Benzylpyridinium Reagents 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 THE INVENTION The present invention relates to compounds suitable for use in mass spectrometry, compositions comprising said compounds, kits comprising said compositions and / or compounds and conjugates. Furthermore, the present invention relates to methods for the mass spectrometric determination of analytes using said compounds. [Background technology]

[0002] Background of the Invention Mass spectrometry (MS) is a widely used technique for the qualitative and quantitative analysis of chemicals ranging from small molecules to macromolecules. Generally, it is a very sensitive and specific method, even capable of analyzing complex biological samples, such as environmental or clinical samples. However, for some analytes, the sensitivity of the measurement remains an issue, especially when analyzed from complex biological matrices such as serum.

[0003] MS is often combined with chromatographic techniques, particularly gas and liquid chromatography, such as HPLC, where the analyzed molecules of interest (analytes) are separated by chromatography and individually subjected to mass spectrometry (Higashi et al. (2016) J. of Pharmaceutical and Biomedical Analysis 130 p.181-190).

[0004] However, there remains a need to increase the sensitivity of MS analytical methods, especially for the analysis of analytes that are low in abundance or where scarce material is available (such as biopsy tissue).

[0005] Several derivatization reagents (compounds) aimed at improving the sensitivity of the measurement of these analytes are known in the art. In particular, reagents containing a charged unit and a neutral loss unit combined in a single functional unit (e.g., WO 2011 / 091436) are known. Other reagents containing separate units are relatively bulky in structure and affect the general workflow of sample preparation and MS measurement (Rahimoff et al. (2017) J. Am. Chem. Soc. 139(30), pp. 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 December, 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 have disadvantages due to poor labeling efficiency, generation of structural isomers due to coupling chemistry, non-optimal ionization efficiency, disadvantages to post-coupling chromatographic separation, non-optimal fragmentation behavior due to multiple fragmentation pathways, and the need for high collision energies.

[0006] Therefore, there is an urgent need in the art for derivatization reagents that not only exhibit chemical structures that do not adversely affect the MS measurement workflow 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 period of time.

[0007] The present invention relates to novel reagents (compounds) that allow for the 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 for individual adaptation for the specific needs arising from the measurement of a particular analyte or for specific workflow adaptations.

[0008] It is an object of the present invention to provide compounds of formula I, kits and compositions each comprising said compounds, for the efficient detection of analytes by mass spectrometric determination. It is also an object of the present invention to provide conjugates and methods for the mass spectrometric determination of analytes.

[0009] This object(s) is / are solved by the subject matter of the independent claims. Further embodiments are the subject matter 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 provides a method for the mass spectrometric determination of an analyte, comprising the steps of formula I: [ka] wherein one of the substituents B1, B2, B3, B4, B5 is a coupling group Q capable of forming a covalent bond with an analyte; Other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, aryloyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotopes or derivatives thereof; 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, or Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, and unsubstituted heteroaromatic. The present invention relates to the compound

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

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

[0014] In a fourth aspect, the present invention relates to a complex for detecting an analyte using mass spectrometry, comprising a binding analyte and a binding compound covalently bound to each other, in particular the complex is formed by chemical reaction of the analyte with a compound of the second aspect of the invention.

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

[0016] In a sixth aspect, the present invention provides a method for mass spectrometric determination of an analyte, comprising: (a) reacting an analyte with a compound of formula I of the first aspect of the invention, whereby a complex of the fourth aspect of the invention is formed; (b) subjecting the complex from step (a) to mass spectrometric analysis; The present invention relates to a method, including:

[0017] In a seventh aspect, the present invention provides a compound of formula V: [ka] wherein one of the substituents B1, B2, B3, B4, B5 is a coupling group Q capable of forming a covalent bond with the analyte; the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, arylyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, arylyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotopes or derivatives thereof; 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, or Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, and unsubstituted heteroaromatic. The present invention relates to the compound [Brief explanation of the drawings]

[0018] [Figure 1]1 shows a schematic diagram of peak "splitting," illustrating the ability of a chromatographic system to separate different isomers from each other resulting from a derivatization reaction of an analyte molecule. [Figure 2] A schematic of the workflow for determining the enhancement factor of a derivatized / labeled analyte compared to an underivatized analyte (e.g., testosterone) or an amplified derivatized analyte (e.g., amplified derivatized testosterone) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0019] Detailed Description of the Invention Before describing the present invention in detail below, it is to be understood that the present invention is not limited to the specific embodiments and examples described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is 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 one of ordinary skill in the art.

[0020] Several documents are cited herein. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. In the event of a conflict between a definition or teaching of such an incorporated reference and a definition or teaching cited herein, the text of the present specification shall control.

[0021] Each element of the present invention is described below. While these elements are listed with specific embodiments, it is understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application unless the context dictates otherwise.

[0022] definition It will be understood that the word "comprise", and variations such as "comprises" and "comprising", imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0023] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0024] Ratios, concentrations, amounts, and other numerical data may be expressed or presented herein in the form of a "range." It is understood that such range format is used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values ​​expressly recited as boundaries of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly recited. By way of example, a numerical range of "4% to 20%" should be interpreted not only to include the explicitly recited value 4% to 20%, but also to include each individual value and subrange within the stated range. Thus, this numerical range includes individual values ​​such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20%, etc., and subranges such as 4-10%, 5-15%, 10-20%, etc. This same principle applies to ranges reciting minimum or maximum values. Moreover, such interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0025] The term "about," when used in connection with a numerical value, is meant to encompass numerical values ​​within a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0026] In the context of the present 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 can perform a different function, and two or more reactive groups can perform the same function. Reactive groups include, but are not limited to, reactive units, charged units, and neutral loss units. The compound may be designated as a label. In the context of the present invention, the term "binding compound" refers to the compound that is bound to an analyte. In principle, the compound and the binding compound can be identical. The compound and the binding compound can 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 is capable of forming a bond to the analyte but is not yet bound to the analyte. The binding compound is bound to the analyte.

[0027] The term "mass spectrometry" ("Mass Spec" or "MS") or "mass spectrometry determination" refers to an analytical technique used to identify compounds by their mass. MS is a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio or "m / z." MS techniques generally involve (1) ionizing compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating their mass-to-charge ratio. Compounds can be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. Generally, one or more molecules of interest are ionized, and the ions are then introduced into a mass spectrometry instrument, where a combination of magnetic and electric fields causes the ions to follow a path in space depending on their mass ("m") and charge ("z"). The term "ionization" or "ionizing" refers to the process of generating ions of an analyte having a net charge equal to one or more electron units. Anions are those with a net negative charge of one or more electron units, while cations are those with a net positive charge of one or more electron units. MS can be performed in either "negative ion mode," where negative ions are generated and detected, or "positive ion mode," where positive ions are generated and detected.

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

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

[0030] Most sample workflows in MS further include a sample preparation and / or enrichment step, where, for example, the analyte of interest is separated from the matrix using gas or liquid chromatography. Typically, for a mass spectrometry measurement, the following three steps are performed:

[0031] 1. A sample containing an analyte of interest is ionized, usually by complexation with a cation, often by protonation to a cation. Ionization sources include, but are not limited to, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).

[0032] 2. The ions are sorted and separated according to their mass and charge. High field asymmetric waveform ion mobility spectrometry (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 down a short length of capillary tube and a high positive or negative potential is applied to the end of the capillary tube. The solution that reaches the end of the tube is vaporized (atomized) into a jet or spray of very small droplets in solvent vapor. This mist of droplets passes through an evaporation chamber, which is slightly heated to prevent condensation and evaporate the solvent. As the droplets become smaller, the electrical surface charge density increases until natural repulsion between like charges causes ions and neutral molecules to be released.

[0035] The term "atmospheric pressure chemical ionization" or "APCI" refers to a mass spectrometry method similar to ESI. However, APCI generates ions through ion-molecule reactions that occur in a plasma at atmospheric pressure. The plasma is maintained by an electrical discharge between the spray capillary and a counter electrode. The ions are then typically extracted into a mass analyzer using a set of differentially pumped skimmer stages. A counterflow of dry, preheated nitrogen gas may be used to improve solvent removal. Gas-phase ionization in APCI can be more effective than ESI for analyzing less polar entities.

[0036] "High-field asymmetric waveform ion mobility spectrometry (FAIMS)" is an atmospheric pressure ion mobility technique that separates gas-phase ions according to their behavior in strong and weak electric fields.

[0037] "Multiple reaction mode" or "MRM" is a detection mode for MS instruments in which a precursor ion and one or more fragment ions are selectively detected.

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

[0039] In the context of this disclosure, the terms "analyte," "analyte molecule," or "analyte of interest" are used interchangeably to refer to chemical species analyzed by mass spectrometry. Chemical species, i.e., analytes, suitable for analysis by mass spectrometry can be any type of molecule present in a living organism, including, but not limited to, nucleic acids (e.g., DNA, mRNA, miRNA, rRNA, etc.), amino acids, peptides, proteins (e.g., cell surface receptors, 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 particular modification of another molecule (e.g., a sugar moiety or phosphoryl residue on a protein, a methyl residue on genomic DNA), or substances internalized by the organism (e.g., therapeutic drugs, drugs of abuse, toxins, etc.), or metabolites of such substances. Such analytes may serve as biomarkers. In the context of the present invention, the term "biomarker" refers to a substance in a biological system that is used as an indicator of the biological state of said system. In the context of the present invention, the term "bound analyte" refers to said analyte that is bound to a compound to form a complex. In principle, the analyte and the bound analyte can be identical. The analyte and the bound analyte can be substantially identical. Substantially identical may mean that both analytes have the same chemical structure, except that the structures of the functional groups differ from each other. Preferably, the analyte is capable of forming 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 indicate that the positive charge of the pyridinium unit is not readily reversible, for example, by washing, dilution, filtration, etc. A permanent positive charge may be the result of, for example, a covalent bond. A permanent positive charge is in contrast to a reversible positive charge (non-permanently positive charge), which may be the result of, for example, an electrostatic interaction.

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

[0042] The analyte may be present in a sample of interest, e.g., a biological sample or a clinical sample. The terms "sample" or "sample of interest" are used interchangeably herein and refer to a portion or piece of a tissue, organ, or individual, and are usually smaller than such tissue, organ, or individual, which is intended to represent the entire tissue, organ, or individual. Upon analysis, the sample yields information regarding 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 lymphatic fluid, or solid samples such as dried blood spots and tissue extracts. A further example of a sample is a cell culture or tissue culture.

[0043] In the context of this disclosure, a sample may be derived from an "individual" or "subject." Typically, the subject is a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0044] Prior to analysis by mass spectrometry, samples may be pretreated in a manner specific to the sample and / or analyte. In the context of the present disclosure, the term "pretreatment" refers to any measure necessary to enable subsequent analysis of the desired analyte by mass spectrometry. Pretreatment measures typically include, but are not limited to, elution of solid samples (e.g., elution of dried blood spots), addition of a hemolyzing reagent (HR) to whole blood samples, and addition of an enzyme reagent to urine samples. Also contemplated as sample pretreatment is the addition of an internal standard (ISTD).

