Imidazolium reagent for mass spectrometry

Abstract

The present invention relates to compounds suitable for use in mass spectrometry and to methods for the mass spectrometric determination of analyte molecules using said compounds.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to compounds suitable for use in mass spectrometry, compositions containing the compounds, and kits containing the compositions and / or compounds and complexes. Furthermore, the present invention relates to a method for determining an analyte by mass spectrometry using the compounds. [Background technology]

[0002] Background of the Invention Mass spectrometry (MS) is a widely used technique for the qualitative and quantitative analysis of chemical substances ranging from small molecules to large molecules. Generally, it is a highly sensitive and specific method, capable of analyzing even complex biological samples, such as environmental or clinical samples. However, the sensitivity of the measurement remains a concern when analyzing certain analytes, particularly 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] However, there is still a need to improve the sensitivity of MS analysis, especially when the amount of analyte is small or the available material is limited (such as biopsy tissue).

[0005] In this technical field, several derivatization reagents (compounds) are known that aim to improve the sensitivity of the measurement of these analytes. In particular, reagents containing charged units and neutral loss units combined in a single functional unit (e.g., International Publication No. 2011 / 091436). Other reagents containing separate units are structurally relatively large and affect the general workflow of sample preparation and MS measurement (Rahimoff et al. (2017) J.Am.Chem.Soc.139(30), p.10359-10364). Known derivatization reagents include, for example, dansyl chloride, RapiFluor-MS (RFMS), Cookson-type reagents, Amplifex Diene, Amplifex Keto, Girard T, Girard P, and pyridylamine (Hong and Wang, Anal Chem., 2007, 79(1):322-326; Frey et al., Steroids, 2016 Dec, 116:60-66; Francis et al., Journal of Pharmaceutical and Biomedical Analysis, 2005, 39(3-4), 411-417; Alley William, 28th International Carbohydrate Symposium, New Orleans, LA, United States, July 17-21 (2016), ICS-209). All of these often result in disadvantages due to insufficient labeling efficiency, formation of structural isomers due to coupling chemistry, suboptimal ionization efficiency, disadvantages to chromatographic separation after coupling, suboptimal fragmentation behavior due to numerous fragmentation pathways, and the need for high collision energies.

[0006] Therefore, there is an urgent need in this field for derivatization reagents that not only exhibit chemical structures that do not negatively impact 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.

[0007] The present invention relates to a novel reagent (compound) that enables highly sensitive quantification 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 individual adaptation for specific needs arising from the measurement of a particular analyte or for adaptation to a specific workflow.

[0008] An object of the present invention is to provide a compound of Formula I, a kit, and a composition each containing the compound for efficient detection of an analyte by mass spectrometry. Furthermore, an object of the present invention is to provide a complex and a method for mass spectrometric determination of an analyte.

[0009] This object(s) is solved by the subject matter of the independent claims. Further embodiments are the subject of the dependent claims.

Summary of the Invention

[0010] Summary of the Invention In the following, the present invention relates to the following aspects.

[0011] In a first aspect, the present invention relates to Formula I for mass spectrometric determination of an analyte:

Chemical formula

[0012] In a second aspect, the present invention relates to a composition comprising a compound according to the first aspect of the present invention.

[0013] In a third aspect, the present invention relates to a kit comprising a compound of the first aspect of the present invention or a composition of the second aspect of the present invention.

[0014] In a fourth aspect, the present invention relates to a complex for detecting an analyte by mass spectrometry, comprising a covalently bonded bonded analyte and a bonded compound, wherein the complex is formed by a chemical reaction between the analyte and a compound according to a second aspect of the present invention.

[0015] In a fifth aspect, the present invention relates to the use of the compound of the first aspect of the present invention for mass spectrometry determination of an analyte.

[0016] In a sixth aspect, the present invention relates to a method for determining the mass spectrometry of an analyte, (a) A step of reacting an analyte with a compound of formula I according to the first aspect of the present invention, wherein a complex according to the fourth aspect of the present invention is formed, (b) A step of subjecting the composite from step (a) to mass spectrometry analysis. This includes methods.

[0017] In the seventh aspect, the present invention relates to formula V: [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. Regarding the compounds. [Brief explanation of the drawing]

[0018] [Figure 1] This diagram illustrates peak "splitting," demonstrating the chromatographic system's ability to separate different isomers resulting from the derivatization reaction of analyte molecules. [Figure 2] This diagram shows a schematic of the workflow for determining the LOD augmentation coefficient. [Modes for carrying out the invention]

[0019] 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. It should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[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 instruction of such incorporated reference and the definition or instruction 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 It should be understood that the word "comprise," as well as variations such as "comprises" and "comprising," means the inclusion of a specified integer or process, or group of integers or processes, but not the exclusion of any other integer or process, or group of integers or processes.

[0023] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to 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.” It should be understood that 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 boundaries of the range, but also all individual numbers or subranges contained within that range, as if each number and subrange were 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 subranges within the indicated range. Thus, this numerical range includes individual values ​​such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20%, and subranges such as 4 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 described.

[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 term “compound” refers to a chemical substance 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. Reactive groups include, but are not limited to, reactive units, charged units, and neutral-loss units. In the context of this invention, the term “conjugated compound” refers to the compound that is conjugated to the analyte. In principle, the compound and the conjugated compound may be identical. The compound and the conjugated compound may be substantially identical. Substantially identical may mean that both compounds have the same chemical structure, except that the structure of the reactive unit K and / or the structure of the coupling group Q are different from each other. Preferably, the compound can form a bond to the analyte but is not yet conjugated to the analyte. The conjugated compound is conjugated to the analyte.

[0027] The terms “mass spectrometry” (“Mass Spec” or “MS”) or “mass spectrometry determination” or “mass spectroscopy” 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 terms “ionization” or “to ionize” refer to the process of generating ions of an analyte that have a net charge equal to one or more electron units. Anions have a net negative charge of one or more electron units, while cations have a net positive charge of one or more electron units. MS (mass spectroscopy) can be performed in either a "negative ion mode," which generates and detects negative ions, or a "positive ion mode," which generates and detects positive ions.

[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-protein analysis 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. Here, for example, the analyte of interest is separated from the matrix using gas or liquid chromatography. Typically, mass spectrometry involves the following three steps:

[0031] 1. A sample containing the target analyte is usually ionized by complex formation with cations, and in many cases by protonation into cations. Ionization sources include, but are not limited to, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).

[0032] 2. The ions are classified and separated according to their mass and charge. High electric field asymmetric waveform ion mobility spectroscopy (FAIMS) can be used as an ion filter.

[0033] 3. The separated ions are detected, for example, in multiple reaction mode (MRM), and the results are displayed on a chart.

[0034] 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 (sprayed) 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.

[0035] 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 nitrogen gas may be used. Gas-phase ionization in APCI can be more effective than ESI for analyzing less polar entities.

[0036] 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.

[0037] "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.

[0038] Mass spectrometry determination may be combined with additional analytical methods, including chromatographic techniques such as gas chromatography (GC), liquid chromatography (LC), particularly HPLC, and / or separation techniques based on ion mobility.

[0039] In the context of this disclosure, the terms “analyte,” “analyte molecule,” or “analyte of interest” are used interchangeably to refer to the chemical species analyzed by mass spectrometry. Chemical species suitable for analysis by mass spectrometry, i.e., analytes, may include, but are not limited to, any type of molecule present in a living organism, including, nucleic acids (e.g., DNA, mRNA, miRNA, rRNA, etc.), amino acids, peptides, proteins (e.g., cell surface receptors, cytoplasmic proteins, etc.), metabolites or hormones (e.g., testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (e.g., vitamin D), molecules characterized by a specific modification of another molecule (e.g., sugar moieties or phosphoryl residues on proteins, methyl residues on genomic DNA), or substances internalized by an organism (e.g., therapeutic drugs, addictive drugs, 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. In the context of this invention, the term "bound analyte" refers to the analyte that is bound to a compound in order to form a complex. In principle, the analyte and the bound analyte may be identical. The analyte and the bound analyte may be substantially identical. Substantially identical may mean that both analytes have the same chemical structure except that the structures of their functional groups are different from each other. Preferably, the analyte can form a bond to a compound but is not yet bound to the compound. The bound analyte is bound to the compound.

[0040] The term "permanently positively charged" is used in the context of this disclosure to mean that the positive charge of an imidazolium unit is not easily reversible by, for example, washing, dilution, or filtration. A permanent positive charge may, for example, be the result of covalent bonding. A permanent positive charge is in contrast to a reversible positive charge (non-permanent positive charge), which may, for example, be the result of electrostatic interactions.

[0041] The term "detection limit" or "LOD" refers to the lowest concentration of an analyte at which a biological analytical method can reliably distinguish the analyte from background noise.

[0042] The analyte may be present in the sample of interest, for example, in a biological or clinical sample. The terms “sample” or “sample of interest” are used interchangeably herein and refer to a part or fragment of a tissue, organ, or individual, which is usually smaller than such a tissue, organ, or individual that is intended to represent the whole. For analysis, the sample provides information about the state of the tissue 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.

[0043] 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, e.g., monkeys), rabbits, and rodents (e.g., mice and rats).

[0044] Prior to analysis by mass spectrometry, the sample may be pretreated in a manner specific to the sample and / or analyte. In the context of this disclosure, the term “pretreatment” refers to any means necessary to enable subsequent analysis of the desired analyte by mass spectrometry. Typical pretreatment means include, but are not limited to, the elution of solid samples (e.g., elution of dried blood spots), the addition of hemolytic reagents (HRs) to whole blood samples, and the addition of enzyme reagents to urine samples. The addition of internal standards (ISTDs) may also be considered as a pretreatment of the sample.

[0045] The term "hemolytic reagent (HR)" refers to a reagent that dissolves cells present in a sample. In the context of this invention, a hemolytic reagent specifically refers to a reagent that dissolves 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.

[0046] Typically, an internal standard (ISTD) is a known quantity of substance that, when applied to a mass spectral detection workflow, exhibits similar properties to the analyte of interest (i.e., including any pretreatment, concentration, and actual detection steps). While the ISTD exhibits similar properties to the analyte of interest, it is distinctly different. For example, during chromatographic separation, such as gas or liquid chromatography, the ISTD has approximately the same retention time as the analyte from the sample. Therefore, both the analyte and the ISTD enter the mass spectrometer simultaneously. The ISTD, however, exhibits a different molecular weight than the analyte from the sample. This allows for the differentiation of ions from the ISTD and ions from the analyte by mass spectrometry using different mass / charge (m / z) ratios. Both are subjected to fragmentation to obtain daughter ions. These daughter ions can be distinguished by their respective m / z ratios and their parent ions. As a result, separate determination and quantification of signals from the ISTD and analyte are possible. Since ISTD is added in known amounts, the signal intensity of the analyte from the sample can be attributed to a specific quantitative amount of the analyte. Thus, the addition of ISTD allows for relative comparison of the amount of detected analyte and enables clear identification and quantification of the target analyte present in the sample when the analyte(s) reach the mass spectrometer. Typically, but not always, ISTD is an isotopically labeled variant of the target analyte (e.g., 2 H, 13 C, or 15 (Including signs such as N)

[0047] 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 step that occurs following sample preparation and provides a sample containing an analyte that is concentrated 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 use, i.e., the intended capture molecule. Which coating is suitable for which analyte is well known to those skilled in the art. The beads can be made from a variety of different materials. The beads may have a variety of sizes and may have porous or non-porous surfaces.

