopant according to a fourth aspect of the present invention may be used in place of DMSO (as a reference) in an eluent stream or in the method according to a first aspect of the present invention or in the diagnostic system according to a third aspect of the present invention.

[0196] In a fourth embodiment of the present invention, the alkyl group may be selected from the group consisting of methyl, ethyl, butyl, pentyl-, and hexyl, and each has iso-alternatives and cyclo-alternatives (e.g., isobutyl or cyclohexyl).

[0197] In a fourth embodiment of the present invention, the aryl is a phenyl group. In an embodiment of the fourth aspect of the present invention, the dopant is selected from substituted sulfolanes, unsubstituted sulfolanes, substituted dibutylsulfones, unsubstituted dibutylsulfones, substituted diphenylsulfones, or unsubstituted diphenylsulfones.

[0198] In an embodiment of a fourth aspect of the present invention, the substituted sulfolane can be selected from the group consisting of 2-phenyl-2,3,4,5-tetrahydrothiophene-S,S-dioxide (13557-28-3), 3-phenyltetrahydrothiophene 1,1-dioxide (16766-64-6), sulfolane-2,2-5,5-d4 (20627-71-8), 3,3,4,4-D4-tetramethylenesulfone (31124-58-0), 2-benzylthiolane 1,1-dioxide (101944-68-7), 2,5-divinylsulfolane (76286-66-3), 3-cyclohexyl-tetrahydrothiophene-1,1-dioxide (71053-08-2), and 2-vinylthiolane 1,1-dioxide (71411-39-7). The numbers in parentheses indicate possible CAS numbers.

[0199] In a fourth embodiment of the present invention, the unsubstituted sulfolane may be selected from pentamethylene sulfone (4988-33-4) or hexamethyl sulfone (6251-33-8). The numbers in parentheses indicate possible CAS numbers.

[0200] In a fourth embodiment of the present invention, the substituted dibutylsulfone may be selected from the group consisting of diisoamylsulfone (2051-06-1), ethyl 3,3-dimethylbutylsulfone (92098-95-8), and 3,3-dimethyl-1-(propane-1-sulfonyl)-butane (91391-45-6). The numbers in parentheses indicate possible CAS numbers.

[0201] In the fourth embodiment of the present invention, the unsubstituted dibutylsulfone is dibutylsulfone.

[0202] In a fourth embodiment of the present invention, the substituted diphenyl sulfone may be selected from the group consisting of phenyl sulfone, (cyclopropylsulfonyl)benzene (17637-57-9), cyclohexylphenyl sulfone (6947-57-5), (cyclohexa-enylsulfonyl)benzene, 1-cyclopenten-1-ylphenyl sulfone (64740-90-5), (cyclopentylsulfonyl)benzene (14633-46-6), 1-(phenylsulfonyl)bicyclo[1.1.0]butane (80989-84-0), and ((cyclopropylmethyl)sulfonyl)benzene (69563-28-6). The numbers in parentheses indicate possible CAS numbers.

[0203] In the fourth embodiment of the present invention, the unsubstituted diphenyl sulfone is a diphenyl sulfone.

[0204] In a fourth embodiment of the present invention, the dopant is the following formula II to IV: [ka] [ka] [ka] Selected from.

[0205] In the fourth embodiment of the present invention, the dopant does not contain a sulfoxide, such as dimethyl sulfoxide (DMSO).

[0206] In a fifth aspect, the present invention relates to the use of the dopant of the fourth aspect of the present invention in a method according to the first aspect of the present invention.

[0207] In further embodiments, the present invention relates to the following aspects.

[0208] 1. A method for determining the presence or level of a target analyte in a sample, a) A step of supplying a first electrospray ionization source for a first stream of analytes containing the target analyte, b) A step of supplying a second electrospray ionization source for a second analyte stream containing a dopant or derivatization reagent, wherein each of the first analyte stream and the second analyte stream is contained in gaseous form or aerosol, c) A step of mixing a stream of a first analyte and a stream of a second analyte to form a mixture or a derivatized analyte of interest, wherein the mixture comprises the analyte of interest and a dopant, and d) A step of determining the presence or level of the target analyte in the sample using mass spectrometry. Methods that include...

[0209] 2. After step c), further step c1) c1) The process of introducing the target mixture or derivatized analyte into a unit for mass spectrometry. The method according to embodiment 1, including the method described in embodiment 1.

[0210] 3. The method according to embodiment 1 or 2, wherein the method is performed to enhance the sensitivity of mass spectrometry detection.