[0045] The term "hemolytic reagent (HR)" refers to a reagent that lyses cells present in a sample, and in the context of the present invention, hemolytic reagent specifically refers to a reagent that lyses cells present in a blood sample, including but not limited to red blood cells present in a whole blood sample. A well-known hemolytic reagent is water (HO). Further examples of hemolytic reagents include, but are not limited to, deionized water, hypertonic liquids (e.g., 8M urea), ionic liquids, and different surfactants.

[0046] Typically, an internal standard (ISTD) is a known quantity of a substance that exhibits similar properties to the analyte of interest when subjected to a mass spectrometric detection workflow (i.e., including any pretreatment, enrichment, and actual detection steps). The ISTD exhibits similar properties to the analyte of interest, but is clearly distinguishable from it. By way of example, during a chromatographic separation, such as gas or liquid chromatography, the ISTD has approximately the same retention time as the analyte of interest from the sample. Thus, both the analyte and the ISTD enter the mass spectrometer simultaneously. The ISTD, however, exhibits a different molecular weight than the analyte of interest from the sample. This allows ions from the ISTD and the analyte to be distinguished in mass spectrometry using their different mass-to-charge (m / z) ratios. Both are subjected to fragmentation to produce daughter ions. These daughter ions can be distinguished by their m / z ratios and their respective parent ions. As a result, the signals from the ISTD and the analyte can be determined and quantified separately. Because the ISTD is added in a known amount, the signal intensity of an analyte from the sample can be attributed to a specific quantitative amount of the analyte. Thus, the addition of the ISTD allows for relative comparison of the amount of analyte detected, allowing for unambiguous identification and quantification of the analyte of interest present in the sample when the analyte(s) reach the mass spectrometer. Typically, although not necessarily, the ISTD is an isotopically labeled variant of the analyte of interest (e.g., 2 H, 13 C, or 15 (including labels such as N).

[0047] In addition to pretreatment, the sample may also be subjected to one or more enrichment steps. In the context of the present disclosure, the term "first enrichment process" or "first enrichment workflow" refers to an enrichment step that occurs following sample pretreatment and provides a sample containing enriched analytes compared to the initial sample. The first enrichment workflow may include chemical precipitation (e.g., using acetonitrile) or the use of a solid phase. Suitable solid phases include, but are not limited to, solid phase extraction (SPE) cartridges and beads. The beads may be nonmagnetic, magnetic, or paramagnetic. The beads may be coated differently to be specific for the analyte of interest. The coating may vary depending on the intended application, i.e., the intended capture molecule. Those skilled in the art will be familiar with which coating is suitable for which analyte. The beads can be made of a variety of different materials. The beads may have various sizes and may include porous or non-porous surfaces.

[0048] In the context of the present disclosure, the term "second enrichment process" or "second enrichment workflow" refers to an enrichment process that occurs following sample pretreatment and a first enrichment process and provides a sample containing an analyte that is enriched relative to the initial sample and the sample after the first enrichment process.

[0049] The term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of the differential distribution of the chemical components as they flow around or over a stationary liquid or solid phase.

[0050] The terms "liquid chromatography" or "LC" refer to the process of selectively retarding one or more components of a fluid solution as the fluid permeates uniformly through a column or capillary passage of finely divided material. Retardation results from the distribution of mixture components between one or more stationary phase(s) and the bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal phase liquid chromatography (NPLC), while methods in which the stationary phase is less polar 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" or "HPLC" refers to a method of liquid chromatography in which the degree of separation is increased by passing a mobile phase under pressure through a stationary phase, typically a densely packed column. Typically, the column is packed with a stationary phase composed of irregular or spherical particles, a porous monolithic layer, or a porous membrane. HPLC has historically been divided into two distinct subclasses based on the polarity of the mobile and stationary phases. Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal-phase liquid chromatography (NPLC), while the opposite (e.g., water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) is called reverse-phase liquid chromatography (RPLC). Micro-LC refers to HPLC methods using columns with narrow internal diameters, typically less than 1 mm, e.g., about 0.5 mm. "Ultra-high performance liquid chromatography" or "UHPLC" refers to an HPLC method using a pressure of 120 MPa (17,405 lbf / in2), or approximately 1200 atmospheres. Rapid LC refers to an LC method using a short column with an internal diameter as described above and a length of less than 2 cm, e.g., 1 cm, at the above flow rate and pressure (micro LC, UHPLC). A short rapid LC protocol involves trapping / washing / elution steps using a single analytical column, achieving LC in a very short time of less than 1 minute.

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

[0053] LC separations may be single-channel LC or multi-channel LC, comprising multiple LC channels arranged in parallel, in which analytes may be separated according to their polarity or log P value, size, or affinity, as is commonly known to those skilled in the art.

[0054] The term "fragmentation" refers to the dissociation of a single molecule into two or more separate molecules. As used herein, the term fragmentation refers to a specific fragmentation event in which the point of destruction of the parent molecule where the fragmentation event occurs is clearly defined and the multiple daughter molecules resulting from the fragmentation event are well characterized. Methods for determining the points of destruction of the parent molecule and the resulting multiple daughter molecules are well known to those skilled in the art. The resulting daughter molecules may be stable or may dissociate during subsequent fragmentation events. For example, if the parent molecule undergoing fragmentation contains an N-benzylpyridinium unit, one skilled in the art can determine, based on the overall structure of the molecule, whether the pyridinium unit fragments to release the benzyl entity or is completely released from the parent molecule; i.e., the resulting daughter molecules are either benzyl molecules or parent molecules lacking the benzyl. Fragmentation can occur via collision-induced dissociation (CID), electron-capture dissociation (ECD), electron-transfer dissociation (ETD), negative electron-transfer dissociation (NETD), electron-detachment dissociation (EDD), photodissociation, particularly infrared multiphoton dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD), surface-induced dissociation (SID), 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, i.e., can form a covalent bond with another molecule, such as an analyte of interest. Typically, such covalent bonds are formed with chemical groups present in other molecules. Thus, 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 serves to react with the reactive unit of the compound, the chemical group present in the analyte molecule is also referred to as 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 an atom of the reactive group and a functional group of the analyte. It is well known to those skilled in the art that when a covalent bond is formed between a reactive group and a functional group of the analyte, an atom is lost during this chemical reaction.

[0056] In the context of the present disclosure, the term "complex" refers to a product produced by the reaction of a compound with an analyte molecule. This reaction results in the formation of a covalent bond between the compound and the analyte. Thus, the term complex refers to a covalently linked reaction product formed by the reaction of a compound with an analyte molecule.

[0057] A "kit" is any article of manufacture (e.g., a package or container) containing at least one reagent, such as a drug for treating a disorder or a probe for specifically detecting a biomarker gene or protein of the present invention. The kit is preferably promoted, distributed, or sold as a unit for carrying out the method of the present invention. Typically, the kit can further include a carrier means compartmentalized to receive in close confinement one or more container means, such as vials, tubes, etc. In particular, each of the container means comprises one of the separate elements used in the method of the first aspect. The kit can 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 additional materials, including, but not limited to, buffers, diluents, filters, needles, syringes, and a package insert with instructions for use. Labeling can be displayed on the container to indicate that the composition is to be used in a particular application and can also indicate instructions for either in vivo or in vitro use. The computer program code can be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disc), or directly to a computer or data processing device. Additionally, the kit may contain standard amounts of biomarkers as described elsewhere herein for calibration purposes.

[0058] Embodiment In a first aspect, the present invention provides a compound of formula I or at least one compound: [ka] wherein one of the substituents B1, B2, B3, B4, B5 is a coupling group Q capable of forming a covalent bond with the analyte; the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, arylyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, arylyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotopes or derivatives thereof; 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, or Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, and unsubstituted heteroaromatic. In particular, the compound(s) of formula I are for the mass spectrometric determination of an analyte.

[0059] The compounds of 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, inclusive). These compounds are characterized by an overall compact structure. This allows for a simple modular design of the labeling reagents, and various issues can be resolved by modifying them with charge-containing moieties (e.g., pyridinium, quaternary ammonium, imidazolium, triphenylphosphonium, stable metal complexes, sulfonic acid, borate, aryltrifluoroborates, sulfate, phosphate, etc.), although permanent charges are preferred. This allows for fine-tuning the hydrophobicity of the reagents to improve separation of sample matrices during the purification process. Furthermore, mass fragmentation can occur at low energy levels to favor primary fragmentation pathways. This can enhance the sensitivity of MS / MS mode. Regardless of the above, further MS experiments can be performed at higher energies to explore additional fragmentation pathways.

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

[0061] In an embodiment of the first aspect of the present invention, alkyl is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, and cyclohexyl. The alkyl can include modified alkyl. Modified alkyl includes the structural unit alkyl-O-alkyl, such as -CH2-O-CH3, -CH2-O-CH2-CH3.

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

[0063] In an embodiment of the first aspect 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 an embodiment of the first aspect of the present invention, alkoxy is selected from the group consisting of methoxy, ethoxy, propoxy, butoxy, phenoxy and benzyloxy.

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

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

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

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

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

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

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

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

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

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

[0075] In an embodiment of the first aspect of the present invention, heteroaryl is selected from the group consisting of imidazole, pyrazole, triazole, tetrazole, oxazole, isoxazole, thiophene, furan, thiazole, pyridine, pyrimidine, benzotriazole, benzofuran and benzimidazole.

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

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

[0078] In an embodiment of the first aspect 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 an embodiment of the first aspect of the present invention, the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, thioacyl, aryloyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, isotopes, or derivatives thereof.

[0080] In an embodiment of the first aspect of the present invention, 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, and unsubstituted heteroaromatic.

[0081] In an embodiment of the first aspect of the invention, the compound of formula I comprises a coupling group Q. At least one of the substituents B1, B2, B3, B4 or B5 of formula I is a coupling group Q. Preferably, B1 or B2 or B3 is Q.

[0082] In an embodiment of the first aspect of the present invention, the coupling group Q has the following formula II: [ka] wherein K is a reactive unit capable of forming a covalent bond with the analyte; n is 0, 1, 2, 3, 4 or 5; X is a carbon atom of the pyridinium cation of formula I, in particular the bonding carbon atom of the pyridinium cation of formula I. is attached to X according to

[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 an embodiment of the first aspect of the present invention, Q is linked to X via a covalent bond.

[0085] In an embodiment of the first aspect of the present invention, Q is selected from the group consisting of methylhydrazide, methylhydrazine, hydrazine, methylhydroxylamine, acetohydrazide, 4-substituted 1,2,4-triazoline-3,5-diones (TADs) and F.

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

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

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

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

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

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

[0092] In an embodiment of the first aspect of the invention, the carbon atom X of the pyridinium cation of formula I is a binding partner of B1, B2, B3, B4 or B5. In an embodiment of the first aspect of the invention, the compounds are of the following groups I-1, I-2, I-3, I-4 and I-5: [ka] (In the formula, A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, K, X and n each have the meaning described above.) Preferably, the compound comprises formula I-1, I-2 or I-3.