[0048] In the context of this disclosure, the terms “second concentration process” or “second concentration workflow” refer to a concentration process that occurs following sample pretreatment and the first concentration process, and provides a sample containing an analyte that is concentrated compared to the initial sample and the sample after the first concentration process.

[0049] 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 its chemical components as it flows around or over a stationary liquid or solid phase.

[0050] 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).

[0051] 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. Methods where the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal-phase liquid chromatography (NPLC), while methods where the opposite is true (e.g., a water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) are called reverse-phase liquid chromatography (RPLC). MicroLC refers to HPLC methods that typically use columns with a narrow (norrow) bore diameter of less than 1 mm, for example, about 0.5 mm. "Ultra-high-performance liquid chromatography" or "UHPLC" refers to an HPLC method using a pressure of 120 MPa (17,405 lbf / in2) or approximately 1200 atmospheres. Rapid LC refers to an LC method (microLC, UHPLC) that uses a short column with the above-mentioned inner diameter and length of less than 2 cm, for example, 1 cm, and applies the above-mentioned flow rate and pressure. Short rapid LC protocols include a trapping / washing / elution process using a single analytical column and achieve LC in a very short time of less than 1 minute.

[0052] Furthermore, hydrophilic interaction chromatography (HILIC), size exclusion LC, ion exchange LC, and affinity LC are well-known.

[0053] 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.

[0054] The term "fragmentation" refers to the dissociation of a single molecule into two or more distinct molecules. As used herein, the term fragmentation refers to a specific fragmentation event in which the breakdown point of the parent molecule in which the fragmentation event occurs is clearly defined, and the resulting daughter molecules are well characterized. Methods for determining the breakdown points of the parent molecule and the resulting 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-benzylimidazolium unit, those skilled in the art can determine, based on the overall structure of the molecule, whether the imidazolium unit fragments and releases a benzyl entity, or is completely released from the parent molecule; that is, the resulting daughter molecules may be either benzyl molecules or a benzyl-less parent molecule. Fragmentation can occur through 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), high-energy C-trap dissociation (HCD), and charge-remote fragmentation.

[0055] 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. 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.

[0056] In the context of this disclosure, the term "complex" refers to the product produced by the reaction of a compound with 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 of a compound with an analyte molecule.

[0057] A “kit” is any product (e.g., a package or container) comprising at least one reagent, such as a drug for treating a disorder, or a probe for specifically detecting the biomarker gene or protein of the present invention. The kit is preferably encouraged, distributed, or sold as a unit for carrying out the method of the present invention. Typically, the kit may further include a compartmentalized carrier means for tightly enclosing and receiving one or more container means, such as vials or tubes. In particular, each container means comprises one of the distinct elements used in the method of the first embodiment. The kit may further include one or more other reagents, including but not limited to a reaction catalyst. The kit may further include one or more other containers containing further materials, including but not limited to a buffer, diluent, filter, needle, syringe, and accompanying documentation with instructions for use. Labels may be presented on the container to indicate that the composition is for use in a particular application, and may also indicate instructions for either in vivo or in vitro use. Computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disk), or directly on a computer or data processing device. Furthermore, the kit may contain standard amounts of biomarkers, as described elsewhere in this specification, for calibration purposes.

[0058] Embodiment In the first aspect, the present invention relates to a compound of formula I or at least one compound of formula I: [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. This relates to the following. In particular, the compound(s) of formula I are for the mass spectrometry determination of the analyte.

[0059] The compounds according to the present invention contain moieties that exhibit mass fragmentation events at low collision energies (e.g., in the range of 10 eV to 50 eV, including boundaries). These compounds are characterized by a generally compact structure. This allows for a simple modular design of the labeling reagent, and various problems can be solved by modifying the charge-containing moiety (e.g., pyridinium, quaternary ammonium, imidazolium, triphenylphosphonium, stable metal complexes, sulfonic acids, borates, aryl trifluoroborates, sulfates, phosphates, etc.), although permanent charges are preferred. This allows for fine-tuning of the hydrophobicity of the reagent to improve the separation of the sample matrix in the purification process. Furthermore, mass fragmentation can occur at low energy levels to favor the primary fragmentation pathway. This can increase the sensitivity of the MS / MS mode. Notwithstanding the above, further MS experiments can be performed at higher energies to obtain further fragmentation pathways.

[0060] In the first embodiment of the present invention, the halogen is selected from the group consisting of F, Cl, Br, and I.

[0061] In the first embodiment of the present invention, the alkyl is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, and cyclohexyl.

[0062] In the first embodiment of the present invention, the N-acylamino is selected from the group consisting of formylamino, acetylamino, propionylamino, and benzoylamino.

[0063] In the first embodiment of the present invention, N,N-dialkylamino is selected from the group consisting of N,N-dimethylamino, N,N-ethylmethylamino, N,N-diethylamino, N,N-methylpropylamino, N,N-ethylpropylamino, N,N-dipropylamino, N,N-butylmethylamino, N,N-butylethylamino, N,N-butylpropylamino, N,N-dibutylamino, N-azetidinyl, N-pyrrolidinyl, N-piperidinyl, and N-piperazinyl.

[0064] In the first embodiment of the present invention, the alkoxy is selected from the group consisting of methoxy, ethoxy, propoxy, butoxy, phenoxy, and benzyloxy.

[0065] In the first embodiment of the present invention, the thioalkoxy is selected from the group consisting of thiomethyl, thioethyl, thiopropyl, thiobutyl, thiopentyl, and thiohexyl.

[0066] In the first embodiment of the present invention, the alkoxycarbonyl is selected from the group consisting of methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, phenoxycarbonyl, and benzyloxycarbonyl.

[0067] In the first embodiment of the present invention, the alkoxythiocarbonyl is selected from the group consisting of methoxythiocarbonyl, ethoxythiocarbonyl, propoxythiocarbonyl, butoxythiocarbonyl, phenoxythiocarbonyl, and benzyloxythiocarbonyl.

[0068] In the first embodiment of the present invention, the acyl is selected from the group consisting of formyl, acetyl, and propionyl.

[0069] In the first embodiment of the present invention, the thioacyl is selected from the group consisting of thioformyl, thioacetyl, thiopropionyl, and thiobenzoyl.

[0070] In the first embodiment of the present invention, allyroyl is selected from the group consisting of benzoyl, naphthoyl, and anthracenoyl.

[0071] In the first embodiment of the present invention, the acyloxy is selected from the group consisting of formyloxy, acetyloxy, and propionyloxy.

[0072] In the first embodiment of the present invention, allyroyloxy is selected from the group consisting of benzoyloxy, naphthoyloxy and anthracenoyloxy.

[0073] In the first embodiment of the present invention, the cycloalkyl is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0074] In the first embodiment of the present invention, the aryl is selected from the group consisting of phenyl, benzyl, naphthyl, anthracenyl, and phenasrenyl.

[0075] In the first embodiment of the present invention, the heteroaryl is selected from the group consisting of imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiophenyl, furanil, thiazolyl, pyridinyl, pyrimidinyl, benzotriazolyl, benzofuranil, and benzimidazolyl.

[0076] In the first embodiment of the present invention, the heterocycloalkyl is selected from the group consisting of N-azetidinyl, N-pyrrolidinyl, N-piperidinyl, and N-piperazinyl.

[0077] In the first embodiment of the present invention, the substituted aromatic is selected from the group consisting of methoxyphenyl, cyanophenyl, and ethylnaphthyl.

[0078] In the first embodiment of the present invention, the ring structure comprising Y1 and Y2 is selected from the group consisting of phenyl, benzyl, methyl, ethyl, propyl, and acetyloxy.

[0079] In the first embodiment of the present invention, the compound is of the following formula: [ka] Includes a structure having the following characteristics.

[0080] The imidazolium ring of formula I exhibits a mesomer effect because the imidazolium structure contains two nitrogen atoms with positive charges, particularly a defined charge on either of the individual N atoms. The mesomer effect is a characteristic of the imidazole ring. It is defined as the polarity that arises in the imidazole molecule due to the interaction between π bonds present on adjacent atoms and lone pairs of electrons. Structures II and I-II are possible arrangements of the π bonds, separately depicted for the compound of formula I.

[0081] In the first embodiment of the present invention, the compound of formula I contains a coupling group Q. At least one of the substituents B1, B2, or B4 of formula I is a coupling group Q. Preferably, B1 or B2 is Q.

[0082] In the first embodiment of the present invention, coupling Q is the following II: [ka] (In the formula, K is a reactive unit that can form a covalent bond with the analyte, n is 0, 1, 2, 3, 4, or 5. X is the carbon atom of the imidazole in formula I. It is combined with X according to the following:

[0083] In an embodiment of the first aspect of the present invention, Q does not exhibit fragmentation at energy levels lower than those that cause mass fragmentation.

[0084] In the first embodiment of the present invention, Q is bonded to X via a covalent bond.

[0085] In the first embodiment of the present invention, Q is selected from the group consisting of methylene hydrazide (the same as acetyl hydrazide), methylene hydrazine, and methylene hydroxylamine.

[0086] In the first embodiment of the present invention, K is directly coupled to X (n=0).

[0087] In the first embodiment of the present invention, K is bonded to X via a methylene group (n=1).

[0088] In the first embodiment of the present invention, K is bonded to X via an ethylene group (n=2).

[0089] In the first embodiment of the present invention, K is bonded to X via a propylene group (n=3).

[0090] In the first embodiment of the present invention, K is bonded to X via a butylene group (n=4).

[0091] In the first embodiment of the present invention, K is bonded to X via a pentylene group (n=5).

[0092] In the first embodiment of the present invention, carbon atom X of imidazolium of formula I is a bonding partner of B1, B2, or B4.

[0093] In the embodiment of the first aspect of the present invention, the compound is from the following groups I-1, I-2, and I-3: [ka] (In the formula, A1, A2, A3, A4, A5, B1, B2, B3, B4, K, X, and n have the meanings described above.) Selected from the above. In an embodiment of the first aspect of the present invention, the compound of formula I according to the present invention comprises a reactive unit K that can react with an analyte or analyte molecule. The reactive unit K can react with the analyte molecule such that a covalent bond is formed between the compound of formula I and the analyte molecule. Preferably, the covalent bond is a single bond or a double bond.

[0094] In an embodiment of the first aspect of the present invention, the reactive unit K forms a covalent bond with the compound of formula I. In particular, the covalent bond is formed between the reactive unit K of the compound of formula I and the functional group present in the analyte molecule.

[0095] In the first embodiment of the present invention, K can react with a carbonyl group, phenol group, amine, hydroxyl group, or diene group of the analyte.

[0096] In the first embodiment of the present invention, K is selected from the group consisting of hydrazides, hydrazines, hydroxylamines, and reactive carbonyls.

[0097] Those skilled in the art select an appropriate reactive unit K for the compound of formula I, depending on the functional groups present in the analyte molecule being measured. Determining which reactive unit K is suitable for binding to the functional groups of the analyte is well known.