[0211] 4. The method according to any one of embodiments 1 to 3, performed to enhance the mass spectrometry signal of the analyte of interest.

[0212] 5. The method according to any one of embodiments 1 to 4, wherein in step b), a derivatizing reagent for a chemical reaction with the target analyte is added.

[0213] 6. The method according to any one of embodiments 1 to 5, wherein a dopant for ion enhancement is added in step b).

[0214] 7. The method according to any one of embodiments 1 to 6, wherein the flow of the first analyte is preferably chromatographically purified before the flow of the first analyte passes through the first electrospray ionization source.

[0215] 8. The method according to any one of embodiments 1 to 7, wherein the flow of the first analyte is mixed with the flow of the second analyte after it has exited the first electrospray ionization source and the second electrospray ionization source.

[0216] 9. The method according to any one of embodiments 1 to 8, wherein the flow of the first analyte and / or the flow of the second analyte has a flow rate in the range of 5 to 1000 μL / min.

[0217] 10. The method according to any one of embodiments 1 to 9, wherein the first electrospray ionization source and / or the second electrospray ionization source is a borosilicate glass capillary and / or a metal-based capillary.

[0218] 11. The method according to any one of embodiments 1 to 10, wherein the first electrospray ionization source and the second electrospray ionization source are arranged at an angle of at least 10° to 180° according to the plan view of the two electrospray ionization sources, and, for the electrospray ionization source, at an angle of at least 10° to 180° according to the front view.

[0219] 12. The method according to any one of embodiments 1 to 11, wherein the flow of a first analyte and the flow of a second analyte are mixed without being obstructed by a rotatable baffle.

[0220] 13. The method according to any one of embodiments 1 to 12, wherein the dopant is DMSO or NH4F.

[0221] 14. The method according to any one of embodiments 1 to 13, wherein the second flow comprises a dopant or derivatizing reagent in a concentration in the range of 5 to 35% (v / v).

[0222] 15. The method according to any one of embodiments 1 to 14, wherein the dopant is DMSO having a concentration in the range of 5 to 35% (v / v).

[0223] 16. The method according to any one of embodiments 1 to 15, wherein the derivatization reagent is selected from the group consisting of dansilchloride, carbamic acid, N-[2-[[[2-(diethylamino)ethyl]amino]carbonyl]-6-quinolinyl]-, 2,5-dioxo-1-pyrrolidinyl ester (RapiFluor-MS), 4-substituted 1,2,4-triazolin-3,5-dione (Cockson type reagent), 4-phenyl-1,2,4-triazolin-3,5-dione derivative (Amplifex Diene), 1-propaneaminium, 3-(aminooxy)-N,N,N-trimethyl compound containing a suitable counterion (Amplifex Keto), acetyldrazide trimethylammonium chloride (Girard T), 1-(carboxymethyl)pyridinium chloride hydrazide (Girard P), and pyridylamine.

[0224] 17. The derivatization reagent is of formula A or B: [ka] (In the formula, X is a reactive unit that can form a covalent bond with the analyte of interest, L1 and L2 are independent of each other, and can be substituted or not substituted. Linkers, especially branched or linear linkers, Y is a neutral loss unit, Z is a charged unit that includes at least one permanently charged portion, in particular one permanently charged portion. The method according to any one of embodiments 1 to 16, comprising the compound or any salt thereof.

[0225] 18. The derivatization reagent is of formula PI: [ka] (In the formula, one of the substituents B1, B2, B3, B4, B5 is a coupling group Q that can form a covalent bond with the analyte, The other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, allyroyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, allyroyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotopes, or derivatives thereof. Y1 and Y2 are independently selected from hydrogen, methyl, ethyl, methoxy, substituted aromatic, unsubstituted aromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heteroaromatic, unsubstituted heteroaromatic, or amine, or Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, or unsubstituted heteroaromatic. The method according to any one of embodiments 1 to 17, comprising the compound.

[0226] 19. Derivatization reagent, formula DI: [ka] (In the formula, one of the substituents B1, B2, and B4 is a coupling group Q that can form a covalent bond with the analyte, The other substituents A1, A2, A3, A4, A5, B1, B2, B4 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, allyroyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, allyroyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotopes, or derivatives thereof. B3 is selected from alkyl, acetyl, vinyl, substituted aromatic, unsubstituted aromatic, substituted benzyl, unsubstituted benzyl, substituted cycloalkyl, unsubstituted cycloalkyl, isotopes and their derivatives. Y1 and Y2 are independently selected from hydrogen, methyl, ethyl, methoxy, substituted aromatic, unsubstituted aromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heteroaromatic, unsubstituted heteroaromatic, or amine, or Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, or unsubstituted heteroaromatic. The method according to any one of embodiments 1 to 18, comprising the compound.