[0093] 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 capable of reacting with an analyte or analyte molecule. The reactive unit K is capable of reacting with an 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 or 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 a functional group present on the analyte molecule.

[0095] In an embodiment of the first aspect of the present invention, K is capable of reacting with a carbonyl group, a phenol group, an amine, a hydroxyl group or a diene group of the analyte.

[0096] In an embodiment of the first aspect of the present invention, K is selected from the group consisting of hydrazide, hydrazine, hydroxylamine, Br, F, 4-substituted-1,2,4-triazoline-3,5-diones (TADs), 4-phenyl-1,2,4-triazoline-3,5-diones (PTADs), 4-pyridinium-1,2,4-triazoline-3,5-diones and reactive carbonyl groups.

[0097] In an embodiment of the first aspect of the present invention, n=0 and K is Br, or n=0 and K is F.

[0098] Depending on the functional groups present in the analyte molecule to be measured, one skilled in the art will select an appropriate reactive unit K for the compound of formula I. It is well known to determine which reactive unit K is suitable for binding to the functional group of the analyte of interest.

[0099] In an embodiment of the first aspect 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 ketone group, an aldehyde group, a thiol group, a diol group, a phenol group, an epoxy group, a disulfide group, a nucleobase group, a carboxylic acid group, a terminal cysteine ​​group, a terminal serine group, and an azide group, each of which is capable of forming a covalent bond with a reactive unit K of a compound of formula I. Furthermore, it is also considered within the scope of the present invention that a functional group present on the analyte molecule is first converted into another group that is readily available by reaction with the reactive unit K of a compound of formula I.

[0100] In an embodiment of the first aspect 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, drugs of abuse, toxins, or their metabolites.

[0101] In an embodiment of the first aspect of the present invention, the analyte molecule comprises a carbonyl group as a functional group selected from the group consisting of a carboxylic acid group, an aldehyde group, a keto group, a masked aldehyde group, a masked keto group, an ester group, an amide group, and an anhydride group. Aldoses (aldehydes and ketos) exist as acetals and hemiacetals, which are masked forms of the parent aldehyde / keto.

[0102] In an embodiment of the first aspect of the present invention, the carbonyl group is an amide group, and those skilled in the art will recognize that, although an amide group itself is a stable group, it can be hydrolyzed to a carboxylic acid group and an amino group. Hydrolysis of the amide group can be achieved via an acid / base catalysis reaction or an enzymatic process, both of which are well known to those skilled in the art. In an embodiment of the first aspect of the present invention, in which the carbonyl group is a masked aldehyde group or a masked keto group, each group is either a hemiacetal group or an acetal group, particularly a cyclic hemiacetal group or an acetal group. In an embodiment of the first aspect 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.

[0103] In an embodiment of the first aspect of the present invention, the carbonyl group is a keto group. In an embodiment of the first aspect of the present invention, the keto group can be transferred to an imine group of the intermediate before reacting with the reactive unit of the compound of formula I. In an embodiment of the first aspect of the present invention, the analyte molecule containing one or more keto groups is a ketosteroid. In certain embodiments of the first aspect of the present invention, the ketosteroid is selected from the group consisting of testosterone, epitestosterone, dihydrotestosterone (DHT), desoxymethyltestosterone (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, allopregnenolone, and aldosterone.

[0104] In an embodiment of the first aspect of the invention, the carbonyl group is a carboxy group. In an embodiment of the first aspect of the invention, the carboxy group is reacted directly with a compound of formula I or converted to an activated ester group prior to reaction with a compound of formula I. In an embodiment of the first aspect of the invention, the analyte molecule comprising one or more carboxy 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 an embodiment of the first aspect of the present invention, the analyte molecule comprising 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.

[0105] In an embodiment of the first aspect of the present invention, the carbonyl group is an aldehyde group. In an embodiment of the first aspect of the present invention, the aldehyde group can be transferred to an imine group of the intermediate before reacting with the reactive unit of the compound of formula I. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more aldehyde groups is selected from the group consisting of pyridoxal, N-acetyl-D-glucosamine, alcaftadine, streptomycin, and josamycin.

[0106] In an embodiment of the first aspect of the present invention, the carbonyl group is a carbonyl ester group. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more ester groups is selected from the group consisting of cocaine, heroin, Ritalin, aceclofenac, acetylcholine, amcinonide, amiloxate, amylocaine, anileridine, aranidipine, artesunate, and pethidine.

[0107] In an embodiment of the first aspect of the present invention, the carbonyl group is an anhydride group. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more anhydride groups is selected from the group consisting of cantharidin, succinic anhydride, trimellitic anhydride, and maleic anhydride.

[0108] In an embodiment of the first aspect of the present invention, the analyte molecule comprises one or more diene groups, particularly conjugated diene groups, as functional groups. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more diene groups is a secosteroid. In an embodiment, the secosteroid is selected from the group consisting of cholecalciferol (vitamin D3), ergocalciferol (vitamin D2), calcifediol, calcitriol, tachysterol, lumisterol, and tacalcitol. In particular, the secosteroid is vitamin D, particularly vitamin D2 or D3, or a derivative 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 invention, the analyte molecule comprising 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.

[0109] In embodiments of the first aspect of the present invention, the analyte molecule comprises one or more hydroxyl groups as functional groups. In embodiments of the first aspect of the present invention, the analyte molecule comprises 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 located adjacent to each other or may be separated by one, two, or three C atoms (1,3-diol, 1,4-diol, 1,5-diol, respectively). In particular embodiments of the first aspect, the analyte molecule comprises a 1,2-diol group. In embodiments where only one hydroxyl group is present, the analyte is selected from the group consisting of primary alcohols, secondary alcohols, and tertiary alcohols. In embodiments of the first aspect of the invention wherein 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, ethylmorphine, morphine, morphine-3-glucuronide, buprenorphine, codeine, dihydrocodeine, p-hydroxypropoxyphene, O-desmethyltramadol, desmetramadol, dihydroquinidine, and quinidine. In an embodiment of the first aspect of the invention, wherein the analyte molecule comprises multiple hydroxyl groups, the analyte is selected from the group consisting of vitamin C, glucosamine, mannitol, tetrahydrobiopterin, cytarabine, azacytidine, ribavirin, floxuridine, gemcitabine, streptozotocin

[0110] , adenosine, vidarabine, cladribine, estriol, trifluridine, clofarabine, nadolol, zanamivir, lactulose, adenosine monophosphate, idoxuridine, regadenoson, lincomycin, clindamycin, canaglifodine, tobramycin, netilmicin, kanamycin, ticagrelor, epirubicin, doxorubicin, arbekacin, streptomycin, ouabain, amikacin, neomycin, framycetin, paromomycin, erythromycin, clarithromycin, azithromycin, vindesine, digitoxin, digoxin, metrizamide, acetyldigitoxin, deslanoside, fludarabine, clofarabine, gemcitabine, cytarabine, capecitabine, vidarabine, and plicamycin.

[0111] In an embodiment of the first aspect 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 an embodiment of the first aspect 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, tiopronin, dimercaprol, and succinimer.

[0112] In an embodiment of the first aspect of the present invention, the analyte molecule comprises one or more disulfide groups as functional groups. In an embodiment of the first aspect of the present invention, the analyte molecule comprising 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. The selenium sulfide can be selenium disulfide, SeS2, or selenium hexasulfide, Se2S6.

[0113] In an embodiment of the first aspect of the present invention, the analyte molecule comprises one or more epoxide groups as functional groups. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more epoxide groups is selected from the group consisting of carbamazepine-10,11-epoxide, carfilzomib, furosemide epoxide, fosfomycin, sevelamer hydrochloride, cerulenin, scopolamine, tiotropium, tiotropium bromide, methylscopolamine bromide, eplerenone, mupirocin, natamycin, and troleandomycin.

[0114] In an embodiment of the first aspect of the invention, the analyte molecule comprises one or more phenolic groups as functional groups. In a particular embodiment of the first aspect of the invention, the analyte molecule comprising one or more phenolic groups is a steroid or steroid-like compound. In an embodiment of the first aspect of the invention, the analyte molecule comprising one or more phenolic groups is sp 2A steroid or steroid-like compound having a hybridized A ring and an OH group at position 3 of the A ring. In certain embodiments of the first aspect of the invention, the steroid or steroid-like analyte molecule is selected from the group consisting of estrogen, an estrogenic compound, estrone (E1), estradiol (E2), 17a-estradiol, 17b-estradiol, estriol (E3), 16-epiestriol, 17-epiestriol, and 16,17-epiestriol, and / or metabolites thereof. In embodiments, the metabolite is estriol, 16-epiestriol (16-epiE3), 17-epiestriol (17-epiE3), 16,17-epiestriol (16,17-epiE3), 16-ketoestradiol (16-ketoE2), 16a-hydroxyestrone (16a-OHEl), 2-methoxyestrone (2-MeOEl), 4-methoxyestrone (4-MeOEl), 2-hydroxyestrone-3-methyl ether (3-MeOEl), 2-methoxyestradiol (2-MeOE2), 4-methoxyestradiol (4-MeOE2), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2-hydroxyestradiol (2-OHE2), estrone (E1), estrone sulfate (E2s), 17a-estradiol (E2a), 17b-estradiol (E2B), estradiol sulfate (E2S), equilin (EQ), 17a-dihydroequilin (EQa), 17b-dihydroequilin (EQb), equilenin (EN), 17-dihydroequilenin (ENa), 17α-dihydroequilenin, 17β-dihydroequilenin (ENb), Δ8,9-dehydroestrone (dE1), Δ8,9-dehydroestrone sulfate (dE1), Δ9-tetrahydrocannabinol, and mycophenolic acid. β and b can be used interchangeably. α and a can be used interchangeably.

[0115] In an embodiment of the first aspect of the present invention, the analyte molecule comprises an amine group as a functional group. In an embodiment of the first aspect of the present invention, the amine group is an alkylamine group or an arylamine group. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more amine groups is selected from the group consisting of proteins and peptides. In an embodiment of the first aspect of the present invention, the analyte molecule comprising an amine group is selected from the group consisting of 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-fluoromethamine, 3-methyl-4-methyl-2-propanol, 3-methyl-2-propanol, 3-methyl-4 ... Cathinone, 4-methoxymethcathinone, 4-methylethcathinone, 4-methylmethcathinone, amfepramone, butyrone, etcathinone, flephedrone, methcathinone, methylone, methylenedioxypyrovalerone, benzoylecgonine, dehydronorketamine, ketamine, norketamine, methadone, normethadone, 6-acetylmorphine, diacetylmorphine, morphine, norhydrocodone, oxycodone, oxymol and 2-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, sisomicin, and 5-methylcytosine.