[0098] In the first embodiment of the present invention, the analyte molecule contains a functional group selected from the group consisting of a carbonyl group, a diene group, a hydroxyl group, an amine group, an imine group, a ketone group, an aldehyde group, a thiol group, a diol group, a phenol group, an epoxy group, a disulfide group, a nucleic acid base group, a carboxylic acid group, a terminal cysteine ​​group, a terminal serine group, and an azide group, each of which can form a covalent bond with the reactive unit K of the compound of formula I. 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 of formula I.

[0099] In the first embodiment of the present invention, the analyte molecule is selected from the group consisting of steroids, ketosteroids, secosteroids, amino acids, peptides, proteins, carbohydrates, fatty acids, lipids, nucleosides, nucleotides, nucleic acids, and other biomolecules, including small molecule metabolites and cofactors, as well as therapeutic agents, addictive drugs, toxins, or their metabolites.

[0100] 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 anhydrides. Aldoses (aldehydes and ketos) exist as acetals and hemiacetals, which are masked forms of the parent aldehyde / keto.

[0101] In the first embodiment of the present invention, the carbonyl group is an amide group, and it is well known to those skilled in the art that the amide group itself is a stable group, but that the amide group can be hydrolyzed to a carboxylic acid group and an amino group. Hydrolysis of the amide group can be achieved via an acid / base catalytic reaction or by an enzymatic process, either of which is 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, in particular 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 reaction with the compound of formula I.

[0102] 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 the imine group of the intermediate before reacting with the reactive unit of the compound of formula I. 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), desoximethyltestosterone (DMT), tetrahydrogestrinone (THG), aldosterone, estrone, 4-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone, 16-ketoestradiol, 16α-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, allopregnenolonone, and aldosterone.

[0103] 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 of formula I or is converted to an activated ester group before reacting with the compound of formula I. 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, isoxepak, 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.

[0104] 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 the imine group of the intermediate before reacting with the reactive unit of the compound of formula I. 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.

[0105] 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, anilelysine, aranidipine, artesunate, and pethidine.

[0106] 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.

[0107] 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, especially 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.

[0108] 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 an embodiment of the first aspect of the present invention, in which the analyte molecule comprises one or more hydroxyl groups, 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, α-hydroxyalprazolam, α-hydroxytriazolam, lorazepam, oxazepam, temazepam, ethyl glucuronide, ethyl morphine, morphine, morphine-3-glucuronide, buprenorphine, codeine, dihydrocodeine, p-hydroxypropoxyfen, O-desmethyltramadol, desmetramadol, dihydroquinidine, and quinidine. In an embodiment of the first aspect of the present invention, the analyte molecule contains multiple hydroxyl groups, and the analyte is vitamin C, glucosamine, mannitol, tetrahydrobiopterin, cytarabine, azacitidine, ribavirin, phloxuridine, gemcitabine, streptozotocin.

[0109] The following are selected from the group consisting of adenosine, vidarabine, cladribine, estriol, trifluridine, clofarabine, nadolol, zanamivir, lactulose, adenosine monophosphate, idoxuridine, regadenosone, lincomycin, clindamycin, canaglifodine, tobramycin, netylmycin, kanamycin, ticagrelol, epirubicin, doxorubicin, arbekacin, streptomycin, ouabain, amikacin, neomycin, flamycetin, paromomycin, erythromycin, clarithromycin, azithromycin, vindesine, digitoxin, digoxin, metrizamide, acetyldigitoxin, deslanoside, fludarabine, clofarabine, gemcitabine, cytarabine, capecitabine, vidarabine, and plicamycin.

[0110] In the first embodiment of the present invention, the analyte molecule comprises 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 comprising 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 suxima.

[0111] 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.

[0112] In the first embodiment of the present invention, the analyte molecule contains one or more epoxide groups as functional groups. In the first embodiment of the present invention, the analyte molecule containing 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.

[0113] 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 is a hybridized 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 (El), 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 The following compounds are selected from the group consisting of (4-OHE1), 2-hydroxyestradiol (2-OHE2), estrone (El), estrone sulfate (Els), 17a-estradiol (E2a), 17b-estradiol (E2B), estradiol sulfate (E2S), equilin (EQ), 17a-dihydroechiline (EQa), 17b-dihydroechiline (EQb), echirenine (EN), 17-dihydroechirenine (ENa), 17α-dihydroechirenine, 17β-dihydroechirenine (ENb), Δ8,9-dehydroestrone (dEl), A8,9-dehydroestrone sulfate (dEl), Δ9-tetrahydrocannabinol, and mycophenolic acid. β or b can be used interchangeably. α and a can be used interchangeably.

[0114] 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, Flephedron, Methcathinone, Methylone, Methylenedioxypyrovalerone, Benzoylecgonine, Dehydronorketamine, Ketamine, Norketamine, Methadone, Normethadone, 6-Acetylmorphine, Diacetylmorphine, Morphine, Norhydrocodone, Oxycodone, Oxymol The following are selected from the group consisting of phon, phencyclidine, norpropoxyfen, 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, desmetramadol, tramadol, lamotrigine, theophylline, amikacin, gentamicin, tobramycin, vancomycin, methotrexate, gabapentin cysomicin, and 5-methylcytosine.

[0115] 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.

[0116] 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.

[0117] 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 molecule(s) are present in a biological or clinical sample 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.

[0118] In an embodiment of the first aspect of the present invention, the reactive unit K is selected from the group consisting of a carbonyl reactive unit, a diene reactive unit, a hydroxyl reactive unit, an amino reactive unit, an imine reactive unit, a thiol reactive unit, a diol reactive unit, a phenol reactive unit, an epoxide reactive unit, a disulfide reactive unit, and an azide reactive unit.

[0119] In an embodiment of the first aspect of the present invention, the reactive unit K is a carbonyl reactive unit, which can react with any kind of molecule having a carbonyl group. In an embodiment of the first aspect of the present invention, the carbonyl reactive unit is selected from the group consisting of a carboxyl reactive unit, a keto reactive unit, an aldehyde reactive unit, an anhydride reactive unit, a carbonyl ester reactive unit, and an imide reactive unit. In an embodiment of the first aspect 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.

[0120] In an embodiment of the first aspect of the present invention, the carbonyl reactive unit is the following group: (i) A hydrazine unit, such as H2N - NH - or H2N - NR 1 - unit (where R 1 is aryl, aryl containing one or more heteroatoms, or C 1~4 alkyl, especially C1 or C2 alkyl, optionally substituted, for example, with halo, hydroxyl, and / or C 1~3 alkoxy), (ii) A hydrazide unit, especially a carbohydrazide or sulfohydrazide unit, especially H2N - NH - C(O)- or H2N - NR 2 - C(O)- unit (where R 2 is aryl, aryl containing one or more heteroatoms, or C 1~4 alkyl, especially C1 or C2 alkyl, optionally substituted, for example, with halo, hydroxyl, and / or C 1~3(substituted with alkoxy), (iii) Hydroxylamino units, e.g., H2N-O- units, (iv) Dithiol units, especially 1,2-dithiol or 1,3-dithiol units Selected from.

[0121] 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 a diazo unit, an alkyl halide, an amine, and a hydrazine unit.

[0122] In the first embodiment of the present invention, the analyte molecule comprises a ketone or aldehyde group, where K is a carbonyl reactive unit, and this belongs to the following group: (i) Hydrazine unit, (ii) Hydrazide unit, (iii) Hydroxylamino unit, and (iv) Dithiol Unit Selected from.

[0123] In the first embodiment of the present invention, the reactive unit K is a diene reactive unit which can react with an analyte having 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.

[0124] In the first embodiment of the present invention, reactive unit K is a hydroxyl-reactive unit that can react with an analyte having a hydroxyl group. In the first embodiment 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 the first embodiment of the present invention, reactive unit K is a diol-reactive unit that reacts with a diol group on the analyte molecule.

[0125] In an embodiment of the first aspect of the present invention, reactive unit H is a 1,2-diol reactive unit, and the 1,2-diol reactive unit contains a boronic acid. In a further embodiment, the diol may be oxidized to the respective ketone or aldehyde, and then reacted with the ketone / aldehyde reactive unit K.

[0126] 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 units.

[0127] 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 sulfonyl chloride units.

[0128] 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 unit. The phenol group present on the analyte molecule can be reacted with a highly reactive electrophile such as triazole dione (TAD, etc.) by reaction (H. Ban et al., "J. Am. Chem. Soc." (2010), Vol. 132, No. 5, pp. 1523-1525), diazotization, or orthonitration, and then reduced to an amine, which can then be reacted with an amine reaction reagent. In the first embodiment of the present invention, the phenol-reactive unit is fluoro-1-pyridinium.

[0129] 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 adjacent O or N atom NH2-N / O molecules. In the first embodiment of the present invention, the epoxide reactive unit is from the following group: (i) 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 including, for example, halo, hydroxyl, and / or C 1~3 (substituted with alkoxy), (ii) Hydrazide units, especially carbohydrazide or sulfohydrazide units, especially H2N-NH-C(O)- or H2N-NR 2 -C(O)-unit (wherein R in the formula) 2 This includes aryls, aryls containing one or more heteroatoms, or C 1~4 Alkyl, particularly C1 or C2 alkyl, optionally including, for example, halo, hydroxyl, and / or C 1~3 (substituted with alkoxy), (iii) Hydroxylamino units, e.g., H2N-O- units Selected from.

[0130] In an embodiment of the first aspect of the present invention, the reactive unit K is a disulfide reactive unit which 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 K.

[0131] In an embodiment of the first aspect of the present invention, the reactive unit K is an azide-reactive unit that reacts with an azide group on the analyte molecule. In an embodiment of the first aspect of the present invention, the azide-reactive unit reacts with the azide group by azide-alkyne cycloaddition. In an embodiment of the first aspect 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 aspect of the present invention, the azide group may be reduced to its respective amino group and then reacted with the amino-reactive unit K.

[0132] 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 K group of the corresponding functional group of the analyte is selected from the groups listed in the right column of Table 1. [Table 1]

[0133] In the first embodiment of the present invention, the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4 that do not form a coupling group Q are each independently selected from hydrogen, methyl, ethyl, propyl, cyclopropyl, isopropyl, butyl, tert-butyl, or methoxy.

[0134] In the first embodiment of the present invention, Q is acetohydrazide.

[0135] In the first embodiment of the present invention, Q is a carbonyl hydrazide.

[0136] In the first embodiment of the present invention, B3 is alkyl.

[0137] In the first embodiment of the present invention, B3 is methyl or ethyl.

[0138] In the first embodiment of the present invention, A1 is H or methoxy.

[0139] In the first embodiment of the present invention, A2 is H or methoxy.

[0140] In the first embodiment of the present invention, A3 is H or methoxy.

[0141] In the first embodiment of the present invention, A4 is H or methoxy.

[0142] In the first embodiment of the present invention, A5 is H or methoxy.

[0143] In the first embodiment of the present invention, Y1 is H, or forms a condensed aromatic or heteroaromatic ring structure with Y2.

[0144] In the first embodiment of the present invention, Y2 is H, or forms a condensed aromatic or heteroaromatic ring structure with Y1.

[0145] In the first embodiment of the present invention, B1 is H. In particular, if B2 is Q, then B1 is H.

[0146] In the first embodiment of the present invention, B4 is H.