[0227] 20. Derivatization reagent, formula CI: [ka] (In the formula, one of the substituents B1, B2, B3, B4, B5 is a coupling group Q that can form a covalent bond with the analyte, The other substituents A1, A2, B1, B2, B3, B4, and B5 are each independently selected from hydrogen, halogen, alkyl, modified alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, allyroyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, allyroyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, sulfur, isotopes, or derivatives thereof. A3 comprises ammonium, pyridinium, phosphonium, or derivatives thereof. If A3 is ammonium and B1 or B5 is coupling group Q, coupling group Q contains four carbon atoms separated from the carbon atom of the CA1A2A3 substituent by single or double bonds, and coupling group Q contains five carbon atoms separated from the carbon atom of the CA1A2A3 substituent by single or double bonds. The method according to any one of embodiments 1 to 19, comprising the compound.

[0228] 21. The method according to any one of embodiments 1 to 20, wherein the analyte of interest is selected from the group consisting of nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids, molecules characterized by specific modifications of other molecules, substances internalized by living organisms, metabolites of such substances, and combinations thereof.

[0229] 22. The method according to any one of embodiments 1 to 21, wherein the target analyte is a peptide selected from the group consisting of preferably a free -NH2 group or a -COOH group.

[0230] 23. The method according to any one of embodiments 1 to 22, wherein the sample is a human sample, preferably a hemolytic whole blood sample, and more particularly a hemolytic human whole blood sample.

[0231] 24. Use of the method described in any one of embodiments 1 to 23 to determine the presence or level of an analyte of interest in a sample.

[0232] 25. A diagnostic system for determining the presence or level of an analyte of interest in a sample, comprising a first electrospray ionization source and a second electrospray ionization source, and a mass spectrometry unit for carrying out the method described in any one of embodiments 1 to 23.

[0233] 26. The diagnostic system according to embodiment 25, wherein the baffle, preferably a rotatable baffle, is located between or would be located between a first electrospray ionization source and a second electrospray ionization source.

[0234] 27. A dopant for enhancing the sensitivity of mass spectrometry detection of an analyte of interest, comprising formula I: R1-SO2-R2 (I) (In the formula, R1 and R2 are each independently selected from alkyl or aryl, where alkyl contains 1 to 6 carbon atoms and aryl contains 1 to 6 carbon atoms.) Dopants, including

[0235] 28. The dopant according to embodiment 27, wherein the dopant is selected from substituted sulfolane, unsubstituted sulfolane, substituted dibutylsulfone, unsubstituted dibutylsulfone, substituted diphenylsulfone, or unsubstituted diphenylsulfone.

[0236] 29. The dopant is expressed in the following formulas II-IV: [ka] [ka] [ka] A dopant selected from the dopant according to embodiment 27 or 28

[0237] 30. A dopant according to any one of embodiments 27 to 29, wherein the dopant does not contain a sulfoxide, preferably DMSO.

[0238] Use of the dopant described in any one of the embodiments 27 to 30 of the method described in 31.1 to 23. [Examples]

[0239] The following examples are provided to illustrate, and not to limit, the invention claimed herein.

[0240] Figure 1 shows a schematic diagram of a diagnostic system 100 according to the present invention. The diagnostic system 100 is used to determine the presence or level of a target analyte in a sample, preferably to carry out a method according to a first aspect of the present invention, and includes a first electrospray ionization source and a second electrospray ionization source 1, 2, and a mass spectrometry unit 14. The diagnostic system 100 does not include a baffle (not shown), which is typically located between the first electrospray ionization source and the second electrospray ionization source 1, 2. The baffle can be removed from commercially available systems so that two independent analyte flows 15, 16 can pass through and intersect in the space in front of the inlet of the mass spectrometry unit. The first electrospray ionization source 1 is for the first analyte flow 15. The second electrospray ionization source is for the second analyte flow 16. The first analyte flow 15 contains the target analyte. Before the first analyte stream 15 passes through the first electrospray ionization source 1, the analyte of interest and / or the first analyte stream 15 may be purified by LC or HPLC. The second analyte stream 16 contains a dopant or derivatizing reagent, and each of the first analyte stream and the second analyte streams 15, 16 is contained in gaseous form or aerosol. The first analyte stream and the second analyte streams 15, 16 are crossed and / or mixed to form the mixture or derivatized analyte of interest. In the case of a mixture, the mixture contains the analyte of interest and the dopant. The presence or level of the analyte of interest is determined in the mass spectrometry unit 14 using mass spectrometry.