[0116] In an embodiment of the first aspect of the invention, the analyte molecule is a carbohydrate or a substance having a carbohydrate moiety, such as a glycoprotein or a nucleoside. In an embodiment of the first aspect of the invention, the analyte molecule is a monosaccharide, in particular selected from the group consisting of ribose, desoxyribose, arabinose, ribulose, glucose, mannose, galactose, fucose, fructose, N-acetylglucosamine, N-acetylgalactosamine, neuraminic acid, N-acetylneuraminic acid, etc. In an embodiment, the analyte molecule is an oligosaccharide, in particular selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and polysaccharides. In an embodiment of the first aspect of the invention, the disaccharide is selected from the group consisting of sucrose, maltose, and lactose. In an embodiment of the first aspect of the invention, the analyte molecule is a substance comprising the above-mentioned monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide moiety.

[0117] In an embodiment of the first aspect of the present invention, the analyte molecule comprises an azide group as a functional group selected from the group consisting of alkyl or aryl azides. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more azide groups is selected from the group consisting of zidovudine and azidocillin.

[0118] Such analyte molecules may be present in biological or clinical samples such as bodily fluids, e.g., blood, serum, plasma, urine, saliva, cerebrospinal fluid, etc., tissue or cell extracts. In embodiments of the first aspect of the 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 invention, the analyte molecule may be present in a purified or partially purified sample, e.g., a sample that is a purified or partially purified protein mixture or extract.

[0119] 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.

[0120] 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 can have either an adjacent O or N atom NH-N / O, or a hypernucleophilic N atom enhanced by the α-effect via a dithiol molecule.

[0121] In an embodiment of the first aspect of the present invention, the carbonyl-reactive unit is selected from the group: (i) a hydrazine unit, such as HN-NH- or HN-NR 1 -unit (wherein R 1 is an aryl, an aryl containing one or more heteroatoms, or C 1~4 alkyl, especially C1 or C2 alkyl, optionally including, for example, halo, hydroxyl, and / or C 1~3 substituted with alkoxy), (ii) a hydrazide unit, in particular a carbohydrazide or sulfohydrazide unit, in particular HN—NH—C(O)— or HN—NR 2 -C(O)- unit (wherein R 2 is an aryl, an aryl containing one or more heteroatoms, or C 1~4 alkyl, especially C1 or C2 alkyl, optionally including, for example, halo, hydroxyl, and / or C 1~3substituted with alkoxy), (iii) a hydroxylamino unit, such as a HN-O- unit; (iv) a dithiol unit, in particular a 1,2-dithiol or 1,3-dithiol unit is selected from.

[0122] In an embodiment of the first aspect of the present invention, the carbonyl-reactive unit is a carboxyl-reactive unit, and the carboxyl-reactive unit reacts with a carboxyl group on an analyte molecule. In an embodiment of the first aspect 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.

[0123] In an embodiment of the first aspect of the present invention, the analyte molecule comprises a ketone or aldehyde group, and K is a carbonyl-reactive unit, which is selected from the group: (i) a hydrazine unit, (ii) a hydrazide unit, (iii) a hydroxylamino unit, and (iv) Dithiol unit is selected from.

[0124] In an embodiment of the first aspect of the present invention, the reactive unit K is a diene-reactive unit, which can react with an analyte having a diene group. In an embodiment of the first aspect of the present invention, the diene-reactive unit is selected from the group consisting of Cookson-type reagents that can act as dienophiles, such as 1,2,4-triazoline-3,5-dione.

[0125] In an embodiment of the first aspect of the present invention, the reactive unit K is a hydroxyl-reactive unit, which can react with an analyte having a hydroxyl group. In an embodiment of the first aspect of the present invention, the hydroxyl-reactive unit is selected from the group consisting of sulfonyl chloride, activated carboxylic acid ester (NHS or imidazolide), and fluoroaromatic / heteroaromatic capable of nucleophilic substitution of fluorine (T. Higashi, J. Steroid Biochem. Mol. Biol. (September 2016) Vol. 162, pp. 57-69). In an embodiment of the first aspect of the present invention, the reactive unit K is a diol-reactive unit that reacts with a diol group on an analyte molecule. In an embodiment of the first aspect of the present invention where the reactive unit is a 1,2-diol-reactive unit, the 1,2-diol-reactive unit comprises a boronic acid. In a further embodiment, the diol can be oxidized to the respective ketone or aldehyde and then reacted with the ketone / aldehyde-reactive unit K.

[0126] In an embodiment of the first aspect of the present invention, the amino-reactive unit reacts with an amino group on an analyte molecule and is selected from the group consisting of an active ester group, such as an N-hydroxysuccinimide (NHS) ester or a sulfo-NHS ester, a pentafluorophenyl ester, a carbonylimidazole ester, a squaric acid ester, a hydroxybenzotriazole (HOBt) ester, a 1-hydroxy-7-azabenzotriazole (HOAt) ester, and a sulfonyl chloride unit.

[0127] In an embodiment of the first aspect of the present invention, the thiol-reactive unit reacts with a thiol group on an analyte molecule. In an embodiment of the first aspect of the present invention, the thiol-reactive unit is selected from the group consisting of haloacetyl groups, in particular 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 an embodiment of the first aspect of the present invention, the phenol-reactive unit reacts with a phenol group on an analyte molecule. In an embodiment of the first aspect of the present invention, the phenol-reactive unit is selected from the group consisting of an N-hydroxysuccinimide (NHS) ester or sulfo-NHS ester, a pentafluorophenyl ester, a carbonylimidazole ester, a squaric acid ester, a hydroxybenzotriazole (HOBt) ester, a 1-hydroxy-7-azabenzotriazole (HOAt) ester, and an active ester unit such as a sulfonyl chloride unit. The phenol group present on the analyte molecule can be reacted with a highly reactive electrophile such as a triazolinedione (e.g., TAD) by reaction (H. Ban et al., J. Am. Chem. Soc. (2010) 132(5):1523-1525), or by diazotization, or alternatively by ortho-nitration, followed by reduction to an amine, which can then react with an amine-reactive reagent. In an embodiment of the first aspect of the present invention, the phenol-reactive unit is fluoro-1-pyridinium.

[0129] In an embodiment of the first aspect of the present invention, the reactive unit K is an epoxide-reactive unit, which can react with an analyte containing an epoxide group. In an embodiment of the first aspect of the present invention, the epoxide-reactive unit is selected from the group consisting of amino, thiol, and a supernucleophilic N atom, enhanced by an α-effect via the adjacent O or N atom NH2-N / O molecule. In an embodiment of the first aspect of the present invention, the epoxide-reactive unit is selected from the group consisting of: (i) a hydrazine unit, such as HN-NH- or HN-NR 1 -unit (wherein R 1 is an aryl, an aryl containing one or more heteroatoms, or C 1~4 alkyl, especially C1 or C2 alkyl, optionally including, for example, halo, hydroxyl, and / or C 1~3 substituted with alkoxy), (ii) a hydrazide unit, in particular a carbohydrazide or sulfohydrazide unit, in particular HN—NH—C(O)— or HN—NR 2 -C(O)- unit (wherein R 2 is an aryl, an aryl containing one or more heteroatoms, or C 1~4 alkyl, especially C1 or C2 alkyl, optionally including, for example, halo, hydroxyl, and / or C 1~3 substituted with alkoxy), (iii) a hydroxylamino unit, such as a HN-O- unit is 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 groups can be reduced to the respective thiol groups 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 an analyte molecule. In an embodiment of the first aspect of the present invention, the azide-reactive unit reacts with the azide group via 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 an alkyne (alkyl or aryl), a linear alkyne, or a cyclic alkyne. 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 can be reduced to the respective amino group and then reacted with the amino-reactive unit K.

[0132] In an embodiment of the first aspect of the present invention, the functional group of the analyte is selected from the options listed in the left column of Table 1. The reactive group of K of the corresponding functional group of the analyte is selected from the options listed in the right column of Table 1. [Table 1]

[0133] In an embodiment of the first aspect of the invention, B1 is Q, or B2 is Q, or B3 is Q, or B4 is Q, or B5 is Q. In particular, B1 is Q, or B2 is Q, or B3 is Q.

[0134] In an embodiment of the first aspect of the present invention, one of B1 or B2 or B3 is Q. Q is selected from the following group: hydrazine unit, hydrazide unit, hydroxylamino unit, F, TAD. Other substituents that are not Q are A1=H, A2=H, A3=H, A4=H, A5=H, Y1=H, Y2=H, B1=H, B2=H, B3=H, B4=H and B5=H.

[0135] In an embodiment of the first aspect of the invention, one of B1 or B2 or B3 is Q. Q is selected from the following group: hydrazine unit, hydrazide unit, hydroxylamino unit, F, TAD. Other substituents that are not Q are A1=alkoxy, in particular methoxy, A2=H, A3=H, A4=H, A5=H, Y1=H, Y2=H, B1=H, B2=H, B3=H, B4=H and B5=H.

[0136] In an embodiment of the first aspect of the present invention, one of B1 or B2 or B3 is Q. Q is selected from the following group: hydrazine unit, hydrazide unit, hydroxylamino unit, F, TAD. Other substituents that are not Q are A1=H, A2=alkoxy, especially methoxy, A3=H, A4=alkoxy, especially methoxy, A5=H, Y1=H, Y2=H, B1=H, B2=H, B3=H, B4=H and B5=H.

[0137] In an embodiment of the first aspect of the present invention, one of B1 or B2 or B3 is Q. Q is selected from the following group: hydrazine unit, hydrazide unit, hydroxylamino unit, F, TAD. Other substituents that are not Q are A1=H, A2=alkoxy, particularly methoxy, A3=alkoxy, particularly methoxy, A4=alkoxy, particularly methoxy, A5=H, Y1=H, Y2=H, B1=H, B2=H, B3=H, B4=H and B5=H.

[0138] In an embodiment of the first aspect of the invention, one of B1 or B2 or B3 is Q. Q is selected from the following group: hydrazine unit, hydrazide unit, hydroxylamino unit, F, TAD. Other substituents that are not Q are A1=H, A2=H, A3=H, A4=H, A5=alkoxy, especially methoxy, Y1=H, Y2=H, B1=H, B2=H, B3=-NMe2, B4=H and B5=H.

[0139] In an embodiment of the first aspect of the present invention, one of B1 or B2 or B3 is Q. Q is selected from the following group: hydrazine unit, hydrazide unit, hydroxylamino unit, F, TAD. Other substituents that are not Q are A1=H, A2=tert-butyl, A3=H, A4=tert-butyl, A5=H, Y1 and Y2 form a fused ring system, B1=H, B2=H, B3=H, B4=H and B5=H.

[0140] In an embodiment of the first aspect of the present invention, one of B1 or B2 or B3 is Q. Q is selected from the following group: hydrazine unit, hydrazide unit, hydroxylamino unit, F, TAD. Other substituents that are not Q are: A1=H, A2=H, A3=H, A4=H, A5=H, Y1 and Y2 form a fused ring system, and B1=H, B2=H, B3=H, B4=H and B5=H.

[0141] In an embodiment of the first aspect of the present invention, Q is acetomethyl hydrazide.