[0147] In the first embodiment of the present invention, B2 is H. In particular, if B1 is Q, then B2 is H.

[0148] In the first embodiment of the present invention, either B1 or B2 is Q. Q is selected from the following group: hydrazine units, hydrazide units, and hydroxylamino units. Other substituents other than Q are A1=H, A2=H, A3=H, A4=H, A5=H, Y1=H, Y2=H, B3=methyl, B4=H, B5=H, and B1=H or B2=H.

[0149] In the first embodiment of the present invention, B1 is Q. Q is selected from the following group: hydrazine units, hydrazide units, and hydroxylamino units. Other substituents other than Q are A1=H, A2=H, A3=H, A4=H, A5=H, Y1=H, Y2=H, B3=methyl, B4=H, B5=H, and B2=H.

[0150] In the first embodiment of the present invention, B2 is Q. Q is selected from the following group: hydrazine units, hydrazide units, and hydroxylamino units. Other substituents other than Q are A1=H, A2=H, A3=H, A4=H, A5=H, Y1=H, Y2=H, B3=methyl, B4=H, B5=H, and B1=H.

[0151] In the first embodiment of the present invention, the compound is charged.

[0152] In the first embodiment of the present invention, the compound is positively charged.

[0153] In the first embodiment of the present invention, the compound is permanently charged. In particular, the compound is positively charged once and permanently. This may mean that the total net charge is positive once and permanently.

[0154] In the first embodiment of the present invention, the positive charge is part of the imidazolium ring.

[0155] In the first embodiment of the present invention, Y1 and Y2 are each independently selected from hydrogen, methyl, ethyl, methoxy, substituted aromatic, unsubstituted aromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heteroaromatic, unsubstituted heteroaromatic, and amine. Hydrogen, alkyl, and / or substituted aromatic are preferred embodiments.

[0156] In the embodiment of the first aspect of the present invention, Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, and unsubstituted heteroaromatic compounds.

[0157] In an embodiment of the first aspect of the present invention, the compound contains a counterion for forming a salt. In particular, the counterion is permanently negatively charged.

[0158] In the first embodiment of the present invention, the compound comprises a counterion for forming a salt, and the counterion is preferably from the following group:Cl - , Br - F - , formic acid, trifluoroacetic acid, PF6 - Selected from sulfonic acid, phosphoric acid, and acetic acid.

[0159] In the first embodiment of the present invention, the compound of formula I is selected from the following group: sign 1 [ka] sign 2 [ka]

[0160] Each of the labels 1 or 2 described above can be formed by the compound alone or in combination with a counterion.

[0161] Stabilization of the carbocation after neutral-loss fragmentation at the benzyl position allows for fragment loss at low energy, providing a highly dominant fragmentation pathway that enables high signal enhancement. In some embodiments, particularly when meta-substitution patterns are present, favorable LC (liquid chromatography) elution characteristics can be observed. Either one of two E / Z stereoisomers, which can be formed with hydrazides or oximes (e.g., for testosterone labeling), may be predominantly produced, or both isomers may co-elute. This results in improved sensitivity due to a better signal-to-noise ratio (S / N).

[0162] Alkylated imidazolium can be fragmented at the N position at intermediate energy. Combining alkylated imidazolium with stabilized benzyl cations as loss fragments yielded a highly dominant fragmentation pathway enabling lower energy fragmentation and high signal enhancement. Either one of two E / Z stereoisomers that can be formed from hydrazides or hydroxylamines (e.g., for testosterone labeling) can be predominantly generated, or both isomers can co-elute. This improves the sensitivity of electron-donating substituents A1, A2, A3, A4 and / or A5 (e.g., OMe). According to embodiments of the first aspect of the present invention, high signal enhancement can be achieved with Y1=H, Y2=H, A1=OMe, A2=H, A3=H, A4=H, A5=H, n=0, and K=hydrazide.

[0163] According to an embodiment of the first aspect of the present invention, high signal enhancement can be achieved with Y1=H, Y2=H, A1=OMe, A2=H, A3=H, A4=H, A5=H, n=1, and K=hydrazide.

[0164] In a second aspect, the present invention relates to compositions comprising compounds of formula I, which are disclosed in detail above or below with respect to a first aspect of the present invention. All embodiments described in relation to a first aspect of the present invention are applicable to a second aspect of the present invention, and vice versa.

[0165] In a third aspect, the present invention relates to a kit comprising a compound of Formula I disclosed in detail above or below with respect to a first aspect of the present invention, or a composition of a second aspect of the present invention disclosed in detail above or below herein. All embodiments described in relation to a first aspect and / or a second aspect of the present invention are applicable to a third aspect of the present invention, and vice versa.

[0166] In a fourth aspect, the present invention relates to a complex for detecting an analyte using mass spectrometry determination, comprising a covalently bound analyte and a bound compound, the complex being formed by a chemical reaction of the analyte and compound of the first aspect of the present invention, and / or, in particular, the analyte 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 another molecule, substances internalized by organisms, metabolites of such substances, and combinations thereof. All embodiments described in the first aspect and / or the second aspect and / or the third aspect of the present invention are applicable to the fourth aspect of the present invention, and vice versa.

[0167] In a fourth embodiment of the present invention, a covalent complex of formula III is formed from the formation of a covalent bond between a compound of formula I and a functional group present in the analyte molecule. Depending on the reactive unit K of the compound of formula I and the functional group of the analyte molecule, those skilled in the art can sufficiently determine the covalent bond formed between the two.

[0168] In a fourth embodiment of the present invention, the bonded compound is formula III and IV: [ka] [ka] (In the formula, one of the substituents B1, B2, and B4 is a coupling group Q that forms a covalent bond with the analyte) * And, The other substituents A1, A2, A3, A4, A5, B1, B2, B4 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, thioacyl, allyroyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, allyroyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, 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, and amine, or Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, benzyl, unsubstituted aromatic, substituted heteroaromatic, and unsubstituted heteroaromatic. n * is 0, 1, 2, 3, 4 or 5, The binding analyte is K * They are covalently bonded via, X * (This is the carbon atom of the imidazole in formula III.) Includes.

[0169] In the fourth embodiment of the present invention, each of A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, Y1, and Y2 in Formula III has the same meaning as described above in the first embodiment of the present invention (A1, A2, A3, A4, A5, B1, B2, B3, B4, Y1, and Y2 in Formula I).

[0170] In an embodiment of a fourth aspect of the present invention, X * The same meaning of X as described above in relation to the first aspect of the present invention. Preferably, X * As shown in equation IV, Q * It is not part of it.

[0171] In an embodiment of a fourth aspect of the present invention, K * This arises from the formation of a covalent bond between the reactive unit K of the compound of formula I and the functional group present in the analyte molecule. Depending on the reactive unit K of the compound of formula I and the functional group of the analyte molecule, a person skilled in the art can sufficiently determine the covalent bond formed between the two.

[0172] Furthermore, it is also considered within the scope of the present invention that functional groups present on the analyte molecule can be readily converted into other usable groups by first reacting with the reactive unit K of the compound of formula I.

[0173] In a fourth embodiment of the present invention, the analyte 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 organisms, metabolites of such substances, and combinations thereof.

[0174] In a fourth embodiment of the present invention, the analyte molecule comprises a functional group selected from the group consisting of a carbonyl group, a diene group, a hydroxyl group, an amine group, an imine group, a thiol group, a diol group, a phenol group, an epoxide group, a disulfide group, and an azide group, each of which can form a covalent bond with the reactive unit K of the compound of formula I.

[0175] In a fourth embodiment of the present invention, the analyte molecule is selected from the group consisting of steroids, ketosteroids, secosteroids, amino acids, peptides, proteins, carbohydrates, fatty acids, lipids, nucleosides, nucleotides, nucleic acids, and other biomolecules, including small molecule metabolites and cofactors, as well as therapeutic agents, addictive drugs, toxins, or their metabolites.

[0176] All embodiments of the analytes described in the first aspect of the present invention are applicable to the fourth aspect of the present invention, and vice versa. In the embodiments of the fourth aspect of the present invention, X *n is a carbon atom of the imidazolium ring of formula III. In the fourth embodiment of the present invention, n * is 0, 1, 2, 3, 4, or 5. Preferably, n * It is either 0 or 1.

[0177] In a fourth embodiment of the present invention, the binding compound is covalently bonded to the carbonyl group, hydroxyl group, or diene group of the analyte to form the complex.

[0178] In the fifth aspect, the present invention relates to the use of compounds of formula I for mass spectrometry determination of an analyte. Preferably, the mass spectrometry determination includes tandem mass spectrometry determination, in particular triple quadrupole mass spectrometry determination. All embodiments described in the first and / or second and / or third and / or fourth aspects of the present invention are applicable to the fifth aspect of the present invention, and vice versa.

[0179] In a fifth embodiment of the present invention, the mass spectrometry determination includes tandem mass spectrometry determination, in particular triple quadrupole mass spectrometry determination.

[0180] In a fifth embodiment of the present invention, the use of the compound of formula I includes use as a derivatizing reagent. In a fifth embodiment of the present invention, the compound of formula I is used to enhance the sensitivity of MS measurements. In the embodiment, the compound of formula I is used to detect the analyte of interest at lower levels of detection, particularly at lower levels of quantification.

[0181] In a fifth embodiment of the present invention, the compound of formula I according to the present invention includes a reactive unit K that can react with an analyte molecule. The reactive unit K can react with the analyte molecule such that a covalent bond is formed between the compound of formula I and the analyte molecule. In the fifth embodiment of the present invention, the reactive unit K forms a covalent bond with the compound of formula I. In particular, the covalent bond is formed between the reactive unit K of the compound of formula I and a functional group present on the analyte molecule.

[0182] Those skilled in the art select an appropriate reactive unit K for the compound of formula I, depending on the functional groups present in the analyte molecule being measured. Determining which reactive unit K is suitable for binding to the functional groups of the analyte is well known.

[0183] The above-described provisions regarding the analyte are applied to the analyte with necessary modifications in the context of the fifth aspect of the present invention.

[0184] The 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 a fifth embodiment of the present invention, the analyte molecule(s) may be present in a biological or clinical sample selected from the group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid, and dried blood spots. In some embodiments of the fifth embodiment 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.

[0185] In a sixth aspect, the present invention relates to a method for determining the mass spectrometry of an analyte. (a) A step of reacting an analyte with a compound of formula I disclosed herein in relation to a first aspect of the present invention, wherein a complex disclosed herein in relation to a fourth aspect of the present invention is formed, (b) A step of subjecting the composite from step (a) to mass spectrometry analysis. Includes.

[0186] Preferably, step (b) is: (i) A step of subjecting the ions of the complex to the first step of mass spectrometry, wherein the ions of the complex are characterized according to their mass / charge (m / z) ratio, (ii) A step of causing ion fragmentation of the complex, thereby generating a first entity, particularly a low molecular weight entity and daughter ions of the complex, wherein the daughter ions of the complex have a different m / z ratio than the ions of the complex. (iii) A step of subjecting the daughter ions of the complex to a second step of mass spectrometry, wherein the daughter ions of the complex are characterized according to their m / z ratio. Including, and / or, (ii) may further include alternative fragmentation of the complex ions, thereby releasing a second entity distinct from the first entity and generating a second daughter ion of the complex, (iii) may further include subjecting the first and second daughter ions of the complex to a second stage of mass spectrometry, thereby characterizing the first and second daughter ions of the complex according to their m / z ratio.