[0241] Figure 2 shows the TIC (total ion count) as a percentage of the retention time for mixtures containing the peptide as the analyte of interest and DMSO as a dopant at various concentrations (0%, 10%, 20%, and 30% (v / v)). The peaks in the various chromatograms are normalized to the highest signal by keeping all other LC-MS parameters I (except the cross-flow concentration of DMSO) constant. Sigma-Aldrich peptide test mixtures were separated by cross-flow LC-MS using a standard gradient with a C18 column using water / acetonitrile as the eluent, by adding various types of dopants in the range of 0–30% (v / v) of DMSO during cross-flow. Therefore, other suitable dopants may be used.

[0242] Signal enhancement can be observed by adding a dopant, such as DMSO. The signal intensity increases by increasing the concentration of the dopant, such as DMSO, according to the method of the present invention. The amount of the analyte of interest remains constant with each injection. LC separation (peak width) is unaffected.

[0243] Figure 3 shows the TIC (Time Indication) in percentages against retention time for mixtures containing tobramycin as the analyte of interest and DMSO as a dopant at various concentrations (0%, 5%, and 30% (v / v)). This illustrates the therapeutic application of dopant spray effects against polysaccharide-based low-molecular-weight substances. In this case, tobramycin is used, and it is shown that the peak chromatography peak width is not altered by the cross-flow technique.

[0244] Signal enhancement can be observed by adding a dopant, such as DMSO. The signal intensity increases by increasing the concentration of the dopant, e.g., DMSO, according to the method of the present invention. The amount of the analyte of interest remains constant with each injection. LC separation (peak width) is unaffected. Therefore, other suitable dopants can be used.

[0245] Figures 4A to 4D show the TIC as a percentage of retention time for mixtures containing estradiol as the analyte of interest, with or without ammonium fluoride as a dopant (Figures 4A and 4D are without NH4F, and Figures 4B and 4C are with NH4F). The present invention uses ammonium fluoride to enhance the [MH] signal of estradiol using a cross-flow method. An average change of approximately 1.04 times (n=2, based on peak area) was obtained as an average of two measurements without NH4F.

[0246] Figure 5 shows the TIC (Time Indication) as a percentage of the retention time of testosterone using a hydrazone derivatization reagent for coupling. Before turning on the cross-spray (mixing the analyte and derivatization reagent), only testosterone is visible, and when the cross-spray is turned on, the derivatization product becomes visible. In this mode, background signals cannot be detected, which means that contamination of the analyte flow in the first and second electrospray ionization sources cannot be observed.

[0247] Figure 6 shows the TIC as a percentage of the retention time of testosterone using the derivatization reagent. The testosterone-MZ2960 reaction product derived from the figure was confirmed by MSMS using a cross-flow spray configuration. The derivatization reaction of testosterone was carried out by cross-spray. The characteristic fragment at 433Da indicates pyridine loss of testosterone-MZ2960 and is proof of a successful derivatization reaction by cross-spray.

[0248] The structure of the MZ2960 is shown in Figure 6 and is as follows: [ka]

[0249] Figure 7 shows the multiplicative changes of each peptide in a peptide mixture (purchased from Sigma Aldrich) against a cross-flow eluent of blank H2O / CAN (CAN = acetonitrile). The peptide mixture is injected by HPLC via a first electrospray ionization source, while the dopants are sprayed via a second electrospray ionization source. As shown, novel dopants, preferably sulfolane derivatives having phenyl and aliphatic structural elements in the C=1 to C=6 range, are shown to be advantageous in signal enhancement. The signal-enhancing effect of the dopant is also observed when the dopants are mixed in front of the mass spectrometer.

[0250] Figure 8 shows a possible schematic experimental setup. The peptide test mixture is mixed with the dopant in the reaction coil after the column and before the mass spectrometer. The results are shown in Figure 9.

[0251] Figure 9 shows the magnification changes of three peptides as analytes of interest with the addition of various dopants, such as DMSO, sulfolane, diphenyl sulfone, 1,4-dioxane, and propylene glycol sulfite. LC-MS with a standard gradient on a C18 column using water / acetonitrile as the eluent was used to separate the peptide test mixture purchased from Sigma-Aldrich. Dopants (DMSO, sulfolane, diphenyl sulfone, dioxane, and propylene glycol sulfite) were added after the column and before ESI-MS and mixed with the eluent using a reaction coil. The plots show the magnification changes of the three peptides with the use of different dopants. DMSO, sulfolane, and diphenyl sulfone exacerbate the peptide signal, while dioxane and propylene glycol do not show any signal-enhancing effect. Therefore, the novel dopants of the fourth aspect of the present invention offer advantages compared to the prior art.