[0142] In an embodiment of the first aspect of the present invention, A1 is H or methoxy.

[0143] In an embodiment of the first aspect of the present invention, A2 is H, tert-butyl or methoxy.

[0144] In an embodiment of the first aspect of the present invention, A3 is H or methoxy.

[0145] In an embodiment of the first aspect of the present invention, A4 is H, tert-butyl or methoxy.

[0146] In an embodiment of the first aspect of the present invention, A5 is H or methoxy.

[0147] In an embodiment of the first aspect of the present invention, Y1 is H or forms a fused aromatic or heteroaromatic ring structure with Y2.

[0148] In an embodiment of the first aspect of the present invention, Y2 is H or forms a fused aromatic or heteroaromatic ring structure with Y12.

[0149] In an embodiment of the first aspect of the present invention, B3 is H or N,N dimethylamine amine.

[0150] In an embodiment of the first aspect of the present invention, B4 is H.

[0151] In an embodiment of the first aspect of the present invention, B5 is H.

[0152] In an embodiment of the first aspect of the present invention, B1 or B2 or B3 is Q, Q is acetomethylhydrazide, A1 is H or methoxy, A2 is H, tert-butyl or methoxy, A3 is H or methoxy, A4 is H, tert-butyl or methoxy, A5 is H or methoxy, Y1 is H or forms a fused aromatic or heteroaromatic ring structure with Y2, Y2 is H or forms a fused aromatic or heteroaromatic ring structure with Y12, B3 is H or N,N dimethylamine, B4 is H, and B5 is H.

[0153] In an embodiment of the first aspect of the present invention, A1 is H.

[0154] In an embodiment of the first aspect of the present invention, A2 is H, tert-butyl or methoxy.

[0155] In an embodiment of the first aspect of the present invention, A3 is H or methoxy.

[0156] In an embodiment of the first aspect of the present invention, A4 is H, tert-butyl or methoxy.

[0157] In an embodiment of the first aspect of the present invention, A5 is H.

[0158] In an embodiment of the first aspect of the present invention, Y1 is H or forms a fused aromatic or heteroaromatic ring structure with Y2.

[0159] In an embodiment of the first aspect of the present invention, Y2 is H or forms a fused aromatic or heteroaromatic ring structure with Y1.

[0160] In an embodiment of the first aspect of the present invention, B3 is H.

[0161] In an embodiment of the first aspect of the present invention, B4 is H.

[0162] In an embodiment of the first aspect of the present invention, B5 is H.

[0163] In an embodiment of the first aspect of the present invention, the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 not forming the coupling group Q are each independently selected from hydrogen, methyl, ethyl, propyl, cyclopropyl, isopropyl, butyl, tert-butyl, N,N-dimethylamine or methoxy.

[0164] In an embodiment of the first aspect of the invention the compound is charged.

[0165] In an embodiment of the first aspect of the invention, the compound is positively charged.

[0166] In an embodiment of the first aspect of the present invention, the compound is permanently charged. In particular, the compound is once and forever positively charged. This may mean that the total net charge is once and forever positive.

[0167] In an embodiment of the first aspect of the present invention, the positive charge is part of a pyridinium.

[0168] In an embodiment of the first aspect of the invention, the permanent positive charge is located on the nitrogen of the pyridinium moiety, and the compound comprises a pyridinium cation.

[0169] In an embodiment of the first aspect 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.

[0170] In an 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.

[0171] In an embodiment of the first aspect of the invention, the compound comprises a counterion to form a salt, in particular the counterion is permanently negatively charged.

[0172] In an embodiment of the first aspect of the present invention, the compound comprises a counterion for forming a salt, the counterion being selected from the following group: Cl - , Br - , F - , formate, trifluoroacetate, PF6 - , sulfonates, phosphates, acetates. In an embodiment of the first aspect of the present invention, the compound of formula I is selected from the group consisting of: [ka] [ka] [ka]

[0173] Each of the above labels 1 to 12 can be formed by a compound alone or in combination with a counterion.

[0174] Stabilization of the carbocation after neutral loss fragmentation at the benzylic position allows for low-energy fragment loss, resulting in a highly favorable fragmentation pathway that allows for high signal enhancement. In some embodiments, particularly with meta-substitution patterns, favorable LC (liquid chromatography) elution characteristics can be observed. Either one of the two E / Z stereoisomers that can be formed with the hydrazide or oxime (e.g., for testosterone labeling) can be predominantly produced, or both isomers can co-elute. This improves sensitivity due to a better S / N (signal-to-noise) ratio.

[0175] Alkylated pyridines can fragment at the N-position with intermediate energy. Combining alkylated pyridines with stabilized benzylic cations as loss fragments resulted in highly favorable fragmentation pathways, allowing for lower energy fragmentation and high signal enhancement. Either one of the two E / Z stereoisomers, which can be formed from hydrazides or hydroxylamines (e.g., for testosterone labeling), can be predominantly produced, or both isomers can co-elute. This enhances sensitivity with electron-donating substituents A1, A2, A3, A4, and / or A5 (e.g., OMe). 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.

[0176] In a second aspect, the present invention relates to a composition comprising a compound of formula I as disclosed in detail above with respect to the first aspect of the invention. All embodiments described with respect to the first aspect of the invention apply to the second aspect of the invention and vice versa.

[0177] In a third aspect, the present invention relates to a kit comprising a compound of formula I as disclosed in detail above with respect to the first aspect of the invention, or a composition of the second aspect of the invention as disclosed in detail hereinabove. All embodiments mentioned with respect to the first aspect of the invention and / or the second aspect of the invention apply to the third aspect of the invention and vice versa.

[0178] In a fourth aspect, the present invention relates to a complex for detecting an analyte using mass spectrometry, comprising a binding analyte and a binding compound covalently bound to each other, in particular the complex is formed by chemical reaction of the analyte with a compound of the first aspect of the 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 an organism, metabolites of such substances and combinations thereof. All embodiments mentioned with respect to the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention apply to the fourth aspect of the invention and vice versa.

[0179] In an embodiment of the fourth aspect of the present invention, a binding complex of formula III results 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, one skilled in the art can fully determine the covalent bond formed between the two. In an embodiment of the fourth aspect of the invention, the binding compound has the formula III and IV: [ka] [ka] wherein one of the substituents B1, B2, B3, B4, and B5 is a coupling group Q that forms a covalent bond with the analyte. * and Other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, thioacyl, aryloyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, isotopes or derivatives thereof; Y1 and Y2 are each independently selected from hydrogen, methyl, ethyl, methoxy, and amine, or Y1 and Y2 form a ring system 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 bound analyte is K * and X * is the bonded carbon atom of the pyridinium cation of formula III In particular, the binding analyte includes K for pyridinium-hydrazide and pyridinium-1,2,4-triazoline-3,5-dione. * Covalently bonded via fluoro-pyridinium: K * =X * and n * =0.

[0180] In an embodiment of the fourth aspect of the present invention, each of A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, Y1 and Y2 of formula III have the same meaning as described above for the first aspect of the present invention (A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, Y1 and Y2 of formula I).

[0181] In an embodiment of the fourth aspect of the present invention, the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, aryloyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotopes or derivatives thereof.

[0182] In an embodiment of the fourth aspect 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; or Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, and unsubstituted heteroaromatic.

[0183] In an embodiment of the fourth aspect of the present invention, X * has the same meaning as X described above for the first aspect of the present invention.

[0184] In an embodiment of the fourth aspect of the present invention, K * results from the formation of a covalent bond between the reactive unit K of the 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, one skilled in the art can fully determine the covalent bond formed between the two.

[0185] Furthermore, it is also considered within the scope of the present invention that a functional group present on the analyte molecule may first be converted to another readily available group by reaction with a reactive unit K of the compound of formula I.

[0186] In an embodiment of the fourth aspect 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 a particular modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

[0187] In an embodiment of the fourth aspect 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 is capable of forming a covalent bond with a reactive unit K of a compound of formula I.

[0188] In an embodiment of the fourth aspect 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, drugs of abuse, toxins, or their metabolites.

[0189] In an embodiment of the fourth aspect of the present invention, the analyte molecule comprises a carbonyl group as a functional group selected from the group consisting of a carboxylic acid group, an aldehyde group, a keto group, a masked aldehyde, a masked keto group, an ester group, an amide group, and an anhydride group.

[0190] In an embodiment of the fourth aspect of the present invention, the carbonyl group is an amide group, and those skilled in the art will recognize that, although an amide group itself is a stable group, it can be hydrolyzed to a carboxylic acid group and an amino group. Hydrolysis of the amide group can be achieved via an acid / base catalysis reaction or an enzymatic process, both of which are well known to those skilled in the art. In an embodiment of the fourth aspect of the present invention, in which the carbonyl group is a masked aldehyde group or a masked keto group, each group is either a hemiacetal group or an acetal group, particularly a cyclic hemiacetal group or an acetal group. In an embodiment of the fourth aspect 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.

[0191] In an embodiment of the first aspect of the present invention, the carbonyl group is a keto group. In an embodiment of the first aspect of the present invention, the keto group can be transferred to an imine group of the intermediate before reacting with the reactive unit of the compound of formula I. In an embodiment of the first aspect of the present invention, the analyte molecule containing one or more keto groups is a ketosteroid. In certain embodiments of the first aspect of the present invention, the ketosteroid is selected from the group consisting of testosterone, epitestosterone, dihydrotestosterone (DHT), desoxymethyltestosterone (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, allopregnenolone, and aldosterone.

[0192] In an embodiment of the fourth aspect of the invention, the carbonyl group is a carboxy group. In an embodiment of the first aspect of the invention, the carboxy group is reacted directly with a compound of formula I or converted to an activated ester group prior to reaction with a compound of formula I. In an embodiment of the first aspect of the invention, the analyte molecule comprising one or more carboxy 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 an embodiment of the first aspect of the present invention, the analyte molecule comprising 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.

[0193] In an embodiment of the fourth aspect of the present invention, the carbonyl group is an aldehyde group. In an embodiment of the first aspect of the present invention, the aldehyde group can be transferred to an imine group of the intermediate before reacting with the reactive unit of the compound of formula I. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more aldehyde groups is selected from the group consisting of pyridoxal, N-acetyl-D-glucosamine, alcaftadine, streptomycin, and josamycin.

[0194] In an embodiment of the fourth aspect of the present invention, the carbonyl group is a carbonyl ester group. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more ester groups is selected from the group consisting of cocaine, heroin, Ritalin, aceclofenac, acetylcholine, amcinonide, amiloxate, amylocaine, anileridine, aranidipine, artesunate, and pethidine.

[0195] In an embodiment of the fourth aspect of the present invention, the carbonyl group is an anhydride group. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more anhydride groups is selected from the group consisting of cantharidin, succinic anhydride, trimellitic anhydride, and maleic anhydride.