[0187] Step (a) may occur at different stages in the sample preparation workflow prior to mass spectrometry determination. Samples containing analyte molecules can be pretreated and / or concentrated by various methods. The pretreatment method depends on the type of sample, such as blood (fresh or dried), plasma, serum, urine, or saliva, while the concentration method depends on the analyte of interest. It is well known to those skilled in the art which pretreatment method is suitable for which sample type. It is also well known to those skilled in the art which concentration method is suitable for which analyte of interest.

[0188] In a sixth embodiment of the present invention, step (a) of the method for determining the mass spectrometry of an analyte molecule is performed following (i) a sample pretreatment step, following (ii) a first concentration of the sample, or following (iii) a second concentration of the sample.

[0189] In a sixth embodiment of the present invention, the sample is a whole blood sample, which is assigned to one of two predetermined sample pretreatment (PT) workflows, both of which include the addition of an internal standard (ISTD) and a hemolytic reagent (HR) followed by a predetermined incubation period (Inc), the difference between the two workflows being the order in which the internal standard (ISTD) and the hemolytic reagent (HR) are added. In the embodiment, water is added as a hemolytic reagent in amounts particularly 0.5:1 to 20:1 mL of water / mL of sample, particularly 1:1 to 10:1 mL of water / mL of sample, and particularly 2:1 to 5:1 mL of water / mL of sample.

[0190] In a sixth embodiment of the present invention, the sample is a urine sample, which is assigned to one of two other predetermined PT workflows, both of which include the addition of an internal standard and an enzyme reagent followed by a predetermined incubation period, the difference between the two workflows being the order in which the internal standard and the enzyme reagent are added. The enzyme reagent is typically a reagent used for glucuronide cleavage or protein cleavage, or any pretreatment of the analyte or matrix. An additional step involves the addition of a derivatizing reagent, such as a compound of the present invention as disclosed above or below herein, followed by an incubation period.

[0191] In a sixth embodiment of the present invention, the enzyme reagent is selected from the group consisting of glucuronidase, (partially) exo or endo-deglycosylase, or exo or endoprotease. In the embodiment, glucuronidase is added in an amount of 0.5 to 10 mg / mL, particularly in an amount of 1 to 8 mg / mL, and particularly in an amount of 2 to 5 mg / mL.

[0192] In a sixth embodiment of the present invention, the sample is plasma or serum and is assigned to another predetermined PT workflow, which includes only the addition of an internal standard (ISTD) prior to a predetermined incubation time.

[0193] As described above or below in this specification, the incubation times and temperatures to be selected for the sample processing, chemical reaction, or process steps under consideration are well known to those skilled in the art. In particular, it is known to those skilled in the art that incubation times and temperatures are interdependent, for example, that higher temperatures typically result in shorter incubation periods, and vice versa. In embodiments of a sixth aspect of the present invention, the incubation temperature is in the range of 4 to 45°C, particularly in the range of 10 to 40°C, particularly in the range of 20 to 37°C, for example, 20, 25, 30, 35, or 37°C. In embodiments, the incubation time is in the range of 30 seconds to 120 minutes, particularly in the range of 30 seconds to 1 minute, 30 seconds to 5 minutes, 30 seconds to 10 minutes, 1 minute to 10 minutes, or 1 minute to 20 minutes, 10 minutes to 30 minutes, 30 minutes to 60 minutes, or 60 minutes to 120 minutes. In certain embodiments, the incubation time is a multiple of 36 seconds, for example, m × 36 seconds, where m = 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0194] Therefore, step (a), which is an embodiment of the present method, is performed following any of the sample pretreatment processes disclosed above.

[0195] In a sixth embodiment of the present invention, the reaction of the compound of formula I with the analyte molecule in step (a) is carried out before any concentration process, and the compound of formula I is added to the pre-treated sample of interest. Thus, a complex of the analyte molecule and the compound of formula I is formed after the pre-treatment and before the first concentration process. The complex is then subjected to the first and second concentration processes before being subjected to mass spectrometry in step (b).

[0196] The pre-treated sample can be further subjected to an analyte concentration workflow. The analyte concentration workflow may include one or more concentration methods. Concentration methods include, but are not limited to, chemical concentration methods, including, but not limited to, chemical precipitation, which are well known in the art, as well as solid-phase concentration methods, including, but not limited to, solid-phase extraction methods, bead workflows, and chromatographic methods (e.g., gas or liquid chromatography).

[0197] In an embodiment of a sixth aspect of the present invention, the first concentration workflow includes adding a solid phase, particularly solid beads, that carries analyte-selective groups to a pre-treated sample. In an embodiment of a sixth aspect of the present invention, the first concentration workflow includes adding magnetic or paramagnetic beads that carry analyte-selective groups to a pre-treated sample. In an embodiment of a sixth aspect of the present invention, the addition of magnetic beads includes stirring or mixing. This is followed by a predetermined incubation period to capture the target analyte(s) on the beads. In an embodiment of a sixth aspect of the present invention, the workflow includes a washing step (W1) after incubation with magnetic beads. Depending on the analyte(s), one or more additional washing steps (W2) are performed. One washing step (W1, W2) consists of a series of steps including magnetic bead separation by a magnetic bead operating unit including a magnet or electromagnet, liquid aspiration, addition of a washing buffer, resuspension of magnetic beads, another magnetic bead separation step, and another liquid aspiration. Furthermore, the washing process may differ in terms of the type of solvent (water / organic / salt / pH) as well as the volume and number or combination of washing cycles. Methods for selecting each parameter are well known to those skilled in the art. The final washing steps (W1, W2) are followed by the addition of an elution reagent, then resuspending of the magnetic beads, and a predetermined incubation period for releasing the target analyte(s). Subsequently, the unbound magnetic beads are separated, and the supernatant containing the target derivatized analyte(s) is captured.

[0198] In an embodiment of the sixth aspect of the present invention, the first concentration workflow includes adding magnetic beads that carry matrix-selective groups to a pre-treated sample. In an embodiment of the sixth aspect of the present invention, the addition of magnetic beads includes stirring or mixing. This is followed by a predetermined incubation period to capture the matrix on the beads. Here, the analyte of interest does not bind to the magnetic beads and remains in the supernatant. The magnetic beads are then separated and the supernatant containing the desired concentrated analyte(s) is collected.

[0199] In a sixth embodiment of the present invention, the supernatant is subjected to a second concentration workflow. Here, the supernatant is transferred to an LC station, or transferred to an LC station after a dilution step by adding a diluent. Different elution procedures / reagents, such as variations in the type (water / organic / salt / pH) and volume of solvent, can also be used. Various parameters are well known to those skilled in the art and can be easily selected.

[0200] In an embodiment of a sixth aspect of the present invention, step (a) of the method was not performed immediately after the pretreatment method. Step (a) may be performed after the first concentration workflow using magnetic beads, as described above in this specification.

[0201] In a sixth embodiment of the present invention, analyte-specific magnetic beads are used, and the compound of formula I disclosed above or below herein is added to the sample of interest after the washing steps (W1, W2) are completed either before, with, or after the elution reagent, followed by an incubation period (defined time and temperature).

[0202] In an embodiment of the sixth aspect of the present invention, the unbonded magnetic beads are then separated and the supernatant containing the complex from step (a) is recovered. In an embodiment of the sixth aspect of the present invention, the supernatant containing the complex from step (a) is transferred to a second concentration workflow, in particular, either directly to an LC station or after a dilution step by adding a diluent.

[0203] In a sixth embodiment of the present invention, matrix-specific magnetic beads are used, and the compound of formula I disclosed above or below herein is added to the sample of interest before or after the magnetic beads are separated. In a sixth embodiment of the present invention, the supernatant containing the complex of step (a) is transferred to a second concentration workflow, in particular, either directly to an LC station or after a dilution step by adding a diluent.

[0204] Accordingly, in an embodiment of the sixth aspect of the present invention, the reaction of the compound of formula I with the analyte molecule in step (a) follows a first concentration process, and the compound of formula I is added to the sample of interest after the first concentration process, particularly after the first concentration process using magnetic beads. Thus, the sample is first pretreated as described above herein, then subjected to a first concentration process, particularly using magnetic beads carrying analyte-selective groups as described above herein, and the compound of formula I is added before, simultaneously with, or after elution from the beads. Thus, a complex of the analyte molecule and the compound of formula I is formed after the first concentration process and before the second concentration process. Thus, the complex is subjected to a second concentration process before being subjected to mass spectrometry in step (b).

[0205] In another embodiment of the sixth aspect of the present invention, step (a) of the method is performed after a second analyte concentration workflow. The second concentration workflow uses chromatographic separation to further concentrate the target analyte in the sample. In the embodiment of the sixth aspect of the present invention, the chromatographic separation is gas or liquid chromatography. Both methods are well known to those skilled in the art. In the embodiment of the sixth aspect of the present invention, the liquid chromatography is selected from the group consisting of HPLC, rapid LC, microLC, flow injection, and trapping and elution.

[0206] In an embodiment of the sixth aspect of the present invention, step (a) of the method is performed simultaneously with or after chromatographic separation. In an embodiment of the sixth aspect of the present invention, the compound of formula I is added to the column together with the elution buffer. In an alternative embodiment, the compound of formula I is added to the post-column.

[0207] In an embodiment of the sixth aspect of the present invention, the first concentration process includes the use of analyte-selective magnetic beads. In an embodiment of the sixth aspect of the present invention, the second concentration process includes the use of chromatographic separation, particularly using liquid chromatography.

[0208] Accordingly, in an embodiment of the sixth aspect of the present invention, the reaction of the compound of formula I with the analyte molecule in step (a) follows a second concentration process, and the compound of formula I is added to the sample of interest after the second concentration process using chromatography, particularly liquid chromatography. In this case, the sample is first pretreated as described above herein, then subjected to a first concentration process using magnetic beads, particularly as described above herein, then subjected to chromatographic separation, particularly using liquid chromatography, and subsequently to the addition of the compound of formula I. Thus, the complex of the bound analyte molecule and the bound compound of formula I is formed after the second concentration process. Therefore, the complex is not subjected to a concentration process before being subjected to mass spectrometry in step (b). Magnetic beads are known to those skilled in the art and are therefore not described in detail.

[0209] In the seventh aspect, the present invention relates to formula V: [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. Regarding the compounds.

[0210] All embodiments described in relation to the first and / or second and / or third and / or fourth and / or fifth and / or sixth aspects of the present invention are applicable to the seventh aspect of the present invention, and vice versa. In particular, all embodiments referred to in relation to the first aspect of the present invention, for example, all embodiments referred to in relation to A1, A2, A3, A4, A5, B1, B2, B3, B4, Y1 and Y2 are applicable to compounds of formula V. In particular, compounds of formula V can be used in mass spectrometry determination. Alternatively or additionally, compounds of formula V can be used in other technical fields, such as chemical labeling and drug delivery.