[0252] This patent application claims priority to European Patent Application No. 20211558.0, the contents of which European Patent Application are incorporated herein by reference.

[0253] List of References 100-Diagnostic System 1. A first electrospray ionization source, e.g., ESI spray 2. A second electrospray ionization source, e.g., ESI spray 3-HPLC or LC 4. Cross-spray or spray-crossing point of the flow of the first analyte and the flow of the second analyte. 5. Ion guide, e.g., T-Wave (trademark) ion guide 6-quadrupole, preferably decomposed quadrupole 7-DRE lenses 8. Collision cells, e.g., T-Wave® collision cells 9-Transfer Optics 10-Scroll pump, preferably an oil-free scroll pump 11. Vacuum pump, preferably a turbomolecular pump 12. Vacuum pump, preferably a turbomolecular pump 13. Vacuum pump, preferably a turbomolecular pump 14. Mass spectrometry units, e.g., collisional quadrupoles 15. Flow of the first analyte 16. Flow of the second analyte 17-Ion guide, TOF 18- Towards a detector, such as an ion detector. 19-Sample cone (not shown)

Claims

1. A method for determining the presence or level of a target analyte in a sample, a) A step of supplying a first electrospray ionization source for a flow of a first analyte containing the target analyte, b) A step of supplying a second electrospray ionization source for a second analyte flow containing a dopant, wherein each of the first analyte flow and the second analyte flow contains the second electrospray ionization source for the second analyte flow in gaseous form or aerosol, c) A step of mixing the first analyte stream and the second analyte stream to form a mixture, wherein the mixture comprises the analyte of the choice and the dopant, and d) A step of determining the presence or level of the target analyte in the sample using mass spectrometry. Includes, The dopant is DMSO or NH4F, or formula I: R1-SO 2-R2 (I) (wherein R1 and R2 are each independently selected from alkyl or aryl, with alkyl containing 1 to 6 carbon atoms and aryl containing 1 to 6 carbon atoms) method.

2. The method according to claim 1, wherein the method is performed to enhance the mass spectrometry signal of the target analyte.

3. The method according to claim 1 or 2, wherein the flow of the first analyte is mixed with the flow of the second analyte after it has exited the first electrospray ionization source and the second electrospray ionization source.

4. The method according to any one of claims 1 to 3, wherein the first electrospray ionization source and the second electrospray ionization source are arranged at an angle of at least 10° to 180° according to the plan view of the two electrospray ionization sources, and, for the electrospray ionization source, at an angle of at least 10° to 180° according to the front view.

5. The method according to any one of claims 1 to 4, wherein the second flow includes the dopant in a concentration in the range of 5 to 35% (v / v).

6. The method according to any one of claims 1 to 5, wherein the analyte for the purpose is selected from the group consisting of nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids, molecules characteristic of specific modifications of other molecules, substances internalized by organisms, metabolites of such substances, and combinations thereof.

7. The method according to claim 6, wherein the target analyte is a peptide selected from the group consisting of a free -NH₂ group or a -COOH group.

8. Use of the method according to any one of claims 1 to 7 for determining the presence or level of a target analyte in a sample.

9. A diagnostic system for determining the presence or level of a target analyte in a sample, comprising a first electrospray ionization source and a second electrospray ionization source and a mass spectrometry unit for carrying out the method according to any one of claims 1 to 8.

10. Use of a dopant in the method according to any one of claims 1 to 8, The dopant is used to enhance the sensitivity of mass spectrometry detection of the target analyte, and is given by formula I: R1-SO 2-R2 (I) (wherein R1 and R2 are each independently selected from alkyl or aryl, with alkyl containing 1 to 6 carbon atoms and aryl containing 1 to 6 carbon atoms) used.

11. The use according to claim 10, wherein the dopant is selected from substituted sulfolane, unsubstituted sulfolane, substituted dibutylsulfone, unsubstituted dibutylsulfone, substituted diphenylsulfone, or unsubstituted diphenylsulfone.

12. The use according to claim 10 or 11, wherein the dopant does not contain a sulfoxide.

13. The use according to any one of claims 10 to 12, wherein the dopant does not contain DMSO.

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