[0196] In an embodiment of the fourth aspect of the present invention, the analyte molecule comprises one or more diene groups, particularly conjugated diene groups, as functional groups. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more diene groups is a secosteroid. In an embodiment, the secosteroid is selected from the group consisting of cholecalciferol (vitamin D3), ergocalciferol (vitamin D2), calcifediol, calcitriol, tachysterol, lumisterol, and tacalcitol. In particular, the secosteroid is vitamin D, particularly vitamin D2 or D3, or a derivative 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 invention, the analyte molecule comprising 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.

[0197] In embodiments of the fourth aspect of the invention, the analyte molecule comprises one or more hydroxyl groups as functional groups. In embodiments of the first aspect of the invention, the analyte molecule comprises 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 located adjacent to each other or may be separated by one, two, or three C atoms (1,3-diol, 1,4-diol, 1,5-diol, respectively). In particular embodiments of the first aspect, the analyte molecule comprises a 1,2-diol group. In embodiments where only one hydroxyl group is present, the analyte is selected from the group consisting of primary alcohols, secondary alcohols, and tertiary alcohols. In embodiments of the first aspect of the invention wherein 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, ethylmorphine, morphine, morphine-3-glucuronide, buprenorphine, codeine, dihydrocodeine, p-hydroxypropoxyphene, O-desmethyltramadol, desmetramadol, dihydroquinidine, and quinidine. In an embodiment of the first aspect of the invention, wherein the analyte molecule comprises multiple hydroxyl groups, the analyte is selected from the group consisting of vitamin C, glucosamine, mannitol, tetrahydrobiopterin, cytarabine, azacytidine, ribavirin, floxuridine, gemcitabine, streptozotocin

[0198] , adenosine, vidarabine, cladribine, estriol, trifluridine, clofarabine, nadolol, zanamivir, lactulose, adenosine monophosphate, idoxuridine, regadenoson, lincomycin, clindamycin, canaglifodine, tobramycin, netilmicin, kanamycin, ticagrelor, epirubicin, doxorubicin, arbekacin, streptomycin, ouabain, amikacin, neomycin, framycetin, paromomycin, erythromycin, clarithromycin, azithromycin, vindesine, digitoxin, digoxin, metrizamide, acetyldigitoxin, deslanoside, fludarabine, clofarabine, gemcitabine, cytarabine, capecitabine, vidarabine, and plicamycin.

[0199] In an embodiment of the fourth aspect 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 an embodiment of the first aspect 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, tiopronin, dimercaprol, and succinimer.

[0200] In an embodiment of the fourth aspect of the present invention, the analyte molecule comprises one or more disulfide groups as functional groups. In an embodiment of the first aspect of the present invention, the analyte molecule comprising 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. The selenium sulfide can be selenium disulfide, SeS2, or selenium hexasulfide, Se2S6.

[0201] In an embodiment of the fourth aspect of the present invention, the analyte molecule comprises one or more epoxide groups as functional groups. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more epoxide groups is selected from the group consisting of carbamazepine-10,11-epoxide, carfilzomib, furosemide epoxide, fosfomycin, sevelamer hydrochloride, cerulenin, scopolamine, tiotropium, tiotropium bromide, methylscopolamine bromide, eplerenone, mupirocin, natamycin, and troleandomycin.

[0202] In an embodiment of the fourth aspect of the invention, the analyte molecule comprises one or more phenolic groups as functional groups. In a particular embodiment of the first aspect of the invention, the analyte molecule comprising one or more phenolic groups is a steroid or steroid-like compound. In an embodiment of the first aspect of the invention, the analyte molecule comprising one or more phenolic groups is sp 2A steroid or steroid-like compound having a hybridized A ring and an OH group at position 3 of the A ring. In certain embodiments of the first aspect of the invention, the steroid or steroid-like analyte molecule is selected from the group consisting of estrogen, an estrogenic compound, estrone (E1), estradiol (E2), 17a-estradiol, 17b-estradiol, estriol (E3), 16-epiestriol, 17-epiestriol, and 16,17-epiestriol, and / or metabolites thereof. In embodiments, the metabolite is estriol, 16-epiestriol (16-epiE3), 17-epiestriol (17-epiE3), 16,17-epiestriol (16,17-epiE3), 16-ketoestradiol (16-ketoE2), 16a-hydroxyestrone (16a-OHEl), 2-methoxyestrone (2-MeOEl), 4-methoxyestrone (4-MeOEl), 2-hydroxyestrone-3-methyl ether (3-MeOEl), 2-methoxyestradiol (2-MeOE2), 4-methoxyestradiol (4-MeOE2), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2-hydroxyestradiol (2-OHE2), estrone (E1), estrone sulfate (E2s), 17a-estradiol (E2a), 17b-estradiol (E2B), estradiol sulfate (E2S), equilin (EQ), 17a-dihydroequilin (EQa), 17b-dihydroequilin (EQb), equilenin (EN), 17-dihydroequilenin (ENa), 17α-dihydroequilenin, 17β-dihydroequilenin (ENb), Δ8,9-dehydroestrone (dE1), Δ8,9-dehydroestrone sulfate (dE1), Δ9-tetrahydrocannabinol, and mycophenolic acid. β and b can be used interchangeably. α and a can be used interchangeably.

[0203] In an embodiment of the fourth aspect of the present invention, the analyte molecule comprises an amine group as a functional group. In an embodiment of the first aspect of the present invention, the amine group is an alkylamine group or an arylamine group. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more amine groups is selected from the group consisting of proteins and peptides. In an embodiment of the first aspect of the present invention, the analyte molecule comprising an amine group is selected from the group consisting of 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-fluoromethamine, 3-methyl-4-methyl-2-propanol, 3 ... Cathinone, 4-methoxymethcathinone, 4-methylethcathinone, 4-methylmethcathinone, amfepramone, butyrone, etcathinone, flephedrone, methcathinone, methylone, methylenedioxypyrovalerone, benzoylecgonine, dehydronorketamine, ketamine, norketamine, methadone, normethadone, 6-acetylmorphine, diacetylmorphine, morphine, norhydrocodone, oxycodone, oxymol and 2-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, sisomicin, and 5-methylcytosine.

[0204] In an embodiment of the fourth aspect of the invention, the analyte molecule is a carbohydrate or a substance having a carbohydrate moiety, such as a glycoprotein or a nucleoside. In an embodiment of the fourth aspect of the invention, the analyte molecule is a monosaccharide, in particular selected from the group consisting of ribose, desoxyribose, arabinose, ribulose, glucose, mannose, galactose, fucose, fructose, N-acetylglucosamine, N-acetylgalactosamine, neuraminic acid, N-acetylneuraminic acid, etc. In an embodiment, the analyte molecule is an oligosaccharide, in particular selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and polysaccharides. In an embodiment of the fourth aspect of the invention, the disaccharide is selected from the group consisting of sucrose, maltose, and lactose. In an embodiment of the fourth aspect of the invention, the analyte molecule is a substance comprising the above-mentioned monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide moiety.

[0205] In an embodiment of the fourth aspect of the present invention, the analyte molecule comprises an azide group as a functional group selected from the group consisting of alkyl or aryl azides. In an embodiment of the fourth aspect of the present invention, the analyte molecule comprising one or more azide groups is selected from the group consisting of zidovudine and azidocillin.

[0206] Such analyte molecules may be present in biological or clinical samples such as bodily fluids, e.g., blood, serum, plasma, urine, saliva, cerebrospinal fluid, etc., tissue or cell extracts. In embodiments of the fourth aspect of the 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 fourth aspect of the invention, the analyte molecule may be present in a purified or partially purified sample, e.g., a sample that is a purified or partially purified protein mixture or extract.

[0207] In an embodiment of the fourth aspect of the present invention, the reactive unit X 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.

[0208] In an embodiment of the fourth 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 fourth 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 fourth aspect of the present invention, the carbonyl-reactive unit can have either an adjacent O or N atom NH2-N / O, or a supernucleophilic N atom enhanced by the alpha effect via a dithiol molecule. In an embodiment of the fourth aspect of the present invention, the carbonyl-reactive unit is selected from the group: (i) a hydrazine unit, such as HN-NH- or HN-NR 1 -unit (wherein R 1 is an aryl, an aryl containing one or more heteroatoms, or C 1~4 alkyl, especially C1 or C2 alkyl, optionally including, for example, halo, hydroxyl, and / or C 1~3 substituted with alkoxy), (ii) a hydrazide unit, in particular a carbohydrazide or sulfohydrazide unit, in particular HN—NH—C(O)— or HN—NR 2 -C(O)- unit (wherein R 2 is an aryl, an aryl containing one or more heteroatoms, or C 1~4 alkyl, especially C1 or C2 alkyl, optionally including, for example, halo, hydroxyl, and / or C 1~3 substituted with alkoxy), (iii) a hydroxylamino unit, such as a HN-O- unit; (iv) a dithiol unit, in particular a 1,2-dithiol or 1,3-dithiol unit is selected from.

[0209] In an embodiment of the fourth aspect of the invention, where the carbonyl-reactive unit is a carboxyl-reactive unit, the carboxyl-reactive unit reacts with a carboxyl group on an analyte molecule. In an embodiment of the fourth aspect of the invention, the carboxyl-reactive unit is selected from the group consisting of a diazo unit, an alkyl halide, an amine, and a hydrazine unit.

[0210] In an embodiment of the fourth aspect of the present invention, the reactive unit K is a diene-reactive unit, which is capable of reacting with an analyte having a diene group. In an embodiment of the fourth aspect of the present invention, the diene-reactive unit is selected from the group consisting of Cookson-type reagents that can act as dienophiles, such as 1,2,4-triazoline-3,5-dione.

[0211] In an embodiment of the fourth aspect of the present invention, the reactive unit K is a hydroxyl-reactive unit, which can react with an analyte having a hydroxyl group. In an embodiment of the fourth aspect of the present invention, the hydroxyl-reactive unit is selected from the group consisting of sulfonyl chlorides, activated carboxylic acid esters (NHS or imidazolides), and fluoroaromatics / heteroaromatics capable of nucleophilic substitution of fluorine (T. Higashi, J. Steroid Biochem. Mol. Biol. (September 2016) Vol. 162, pp. 57-69). In an embodiment of the fourth aspect of the present invention, the reactive unit K is a diol-reactive unit that reacts with a diol group on an analyte molecule. In an embodiment of the fourth aspect of the present invention, where the reactive unit is a 1,2-diol-reactive unit, the 1,2-diol-reactive unit comprises a boronic acid. In a further embodiment, the diol can be oxidized to the respective ketone or aldehyde and then reacted with the ketone / aldehyde-reactive unit K.

[0212] In an embodiment of the fourth aspect of the present invention, the amino-reactive unit reacts with an amino group on an analyte molecule. In an embodiment of the fourth aspect 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, squaric acid ester, hydroxybenzotriazole (HOBt) ester, 1-hydroxy-7-azabenzotriazole (HOAt) ester, and sulfonyl chloride unit.

[0213] In an embodiment of the fourth aspect of the present invention, the thiol-reactive unit reacts with a thiol group on an analyte molecule. In an embodiment of the fourth aspect of the present invention, the thiol-reactive unit is selected from the group consisting of haloacetyl groups, in particular 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.