[0211] In embodiments of the present invention, the clinical diagnostic system comprises a compound of the first aspect of the present invention and / or a composition of the second aspect of the present invention and / or a kit of the third aspect of the present invention and / or a complex of the fourth aspect of the present invention. Additionally or optionally, the compound of the first aspect of the present invention is used for mass spectrometry determination of the analyte, and the clinical diagnostic system includes mass spectrometry determination. Additionally or optionally, the mass spectrometry determination method of the analyte according to the sixth aspect of the present invention is performed by the clinical diagnostic system.

[0212] A "clinical diagnostic system" is an automated laboratory device dedicated to the analysis of samples for in vitro diagnostics. A clinical diagnostic system can have different configurations depending on the needs and / or desired laboratory workflow. Further configurations can be obtained by combining multiple devices 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 can be analytical, pre-analytical, or post-analytical, or it can be an auxiliary function to any of the pre-analytical, analytical, or post-analytical functions. Specifically, for example, by performing one or more pre-analytical and / or post-analytical steps, a module can be configured to work in conjunction with one or more other modules to perform a dedicated task in the sample processing workflow. In particular, a clinical diagnostic system may include one or more analytical devices designed to perform their respective workflows optimized for specific types of analysis, such as clinical chemistry, immunochemistry, coagulation, hematology, liquid chromatography separation, mass spectrometry, etc. Therefore, a clinical diagnostic system may include either a single analyzer or a combination of such analyzers, each with 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 include 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. 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 with multiple LC channels, and / or a sample preparation / LC interface for inputting the prepared sample into any one of the LC channels.The clinical diagnostic system may further comprise a controller programmed to assign samples to a predefined sample preparation workflow, each comprising a predefined set of sample preparation steps and requiring a predefined time for completion depending on the analyte of interest. The clinical diagnostic system may further comprise a mass spectrometer (MS) and an LC / MS interface for connecting an LC separation station to the mass spectrometer. As used herein, the terms “automatically” or “automated” are broad terms and should be given their usual customary meanings to those skilled in the art, and should not be limited to any special or customized meanings. Specifically, the terms can refer to a process that is performed entirely by at least one computer and / or computer network and / or machine, without any manual action and / or user interaction.

[0213] A "sample preparation station" may be a pre-analytical module coupled to one or more analytical instruments or units within an analytical instrument, and is designed to perform a series of sample preparation 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 preparation steps may include one or more of the following operations performed sequentially, in parallel, or with a time delay 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.

[0214] A liquid chromatography (LC) separation station is an analyzer, module, or unit within an analyzer designed to subject a prepared sample to chromatographic separation, for example, to separate an analyte of interest from matrix components, such as residual matrix components after sample preparation, which may still interfere with subsequent detection, such as mass spectrometry detection, and / or to separate the analytes of interest from each other to enable individual detection. According to one embodiment, an 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, an LC separation station is a multi-channel LC station having multiple LC channels.

[0215] 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 each preparation being initiated according to the sample preparation initiation sequence.

[0216] Clinical diagnostic systems utilize LC connected to mass spectrometry more conveniently and reliably, making them suitable for clinical diagnosis. In particular, they enable high throughput, e.g., up to 100 samples / hour or more, using random-access sample preparation and LC separation while allowing online connection to mass spectrometry. Furthermore, the process can be fully automated, increasing walk-away time and reducing the required skill level.

[0217] In further embodiments, the present invention relates to the following aspects.

[0218] 1. Equation I for determining the mass spectrometry of the analyte: [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. A compound of [this].

[0219] 2. The following formula: [ka] A compound according to embodiment 1, comprising a structure having the above characteristics.

[0220] 3. The coupling group Q is given by the following equation II: [ka] (In the formula, K is a reactive unit that can form a covalent bond with the analyte, n is 0, 1, 2, 3, 4, or 5. X is the carbon atom of the imidazole in formula I. The compound according to embodiment 1 or 2, which is bonded to X according to the following.

[0221] 4. A compound according to any one of embodiments 1 to 3, wherein B1 is Q.

[0222] 5. A compound according to any one of embodiments 1 to 4, wherein B2 is Q.

[0223] 6. A compound according to any one of embodiments 1 to 5, wherein K can react with a carbonyl group, phenol group, amine, hydroxyl group, or diene group of the analyte.

[0224] 7. The compound according to any one of embodiments 1 to 6, wherein K is selected from the group consisting of hydrazides, hydrazines, hydroxylamines, and reactive carbonyls.

[0225] 8. The compound according to any one of embodiments 1 to 7, wherein Q is selected from the group consisting of methylene hydrazide, methylene hydrazine, and methylene hydroxylamine.

[0226] 9. The compound according to any one of embodiments 1 to 8, wherein the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4 that do not form a coupling group Q are each independently selected from hydrogen, methyl, ethyl, propyl, cyclopropyl, isopropyl, butyl, tert-butyl, or methoxy.

[0227] 10. The compound according to any one of embodiments 1 to 9, wherein Q is acetohydrazide or carbonylhydrazide.

[0228] 11. The compound according to any one of embodiments 1 to 10, wherein B3 is alkyl.

[0229] 12. The compound according to any one of embodiments 1 to 11, wherein B3 is methyl or B3 is ethyl.

[0230] The compound according to any one of aspects 1 to 12, wherein A1 is H or A1 is methoxy.

[0231] The compound according to any one of aspects 1 to 13, wherein A2 is H or A2 is methoxy.

[0232] The compound according to any one of aspects 1 to 14, wherein A3 is H or A3 is methoxy.

[0233] The compound according to any one of aspects 1 to 15, wherein A4 is H or A4 is methoxy.

[0234] The compound according to any one of aspects 1 to 16, wherein A5 is H or A5 is methoxy.

[0235] The compound according to any one of aspects 1 to 17, wherein Y1 is H or Y1 forms a condensed aromatic or heteroaromatic ring structure with Y2.

[0236] The compound according to any one of aspects 1 to 18, wherein Y2 is H or Y2 forms a condensed aromatic or heteroaromatic ring structure with Y1.

[0237] The compound according to any one of aspects 1 to 19, wherein B1 is H, particularly when B2 is Q.

[0238] The compound according to any one of aspects 1 to 20, wherein B4 is H.

[0239] The compound according to any one of aspects 1 to 21, wherein B2 is H, particularly when B1 is Q.

[0240] The compound according to any one of aspects 1 to 22, which is permanently positively charged.

[0241] 24. Comprising a counterion for forming a salt, the counterion preferably being in the following group: Cl - , Br- F - , formic acid, trifluoroacetic acid, PF6 - A compound according to any one of embodiments 1 to 23, selected from sulfonic acid, phosphoric acid, and acetic acid.

[0242] 25. A composition comprising the compound described in any one of embodiments 1 to 24.

[0243] 26. A kit comprising a compound described in any one of embodiments 1 to 24, or a composition according to embodiment 25.

[0244] 27. A complex for detecting an analyte by mass spectrometry, comprising a covalently bonded analyte and a bonded compound, wherein the complex is formed by a chemical reaction between the analyte and a compound described in any one of embodiments 1 to 24.

[0245] 28. The compound is formed by formulas III and IV: [ka] [ka] (In the formula, one of the substituents B1, B2, and B4 is a coupling group Q that forms a covalent bond with the analyte) * And, The other substituents A1, A2, A3, A4, A5, B1, B2, B4 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, thioacyl, allyroyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, allyroyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, 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 derivatives thereof, Y1 and Y2 are each independently selected from hydrogen, methyl, ethyl, methoxy, amine, or Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, benzyl, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, n * is 0, 1, 2, 3, 4 or 5, The binding analyte is covalently bonded via K * and, X * is a carbon atom of the imidazole of formula III) A complex according to embodiment 27, comprising

[0246] 29. The complex according to embodiment 27 or 28, wherein the 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 an organism, metabolites of such substances, and combinations thereof.

[0247] 30. The complex according to any one of embodiments 27 to 29, wherein the binding compound covalently binds via a carbonyl group, a hydroxyl group or a diene group of the analyte to form the complex.

[0248] 31. Use of a compound according to any one of embodiments 1 to 24 for mass spectrometric determination of an analyte.

[0249] 32. Use of a compound according to embodiment 31, wherein the mass spectrometric determination comprises tandem mass spectrometric determination, particularly triple quadrupole mass spectrometric determination.

[0250] 33. A method for mass spectrometric determination of an analyte, comprising (a) A step of reacting an analyte with a compound of formula I defined in any one of embodiments 1 to 24, wherein a complex defined in any one of embodiments 27 to 30 is formed, (b) A step of subjecting the composite from step (a) to mass spectrometry analysis. Methods that include...

[0251] 34. Mass spectrometry step (b) (i) A step of subjecting the ions of the complex to the first step of mass spectrometry, wherein the ions of the complex are characterized according to their mass / charge (m / z) ratio, (ii) A step of causing ion fragmentation of the complex, wherein a first entity, particularly a low molecular weight entity, is released and daughter ions of the complex are generated, the daughter ions of the complex having a m / z ratio different from that of the complex ions, (iii) A step of subjecting the daughter ions of the complex to a second step of mass spectrometry, wherein the daughter ions of the complex are characterized according to their m / z ratio. Including, and / or, (ii) may further include alternative fragmentation of the complex ions, thereby releasing a second entity distinct from the first entity and generating a second daughter ion of the complex, (iii) further comprises subjecting the first daughter ion and the second daughter ion of the complex to a second step of mass spectrometry, thereby characterizing the first daughter ion and the second daughter ion of the complex according to their m / z ratio, the method of embodiment 33.

[0252] 35.Formula V: [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. A compound of [this]. [Examples]

[0253] The following examples are provided to illustrate, and not to limit, the invention claimed herein.

[0254] Example 1: Synthesis of Label 1 [ka] Step 1: Imidazole alkylation: Synthesis of methyl 3-benzylimidazole-4-carboxylate [ka]

[0255] A mixture of methyl 4-imidazole carboxylate (0.622 mmol), benzyl chloride (1.130 mmol), and potassium carbonate (1.198 mmol) was dissolved in CHCl3 (1 mL). The solution was stirred at 70°C for 7 hours. The reaction mixture was filtered and washed with some chloroform. The solvent was removed under vacuum. The crude mixture was purified by column chromatography (hexane / siRNA (2:3) - pure siRNA) on silica, yielding 20.4 mg (15%) of the desired product as a colorless oil. 1 ¹H NMR (600 MHz, chloroform-d) δ ppm 3.82 (s, 3 H) 5.53 (s, 2 H) 7.18 (d, J=7.12 Hz, 2 H) 7.29-7.36 (m, 3 H) 7.63 (s, 1 H) 7.78 (s, 1 H). 13 C NMR(150 MHz,chloroform-d)δ ppm 50.02(1 C)51.47(1 C)122.44(1 C)127.29(2 C)128.11(1 C)128.88(2 C)136.15(1 C)138.08(1 C)142.22(1 C)160.62(1 C). HPLC-MS (m / z) [M+H]+ calculated value 217.10, detected value 217.33.

[0256] Step 2: Methylation: Synthesis of methyl 3-benzyl-1-methylimidazole-1-ium-4-carboxylate trifluoroacetate [ka]

[0257] A mixture of methylbenzylimidazole-4-carboxylate (0.094 mmol) was dissolved in dry DMF (1 mL), and iodomethane (0.321 mmol) was added. The solution was stirred at 80°C for 24 hours. The reaction mixture was quenched with MeOH, and the solvent was removed under vacuum. The crude mixture was purified by preparative HPLC, and 24.8 mg (77%) of the product was obtained as a colorless oily substance as the TFA salt.