[0214] In an embodiment of the fourth aspect of the present invention, the phenol-reactive unit reacts with a phenol group on an analyte molecule and is selected from the group consisting of an active ester unit, such as an N-hydroxysuccinimide (NHS) ester or a sulfo-NHS ester, a pentafluorophenyl ester, a carbonylimidazole ester, a squaric acid ester, a hydroxybenzotriazole (HOBt) ester, a 1-hydroxy-7-azabenzotriazole (HOAt) ester, and a sulfonyl chloride unit.

[0215] Phenol groups present on analyte molecules can be reacted with highly reactive electrophiles such as triazolinediones (e.g., TAD) by reaction (H. Ban et al., J. Am. Chem. Soc. (2010) Vol. 132, No. 5, pp. 1523-1525), or by diazotization, or alternatively by ortho-nitration, followed by reduction to amines, which can then react with amine-reactive reagents. In an embodiment of the first aspect of the present invention, the phenol-reactive unit is selected from the group consisting of fluoro-1-pyridinium.

[0216] In an embodiment of the fourth aspect of the present invention, the reactive unit K is an epoxide-reactive unit, which can react with an analyte containing an epoxide group. In an embodiment of the fourth aspect of the present invention, the epoxide-reactive unit is selected from the group consisting of amino, thiol, and a supernucleophilic N atom, enhanced by an α-effect via the adjacent O or N atom NH2-N / O molecule. In an embodiment of the fourth aspect of the present invention, the epoxide-reactive unit is selected from the group consisting of: (i) a hydrazine unit, such as HN-NH- or HN-NR 1 -unit (wherein R 1 is an aryl, an aryl containing one or more heteroatoms, or C 1~4 alkyl, especially C1 or C2 alkyl, optionally including, for example, halo, hydroxyl, and / or C 1~3 substituted with alkoxy), (ii) a hydrazide unit, in particular a carbohydrazide or sulfohydrazide unit, in particular HN—NH—C(O)— or HN—NR 2 -C(O)- unit (wherein R 2 is an aryl, an aryl containing one or more heteroatoms, or C 1~4 alkyl, especially C1 or C2 alkyl, optionally including, for example, halo, hydroxyl, and / or C 1~3 substituted with alkoxy), (iii) a hydroxylamino unit, such as a HN-O- unit is selected from.

[0217] In an embodiment of the fourth 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 fourth aspect of the present invention, the disulfide-reactive unit is selected from the group consisting of thiols. In a further embodiment, the disulfide groups can be reduced to the respective thiol groups and then reacted with the thiol-reactive unit K.

[0218] In an embodiment of the fourth aspect of the present invention, the reactive unit K is an azide-reactive unit that reacts with an azide group on an analyte molecule. In an embodiment of the fourth aspect of the present invention, the azide-reactive unit reacts with the azide group via azide-alkyne cycloaddition. In an embodiment of the fourth aspect of the present invention, the azide-reactive unit is selected from the group consisting of an alkyne (alkyl or aryl), a linear alkyne, or a cyclic alkyne. The reaction between the azide and the alkyne can proceed with or without the use of a catalyst. In a further embodiment of the fourth aspect of the present invention, the azide group can be reduced to the respective amino group and then reacted with the amino-reactive unit K.

[0219] In an embodiment of the fourth aspect of the invention, the binding analyte is K * In particular, in the case of pyridinium hydrazides, K * is a hydrazide, and the nitrogen from the hydrazide covalently binds to the analyte. In the case of 4-pyridinium 1,2,4-triazoline-3,5-dione, K * is 1,2,4-triazoline-3,5-dione, and the nitrogen(s) from the 1,2,4-triazoline-3,5-dione covalently bind to the analyte. In the case of fluoropyridinium, K * is K * =X * and n * = 0, and carbon X * is covalently bound to the analyte.

[0220] In an embodiment of the fourth aspect of the present invention, K *is selected from the group consisting of hydrazide, hydrazine, hydroxylamine, Br, F, 4-substituted-1,2,4-triazoline-3,5-diones (TADs), 4-phenyl-1,2,4-triazoline-3,5-diones (PTADs), 4-pyridinium-1,2,4-triazoline-3,5-diones, and reactive carbonyl groups.

[0221] In an embodiment of the fourth aspect of the present invention, X * is the bonding carbon atom of the pyridinium cation of formula III to a group selected from the group consisting of B1, B2, B3, B4, and B5.

[0222] In an embodiment of the fourth aspect of the present invention, n * is 0, 1, 2, 3, 4 or 5. Preferably, n * is 0 or 1.

[0223] In an embodiment of the fourth aspect of the invention, the binding compound is covalently bound via a carbonyl, hydroxyl or diene group of the analyte to form the complex.

[0224] In a fifth aspect, the present invention relates to the use of a compound of formula I for mass spectrometric determination of an analyte. Preferably, the mass spectrometric determination comprises tandem mass spectrometric determination, in particular triple quadrupole mass spectrometric determination. All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention and / or the fourth aspect of the invention apply to the fifth aspect of the invention, and vice versa.

[0225] In an embodiment of the fifth aspect of the present invention, the mass spectrometric determination comprises a tandem mass spectrometric determination, in particular a triple quadrupole mass spectrometric determination.

[0226] In an embodiment of the fifth aspect of the invention, the use of a compound of formula I comprises use as a derivatization reagent. In an embodiment of the fifth aspect of the invention, a compound of formula I is used to increase the sensitivity of MS measurements. In an embodiment, a compound of formula I is used to detect an analyte of interest at lower levels of detection, particularly lower levels of quantification.

[0227] In an embodiment of the fifth aspect of the invention, the compound of formula I according to the invention comprises a reactive unit K capable of reacting with an analyte molecule. The reactive unit K is capable of reacting with an analyte molecule such that a covalent bond is formed between the compound of formula I and the analyte molecule. In an embodiment of the fifth aspect of the 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.

[0228] Depending on the functional groups present in the analyte molecule to be measured, one skilled in the art will select an appropriate reactive unit K for the compound of formula I. It is well known to determine which reactive unit K is suitable for binding to the functional group of the analyte of interest.

[0229] What has been said above with respect to analytes applies mutatis mutandis to analytes in the context of the fifth aspect of the invention.

[0230] The analyte molecule may be present in a biological or clinical sample, such as a bodily fluid, e.g., blood, serum, plasma, urine, saliva, cerebrospinal fluid, etc., a tissue or cell extract. In embodiments of the fifth aspect of the 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 a dried blood spot. In some embodiments of the fifth aspect of the invention, the analyte molecule may be present in a purified or partially purified sample, e.g., a sample that is a purified or partially purified protein mixture or extract.

[0231] In a sixth aspect, the present invention relates to a method for the mass spectrometric determination of an analyte, comprising: (a) reacting an analyte with a compound of formula I as disclosed hereinabove with respect to the first aspect of the invention, whereby a complex as disclosed hereinabove with respect to the fourth aspect of the invention is formed; (b) subjecting the complex from step (a) to mass spectrometry analysis; Includes.

[0232] Preferably, step (b) comprises: (i) subjecting ions of the complex to a first stage of mass spectrometry, whereby the ions of the complex are characterized according to their mass / charge (m / z) ratio; (ii) causing fragmentation of ions of the complex, whereby daughter ions of the first entity, particularly the low molecular weight entity, and the complex are produced, the daughter ions of the complex having an m / z ratio different from that of the ion of the complex; (iii) subjecting daughter ions of the complex to a second stage of mass spectrometry, whereby the daughter ions of the complex are characterized according to their m / z ratio; and / or (ii) may further comprise alternative fragmentation of the ion of the complex, whereby a second entity different from the first entity is released and a second daughter ion of the complex is produced; and (iii) may further comprise subjecting the first daughter ion and second daughter ion of the complex to a second stage of mass spectrometry, whereby the first daughter ion and second daughter ion of the complex are characterized according to their m / z ratios.

[0233] Step (a) can occur at different stages in the sample preparation workflow prior to mass spectrometric determination. Samples containing analyte molecules can be pretreated and / or enriched 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 enrichment method depends on the analyte of interest. Those skilled in the art will know which pretreated sample types are suitable for which sample types. Those skilled in the art will also know which enrichment methods are suitable for which analytes of interest.

[0234] In an embodiment of the sixth aspect of the present invention, step (a) of the method for mass spectrometric determination of analyte molecules is performed (i) following a sample pretreatment step, (ii) following a first enrichment of the sample, or (iii) following a second enrichment of the sample.

[0235] In an embodiment of the sixth aspect of the present invention, where the sample is a whole blood sample, the sample is assigned to one of two predetermined sample preparation (PT) workflows, both of which include the addition of an internal standard (ISTD) and a hemolysis reagent (HR) followed by a predetermined incubation period (Inc), where the difference between the two workflows is the order in which the internal standard (ISTD) and the hemolysis reagent (HR) are added. In an embodiment, water is added as the hemolysis reagent in an amount of 0.5:1 to 20:1 ml water / ml sample, particularly 1:1 to 10:1 ml water / ml sample, particularly 2:1 to 5:1 ml water / ml sample.

[0236] In an embodiment of the sixth aspect of the present invention, the sample is a urine sample, and the sample is assigned to one of two other predetermined PT workflows, both of which include the addition of an internal standard and an enzymatic reagent followed by a predetermined incubation period, where the difference between the two workflows is the order in which the internal standard and the enzymatic reagent are added. The enzymatic reagent is typically a reagent used for glucuronide cleavage or protein cleavage, or any pretreatment of the analyte or matrix. In an additional step, a derivatization reagent, such as a compound of the present invention as disclosed herein above or below, is added, followed by an incubation period.

[0237] In an embodiment of the sixth aspect of the present invention, the enzyme reagent is selected from the group consisting of glucuronidase, (partial) exo- or endo-deglycosylating enzyme, or exo- or endo-preoteases. In an embodiment, the glucuronidase is added in an amount of 0.5 to 10 mg / mL, in particular in an amount of 1 to 8 mg / mL, in particular in an amount of 2 to 5 mg / mL.

[0238] In an embodiment of the sixth aspect of the invention, the sample is plasma or serum and is assigned to another predefined PT workflow which only involves the addition of an internal standard (ISTD) prior to a predefined incubation time.

[0239] As described hereinabove or below, the incubation time and temperature to be selected for the sample treatment, chemical reaction, or method step under consideration are well known to those skilled in the art. In particular, those skilled in the art know that incubation time and temperature are interdependent, e.g., higher temperatures typically result in shorter incubation periods, and vice versa. In embodiments of the 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, and particularly in the range of 20 to 37°C. In embodiments, the incubation time is 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.

[0240] Thus, step (a) of this method embodiment is carried out following any of the sample pretreatment processes disclosed above.