[0258] HPLC method C-18 column: 0 min: 98% H2O 0.1% TFA,2% CH3CN 0.1% TFA; 0~40 min: 2% H2O 0.1% TFA,98% CH3CN 0.1% TFA; 40~45 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 45~46 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 46~49 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 49~50 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. HPLC-MS (m / z) [M]+: Calculated value 231.11, Detected value 231.43.

[0259] Step 3: Hydrazide formation: Synthesis of [(3-benzyl-1-methylimidazole-1-ium-4-carbonyl)amino]ammonium bistrifluoroacetate [ka]

[0260] An imidazolium derivative (0.072 mmol) was dissolved in 1 mL of MeOH. Hydrazine monohydrate (0.720 mmol) was added, and the reaction mixture was stirred at 50°C for 3 hours. The crude reaction mixture was used in the next step without purification. 1¹H NMR (400 MHz, D2O) δ ppm 3.77 (s,3 H) 5.47 (s,2 H) 7.15-7.17 (m,2 H) 7.29-7.32 (m,3 H) 7.77 (s,1 H). One proton signal (s,1 H) on the imidazole unit is lost due to H / D exchange. 13 C NMR(101 MHz,D2O)δ ppm 36.09(1 C)51.92(1 C)125.25(1 C)125.73(1 C)128.03(2 C)129.04(1 C)129.12(2 C)133.31(1 C)138.83(1 C)158.00(1 C).

[0261] Example 2: Synthesis of Label 2 [ka] Step 1: Imidazole alkylation: Synthesis of methyl 1-benzylimidazole-4-carboxylate [ka]

[0262] A mixture of methyl 4-imidazole carboxylate (0.622 mmol), benzyl chloride (1.130 mmol), and potassium carbonate (1.198 mmol) was dissolved in CHCl3 (1 mL). The solution was stirred at 70°C for 7 hours. The reaction mixture was filtered and washed with some chloroform. The solvent was removed under vacuum. The crude mixture was purified by column chromatography on silica (hexane / siRNA (2:3) - pure siRNA) to obtain 54.4 mg (40%) of the desired product as a colorless oil. 1 ¹H NMR (600 MHz, chloroform-d) δ ppm 3.83 (s, 3 H) 5.10 (s, 2 H) 7.15 (d, J=6.41 Hz, 2 H) 7.30-7.36 (m, 3 H) 7.52 (s, 1 H) 7.56 (s, 1 H). 13C NMR(150 MHz,chloroform-d)δ ppm 51.28(1 C)51.57(1 C)125.31(1 C)127.49(2 C)128.67(1 C)129.14(2 C)134.06(1 C)134.86(1 C)138.00(1 C)163.13(1 C). HPLC-MS (m / z) [M+H]+ calculated value 217.10, detected value 217.33.

[0263] Step 2: Methylation: Synthesis of methyl 1-benzyl-3-methylimidazole-3-ium-4-carboxylate trifluoroacetate [ka]

[0264] A mixture of methylbenzylimidazole-4-carboxylate (0.252 mmol) was dissolved in dry DMF (1 mL), and iodomethane (0.723 mmol) was added. The solution was stirred at 80°C for 24 hours. The reaction mixture was quenched with MeOH, and the solvent was removed under vacuum. The crude mixture was purified by preparative HPLC, and 74.9 mg (86%) of the product was obtained as a colorless oily substance as the TFA salt.

[0265] HPLC method C-18 column: 0 min: 98% H2O 0.1% TFA,2% CH3CN 0.1% TFA; 0~40 min: 2% H2O 0.1% TFA,98% CH3CN 0.1% TFA; 40~45 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 45~46 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 46~49 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 49~50 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. HPLC-MS (m / z) [M]+: Calculated value 231.11, Detected value 231.43.

[0266] Step 3: Hydrazide formation: Synthesis of [(1-benzyl-3-methylimidazole-3-ium-4-carbonyl)amino]ammonium bistrifluoroacetate [ka]

[0267] An imidazolium derivative (0.218 mmol) was dissolved in 1 mL of MeOH. Hydrazine monohydrate (2.058 mmol) was added, and the reaction mixture was stirred at 50°C for 3 hours. The crude reaction mixture was used in the next step without purification. 1 H NMR(400 MHz,D2O)δ ppm 3.83(s,3 H)5.26(s,2 H)7.25-7.35(m,5 H)7.77(s,1 H)8.77(s,1 H). 13 C NMR(101 MHz,D2O)δ ppm 35.56(1 C)53.21(1 C)123.54(1 C)128.72(2 C)129.32(2 C)129.44(1 C)132.68(1 C)138.82(1 C)158.10(1 C).

[0268] Example 3: General protocol for the reaction of testosterone and hydrazide derivatives: [ka] A hydrazide derivative (0.072 mmol) was dissolved in dry MeOH (1 mL), and testosterone (0.052 mmol) was added. The solution was stirred overnight at room temperature. The solvent was removed under vacuum, and the crude mixture was purified by preparative HPLC to obtain the desired product as a white solid.

[0269] Example 3.1: Preparation of labeled 1-testosterone derivative [ka] A mixture of crude [(3-benzyl-1-methylimidazole-1-ium-4-carbonyl)amino]ammonium bistrifluoroacetate (0.072 mmol) was dissolved in dry MeOH (1 mL), and testosterone (0.052 mmol) was added. The solution was stirred at room temperature for 20 hours. The solvent was removed under vacuum, and the crude mixture was purified by preparative HPLC, yielding 31.8 mg (97%) of the desired product as a colorless solid.

[0270] HPLC method C-18 column: 0 min: 98% H2O, 2% CH3CN; 0-45 minutes: 2% H2O, 98% CH3CN; 45-46 minutes: 0% H2O; 100% CH3CN; 46-55 minutes: 0% H2O; 100% CH3CN; 55-56 minutes: 50% H2O; 50% CH3CN. HPLC-MS (m / z) [M]+: Calculated value 501.32, Detected value 501.67.

[0271] Example 3.2: Preparation of labeled 2-testosterone derivative [ka] A mixture of crude [(1-benzyl-3-methylimidazole-3-ium-4-carbonyl)amino]ammonium bistrifluoroacetate (0.218 mmol) was dissolved in dry MeOH (1 mL), and testosterone (0.177 mmol) was added. The solution was stirred at room temperature for 20 hours. The solvent was removed under vacuum, and the crude mixture was purified by preparative HPLC, yielding 16.3 mg (12%) of the desired product as a colorless solid. HPLC method C-18 column: 0 min: 98% H2O, 2% CH3CN; 0-10 minutes: 98% H2O, 2% CH3CN; 10-60 minutes: 30% H2O; 70% CH3CN; 60-70 minutes: 0% H2O; 100% CH3CN; 70-71 minutes: 50% H2O; 50% CH3CN. HHPLC-MS (m / z) [M]+: Calculated value 501.32, Detected value 501.67.

[0272] Example 4: Analytical derivatization of testosterone using label 1 or label 2 A 500 ng / mL solution of testosterone (S1) was prepared in methanol. A solution (S2) containing either the derivatization reagent, label 1, or label 2 in excess compared to solution (S1), diluted in methanol (molar ratio > 1000), was added, and the solution was acidified with glacial acidic acid (20% v / v). Solutions S1 and S2 were mixed to obtain solution S3, which was held at 65°C for 2 hours, followed by 12 hours at room temperature. After 12 hours, solution S3 was diluted with methanol to obtain five independent concentration levels based on the molecular weight of testosterone at 1 veq; 1 / 20 veq; 1 / 40 veq; 1 / 100 veq; and 1 / 200 veq. 1 veq is within the proportional range of the detector, and 1 / 200 veq is selected so that it falls below the detection limit of the instrument used. For example, the following concentrations are used in the Waters Quattro micro system (100 ng / mL; 5 ng / mL; 2.5 ng / mL; 1 ng / mL; 500 pg / mL). A 0 ng / mL blank solution was prepared using solution S2.

[0273] Following the derivatization of the analyte molecule, isomers may be induced, potentially resulting in multiple peaks. The chromatographic behavior of these isomers needs to be addressed by methods that quantify their properties relatively. Figure 1 illustrates the signal distribution over time (%) when a specific chromatographic method is applied at 5% of the absolute peak height. The "separation" parameter describes the chromatographic system's ability to separate isomers arising from the derivatization reaction of the analyte molecule. The retention time, measured at the centroid of the isomer peaks, can describe the polarity of the resulting derivatives when reverse-phase chromatographic material is used for separation. Areas A1 and A2 represent the number of signals. *The measurements are taken in minutes and reported as the ratio of the derived isomers obtained. If the ratio of peaks A1 to A2 is 50 / 50, the resulting isomers are produced in equal amounts. If the label can affect this ratio, which is not equal to 50 / 50, one isomer is mainly produced. The signal intensity is therefore heterogeneously distributed, increasing the signal height against unaffected background noise.

[0274] For each labeled substance, the respective testosterone derivative was measured by electrospray ionization mass spectrometry after liquid chromatography separation, and different fragmentation scans were performed with collision energies of 15V, 20V, 25V, 30V, 35V, and 40V.

[0275] Fragmentation was optimized using the mass signals of the molecular ion peak and the most abundant fragment ion peak (neutral loss). Fragmentation energy is one of the most important tuning parameters for the signal gain of charged molecules with neutral loss units. Fragmentation energy is important because the molecule must not be cleaved during source and further ion pathway conditions. Cleavage or cleavage of the neutral loss fragment should occur only in the collision cell (and / or associated apparatus) to form a specific target fragment. If the collision energy is too high for the cleavage of the neutral loss fragment, other undesirable fragmentation pathways may also occur, resulting in a loss of signal intensity for the neutral loss fragmentation pathway. Therefore, the collision energy is considered to be neither too low nor too high to obtain optimal fragmentation behavior. The maximum area count for each collision energy was used in a further detection limit quantification approach.

[0276] For each labeled substance, the respective testosterone derivative was tuned in a triple quadrupole mass spectrometer by injecting it into the mass spectrometer via liquid chromatography. The tuning parameter was the collision energy that yielded the highest signal from the chromatographic peak in each of the five independent collision energy experiments.

[0277] Chromatography and MS parameters Polarity ES+ Calibration Static 2 Soft transmission mode disabled Capillary (kV) 3.00 3.14 Cone (V) 50.00 144.92 Source offset (V) 30.0 Sauce temperature (°C) 140 140 Desolvation temperature (℃)350 350 Cone gas flow rate (L / Hr) 150 149 Desolvation gas flow rate (L / Hr) 1000 990 Impinging gas flow (mL / min) 0.15 0.14 Nebulizer gas flow (Bar): 7.00 6.52 LM1 resolution 3.0 HM1 resolution 15.0 Ionic energy 1-0.2 MS mode collision energy 4.00 MSMS mode collision energy 2.00 MS Mode Entry 1.00 MS mode exit 1.00 Gas-on MS mode inlet 1.00 Gas-on MS mode outlet 1.00 Gas-on MSMS mode entry 1.00 Gas-on MSMS mode outlet 1.00 Gas off MS mode entry 30.00 Gas off MS mode exit 30.00 Gas off MSMS mode entry 30.00 Gas off MSMS mode exit 30.00 ScanWave MS Mode Entry 1.00 ScanWave MS mode output 1.00 ScanWave MSMS mode entry 1.00 ScanWave MSMS mode exit 1.00 LM2 resolution 3.0 HM2 resolution 15.0 Ion energy 2 0.2 Gain 1.00 Multiplier 513.80 Active Reservoir C Corn energy lamp: Disabled Probe temperature lamp: Disabled Collision energy lamp: Disabled Equipment Parameters - Function 2: Parameter file - E:\Regulated projects\ESI-Derivatization_PDA.PRO\ACQUDB\cz20Mrz2019-testo-label-general_tuning.IPR Polarity ES+ Calibration Static 2 Soft transmission mode disabled Capillary (kV) 3.00 3.14 Cone (V) 50.00 144.92 Source offset (V) 30.0 Sauce temperature (°C) 140 140 Desolvation temperature (℃)350 350 Cone gas flow rate (L / Hr) 150 149 Desolvation gas flow rate (L / Hr) 1000 990 Impinging gas flow (mL / min) 0.15 0.14 Nebulizer gas flow (Bar): 7.00 6.52 LM1 resolution 3.0 HM1 resolution 15.0 Ionic energy 1-0.2 MS mode collision energy 4.00 MSMS mode collision energy 2.00 MS Mode Entry 1.00 MS mode exit 1.00 Gas-on MS mode inlet 1.00 Gas-on MS mode outlet 1.00 Gas-on MSMS mode entry 1.00 Gas-on MSMS mode outlet 1.00 Gas off MS mode entry 30.00 Gas off MS mode exit 30.00 Gas off MSMS mode entry 30.00 Gas off MSMS mode exit 30.00 ScanWave MS Mode Entry 1.00 ScanWave MS mode output 1.00 ScanWave MSMS mode entry 1.00 ScanWave MSMS mode exit 1.00 LM2 resolution 3.0 HM2 resolution 15.0 Ion energy 2 0.2 Gain 1.00 Multiplier 513.80 Active Reservoir C Corn energy lamp: Disabled Probe temperature lamp: Disabled Collision energy lamp: Disabled Engineer settings: MS1 low mass position 673 MS1 high mass position 335 MS1 low mass resolution 215 MS1 high mass resolution 2152 MS1 resolution linearity 531 MS1 High Mass DC Balance 0.07 MS1 DC polarity negative MS2 low mass position 672 MS2 high mass position 291 MS2 low mass resolution 219 MS2 high mass resolution 2162 MS2 resolution linearity 528 MS2 High Mass DC Balance 0.50 MS2 DC polarity negative Inter-scan delay: Automatic mode MS scan delay (seconds): 0.003 Polarity / Mode Switch Scan Delay (seconds): 0.020 Enhanced inter-scan delay (seconds) 0.020 Inter-channel delay - See table. MS1 delay table: R delay <=1.250 0.001 <=4.000 0.002 <=10.000 0.003 <=20.000 0.004 > 20.000 0.005 ---------------Execution of Method Parameters-------------- Waters Acquity SDS Duration: 6.50 minutes comment: Solvent selection A: A2 Westen selection B:B1 Low pressure limit: 0.000 bar High pressure limit: 1034.200 bar Solvent name A: Water + NH4Ac + 0.1% Formic Acid Solvent name B: MeOH + NH4Ac + 0.1% formic acid Switch 1: No change Switch 2: No changes Switch 3: No changes Seal cleaning: 5.0 minutes Chart Out 1: System Pressure Chart Out 2: %B System pressure data channel: Yes Flow data channel: None %A data channel: None %B data channel: Yes Primary A pressure data channel: None Accumulator A pressure data channel: None Primary B pressure data channel: None Accumulator B pressure data channel: None Degasser pressure data channel: None [Slope Table] Time (minutes) Flow rate %A %B curve 1.Initial 0.400 60.0 40.0Initial 2.0.50 0.400 60.0 40.0 6 3.3.00 0.400 10.0 90.0 6 4.5.00 0.400 10.0 90.0 6 5.5.10 0.400 60.0 40.0 6 6.6.50 0.400 60.0 40.0 6 Ran event: Yes Gradient start (relative to injection): 0 μL 2D iteration: None Waters Acquity PDA Duration: 6.50 minutes PDA detector type: ULC LG 500nm Lamp: On Sampling rate: 10 points / second Filter time constant: 0.2000 seconds Exposure time: automatic msec Interpolation in the second-order filter region: None Use of UV blocking filter: None 3D channel... Range: 200-400 Resolution: 2.4nm Initial switch 1: No change Initial Switch 2: No changes Waters ACQUITY FTN AutoSampler Duration: 6.50 minutes comment: Preload: Disabled Offline loop: Automatic min Washing solvent name: ACN: Water Pre-injection rinsing time: 15.0 seconds Post-injection rinsing time: 15.0 seconds Purge solvent name: ACN: Water Dilution: Ineffective Dilution volume: 0 uL Delay time: 0 minutes Dilution needle placement: Automatic mm Target column temperature: 40.0℃ Column temperature alarm band: Disabled Target sample temperature: 6.0°C Sample temperature alarm band: Disabled Syringe withdrawal speed: Automatic Needle placement: 0.5mm Air gap before suction: Automatic Air gap after suction: Automatic Column temperature data channel: None Room temperature data channel: Yes Sample temperature data channel: Yes Sample organizer temperature data channel: None Sample pressure data channel: None Preheater temperature data channel: None Seal force data channel: None No spray mode: None Automatic addition and mixing stroke cycle: Automatic Automatic addition and mixing stroke volume: Automatic uL Active preheater: Use console configuration Run event: None Sample run injection parameters Injection volume (ul)-5.00 Function 1 Scans during operation: 738 Cycle time (seconds): Automatic Scan delay (seconds): Automatic Inter-channel delay (seconds): Automatic Span (Da): 0.500 Start and end times (minutes): 0.000~5.000 Ionization mode: ES+ Data type: SIR or MRM augmentation Function type: 1-channel MRM Chan reaction residence time (seconds) Cone Bolt. Col. Energy delay (seconds) compound 1:289.25 > 108.78 0.200 Tune 25.0 Auto Testosterone Function 2 Scans during operation: 737 Cycle time (seconds): Automatic Scan delay (seconds): Automatic Inter-channel delay (seconds): Automatic Span (Da): 0.500 Start and end times (minutes): 0.000~5.000 Ionization mode: ES+ Data type: SIR or MRM augmentation Function type: 1-channel MRM Chan reaction residence time (seconds) Cohnbolt. Col. Energy delay (seconds) Compound formula | Mass 1:624.40 > 203.00 0.200 Tune 40.0 Automatic DMA041 CE40 420.2 Function 3 Scanning during function: 3901 Function type: Diode array Wavelength range (nm): 200~400

[0278] The detection limits were obtained from the linear calibration curves using the procedure described in DIN EN ISO 32645. The enhancement factor was calculated based on the labeled analyte, e.g., testosterone LOD, compared to the underivative analyte, e.g., testosterone LOD. For example, an LOD of 6 pg / mL for testosterone and an enhancement factor of 10 means an LOD of 0.6 pg / mL for reagent-testosterone. As the enhancement factor increases, the LOD of the reagent-analyte decreases. The workflow is shown in Figure 2. The results are shown in Table 2 below. [Table 2]

[0279] As can be seen from Table 2, labels 1 and 2 and their complexes show signal enhancement compared to the underivative analytes. Label 2 and its complexes show better enhancement factors than state-of-the-art inductors, such as Sciex's Girard T and Amplifex.

[0280] In addition to testosterone, other analytes, such as estradiol or vitamin D, can be selected as the target analyte. Complexes with these other analytes show signal enhancement compared to non-derivative analytes.

[0281] This patent application claims priority to European Patent Application No. 20175799.4, the contents of which are incorporated herein by reference.

Claims

1. Equation (I) for determining the mass spectrometry of the analyte: 【Chemistry 1】 (In the formula, one of the substituents B1, B2, and B4 is a coupling group Q that can form a covalent bond with the functional group of the analyte, The other substituents A1, A2, A3, A4, A5, B1, B2, and B4 are each independently selected from hydrogen, methyl, ethyl, propyl, cyclopropyl, isopropyl, butyl, tert-butyl, and methoxy. B3 is selected from alkyl, acetyl, vinyl, unsubstituted aromatic, unsubstituted benzyl, and unsubstituted cycloalkyl. Y1 and Y2 are independently selected from hydrogen, methyl, ethyl, methoxy, unsubstituted aromatic, unsubstituted cycloalkyl, unsubstituted heteroaromatic, and amine, or Y1 and Y2 form a ring structure selected from unsubstituted cycloalkyl, unsubstituted aromatic, and unsubstituted heteroaromatic. The coupling group Q is given by the following formula (II): 【Chemistry 2】 (In the formula, K is a reactive unit that can form a covalent bond with the analyte, and K is selected from hydrazide, hydrazine, and hydroxylamine.) n is 0, 1, 2, 3, 4, or 5. X is the carbon atom of the imidazole in formula (I) (bonded to X by the formula (I)). A compound of [this].

2. The compound according to claim 1, wherein B1 is Q, or B2 is Q.

3. The compound according to claim 1 or 2, which is permanently positively charged.

4. The compound according to any one of claims 1 to 3, wherein the compound comprises a counterion for forming a salt.

5. The aforementioned counterions belong to the following group: Cl － , Br － F － , formic acid, trifluoroacetic acid, PF ６ － The compound according to claim 4, selected from sulfonic acid, phosphoric acid, and acetic acid.

6. A composition comprising the compound described in any one of claims 1 to 5.

7. A kit comprising a compound according to any one of claims 1 to 5, or a composition according to claim 6.

8. Use of the compound according to any one of claims 1 to 5 for determining the mass spectrometry of the analyte.

9. The use according to claim 8, wherein the mass spectrometry determination is a tandem mass spectrometry determination or a triple quadrupole mass spectrometry determination.

10. A method for determining the mass spectrometry of an analyte, (a) A step of reacting the analyte with a compound of formula (I) according to any one of claims 1 to 5, wherein a complex is formed thereon, (b) A step of subjecting the composite from step (a) to mass spectrometry analysis. Methods that include...

11. The mass spectroscopic analysis step (b) (i) A step of subjecting the ions of the complex to a first step of mass spectrometry, wherein the ions of the complex are characterized according to their mass / charge (m / z) ratio, (ii) A step of causing ion fragmentation of the complex, wherein a first entity is released and daughter ions of the complex are generated, and the m / z ratio of the daughter ions of the complex is different from that of the ions of the complex. (iii) a step of subjecting the daughter ions of the complex to a second step of mass spectrometry, wherein the daughter ions of the complex are characterized according to their m / z ratio, and / or (ii) may further include alternative fragmentation of the ions of the complex, thereby releasing a second entity different from the first entity and generating a second daughter ion of the complex, (iii) further comprises subjecting the first daughter ion and the second daughter ion of the complex to a second step of mass spectrometry, thereby characterizing the first daughter ion and the second daughter ion of the complex according to their m / z ratio. The method according to claim 10.

12. The method according to claim 11, wherein the first entity released in (ii) is a low molecular weight entity.

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