[0241] 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) is carried out before any enrichment process, and the compound of formula I is added to the pretreated sample of interest. Thus, a complex between the analyte molecule and the compound of formula I is formed after pretreatment and before the first enrichment process. Thus, the complex is subjected to the first and second enrichment processes before being subjected to mass spectrometric analysis in step (b).

[0242] The pretreated sample can be further subjected to an analyte enrichment workflow. The analyte enrichment workflow can include one or more enrichment methods. Enrichment methods are well known in the art and include, but are not limited to, chemical enrichment methods, including but not limited to chemical precipitation, and enrichment methods using solid phases, including but not limited to solid-phase extraction methods, bead workflows, and chromatographic methods (e.g., gas or liquid chromatography).

[0243] In an embodiment of the sixth aspect of the present invention, the first enrichment workflow comprises adding a solid phase, in particular solid beads, carrying an analyte-selective group to the pretreated sample. In an embodiment of the sixth aspect of the present invention, the first enrichment workflow comprises adding magnetic or paramagnetic beads carrying an analyte-selective group to the pretreated sample. In an embodiment of the sixth aspect of the present invention, the addition of the magnetic beads comprises stirring or mixing. This is followed by a predetermined incubation period for capturing the analyte(s) of interest on the beads. In an embodiment of the sixth aspect of the present invention, the workflow comprises a washing step (W1) after incubation with the 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 manipulation unit including a magnet or electromagnet, liquid aspiration, addition of a washing buffer, resuspension of the magnetic beads, another magnetic bead separation step, and another liquid aspiration. Furthermore, the washing steps may differ in terms of the type of solvent (water / organic / salt / pH), apart from the volume and number or combination of washing cycles. The selection of each parameter is well known to those skilled in the art. The final washing step (W1, W2) is followed by the addition of an elution reagent, followed by resuspension of the magnetic beads and a predetermined incubation period to release the analyte(s) of interest from the magnetic beads. Subsequently, the unbound magnetic beads are separated, and the supernatant containing the derivatized analyte(s) of interest is captured.

[0244] In an embodiment of the sixth aspect of the present invention, a first enrichment workflow comprises adding magnetic beads carrying a matrix-selective group to a pretreated sample. In an embodiment of the sixth aspect of the present invention, the addition of the magnetic beads comprises stirring or mixing. This is followed by a predetermined incubation period to capture the matrix on the beads. Here, the analytes of interest do not bind to the magnetic beads and remain in the supernatant. The magnetic beads are then separated, and the supernatant containing the enriched analyte(s) of interest is collected.

[0245] In an embodiment of the sixth aspect of the present invention, the supernatant is subjected to a second concentration workflow, where it is transferred to an LC station or transferred to an LC station after a dilution step by adding a diluent. Different elution procedures / reagents can also be used, such as varying the type of solvent (water / organic / salt / pH) and volume. The various parameters are well known to those skilled in the art and can be easily selected.

[0246] In an embodiment of the sixth aspect of the present invention, step (a) of the method is not carried out immediately after the pretreatment method. Step a) may be carried out after the first enrichment workflow using magnetic beads as described herein above.

[0247] In an embodiment of the sixth aspect of the present invention, analyte-specific magnetic beads are used and a compound of formula I as disclosed herein above or below is added to the sample of interest after the washing steps (W1, W2) have been completed either before, together with or following the elution reagent, followed by an incubation period (defined time and temperature).

[0248] In an embodiment of the sixth aspect of the present invention, the unbound magnetic beads are then separated and the supernatant containing the complexes of step (a) is collected. In an embodiment of the sixth aspect of the present invention, the supernatant containing the complexes of step (a) is transferred to a second enrichment workflow, in particular either directly to an LC station or after a dilution step by adding a diluent.

[0249] In an embodiment of the sixth aspect of the invention, matrix-specific magnetic beads are used and a compound of formula I as disclosed herein above or below is added to the sample of interest before or after the magnetic beads are separated. In an embodiment of the sixth aspect of the invention, the supernatant containing the complexes of step (a) is transferred to a second enrichment workflow, in particular either directly to an LC station or after a dilution step by adding a diluent.

[0250] Therefore, in an embodiment of the sixth aspect of the present invention, it is concluded that the reaction between the compound of formula I and the analyte molecule in step (a) is carried out following a first enrichment process, and the compound of formula I is added to the sample of interest after the first enrichment process, particularly the first enrichment process using magnetic beads. Thus, the sample is first pretreated as described hereinabove, and then subjected to a first enrichment process, particularly the first enrichment process using magnetic beads carrying an analyte-selective group as described hereinabove, and the compound of formula I is added before, simultaneously with, or after elution from the beads. Thus, a complex between the analyte molecule and the compound of formula I is formed after the first enrichment process and before the second enrichment process. Thus, the complex is subjected to a second enrichment process before being subjected to mass spectrometric analysis in step (b).

[0251] In another embodiment of the sixth aspect of the invention, step (a) of the method is performed after a second analyte enrichment workflow. The second enrichment workflow uses chromatographic separation to further enrich the analyte of interest in the sample. In an embodiment of the sixth aspect of the invention, the chromatographic separation is gas or liquid chromatography, both methods being familiar to those skilled in the art. In an embodiment of the sixth aspect of the invention, the liquid chromatography is selected from the group consisting of HPLC, rapid LC, micro LC, flow injection, and trapping and elution.

[0252] In an embodiment of the sixth aspect of the invention, step (a) of the method is carried out simultaneously with or after chromatographic separation. In an embodiment of the sixth aspect of the invention, the compound of formula I is added to the column with the elution buffer. In an alternative embodiment, the compound of formula I is added post-column.

[0253] In an embodiment of the sixth aspect of the invention, the first enrichment process comprises the use of analyte-selective magnetic beads. In an embodiment of the sixth aspect of the invention, the second enrichment process comprises the use of chromatographic separation, in particular using liquid chromatography.

[0254] Therefore, in an embodiment of the sixth aspect of the present invention, it is concluded that the reaction of the compound of formula I with the analyte molecule in step (a) is followed by a second enrichment process, and the compound of formula I is added to the sample of interest after the second enrichment process using chromatography, particularly liquid chromatography.Therefore, in this case, the sample is first pretreated as described hereinabove, then subjected to a first enrichment process, particularly using magnetic beads as described hereinabove, and then subjected to chromatographic separation, particularly using liquid chromatography, followed by the addition of the compound of formula I.Therefore, the complex between the bound analyte molecule and the bound compound of formula I is formed after the second enrichment process.Therefore, the complex is not subjected to an enrichment process before being subjected to mass spectrometric analysis in step (b).

[0255] In a seventh aspect, the present invention provides a compound of formula V: [ka] wherein one of the substituents B1, B2, B3, B4, B5 is a coupling group Q capable of forming a covalent bond with the analyte; the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, arylyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, arylyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotopes or derivatives thereof; 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, or Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, and unsubstituted heteroaromatic. Regarding.

[0256] All embodiments mentioned in the first aspect of the present invention and / or the second aspect of the present invention and / or the third aspect of the present invention and / or the fourth aspect of the present invention and / or the fifth aspect of the present invention and / or the sixth aspect of the present invention also apply to the seventh aspect of the present invention, and vice versa. In particular, all embodiments mentioned in the first aspect of the present invention, such as A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, Y1 and Y2, apply to compounds of formula V. In particular, compounds of formula V can be used for mass spectrometry determination. Alternatively or additionally, compounds of formula V can be used in other technical fields, such as chemical labeling and drug delivery.

[0257] In an embodiment of the seventh aspect of the present invention, the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, thioacyl, aryloyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, isotopes or derivatives thereof.

[0258] In an embodiment of the seventh aspect of the present invention, 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, and unsubstituted heteroaromatic.

[0259] In an eighth aspect, the present invention provides a compound of formula VI: [ka] The compounds of the present invention exhibit good stability, binding affinity to the analyte, and / or provide enhanced MS signal.

[0260] In a ninth aspect, the present invention provides a compound of formula VII: [ka] (In the formula, Y1=H, Y2=H, Y3 is H, or [ka] and B3 is [ka] or its oxidized and reactive forms: [ka] and The other substituents A1, A2, A3, A4, A5, B1, B2, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, aryloyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotopes, or derivatives thereof. All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention and / or the fourth aspect of the invention and / or the fifth aspect of the invention and / or the sixth aspect of the invention and / or the seventh aspect of the invention and / or the eighth aspect of the invention apply to the seventh aspect of the invention and vice versa.

[0261] In an embodiment of the invention, a clinical diagnostic system comprises a compound of the first aspect of the invention and / or a composition of the second aspect of the invention and / or a kit of the third aspect of the invention and / or a complex of the fourth aspect of the invention and / or a compound of the seventh aspect of the invention. Additionally or optionally, the compound of the first aspect of the invention is used for mass spectrometric determination of an analyte, and the clinical diagnostic system comprises mass spectrometric determination. Additionally or optionally, the method for mass spectrometric determination of an analyte of the sixth aspect of the invention is carried out by the clinical diagnostic system.

[0262] A "clinical diagnostic system" is a laboratory automation device dedicated to analyzing samples for in vitro diagnostics. Clinical diagnostic systems can have different configurations depending on the need and / or desired laboratory workflow. Further configurations can be achieved by combining multiple devices and / or modules together. A "module" is a work cell, typically smaller in size than the entire clinical diagnostic system, with a dedicated function. This function can be analytical, but can also be pre-analytical or post-analytical, or the function can be a support function for any of the pre-analytical, analytical, or post-analytical functions. Specifically, a module can be configured to cooperate with one or more other modules to perform a dedicated task in a sample processing workflow, for example, by performing one or more pre-analytical and / or post-analytical steps. In particular, a clinical diagnostic system can include one or more analytical devices designed to perform respective workflows optimized for specific types of analysis, such as clinical chemistry, immunochemistry, coagulation, hematology, liquid chromatography separations, mass spectrometry, etc. Thus, a clinical diagnostic system can include one analytical device or any combination of such analytical devices with their respective workflows, and pre-analysis and / or post-analysis modules can be coupled to individual analytical devices or shared by multiple analytical devices. Alternatively, pre-analysis and / or post-analysis functions can be performed by units integrated into the analytical devices. A clinical diagnostic system can include functional units such as a liquid handling unit for pipetting, pumping, and / or mixing samples and / or reagents and / or system fluids, as well as functional units for sorting, storing, transporting, identifying, separating, and detecting. A clinical diagnostic system can include a sample preparation station for automated preparation of samples containing analytes 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 include a controller programmed to assign samples to predefined sample preparation workflows, each of which includes a predefined series of sample preparation steps and requires a predefined time for completion depending on the analyte of interest. The clinical diagnostic system may further include a mass spectrometer (MS) and an LC / MS interface for connecting the LC separation station to the mass spectrometer. The terms "automatically" and "automated" as used herein are broad terms and should be given their ordinary and customary meaning to those skilled in the art and should not be limited to a specific or customized meaning. The terms may specifically, but are not limited to, refer to a process performed entirely by at least one computer and / or computer network and / or machine, particularly without manual action and / or user interaction.

[0263] A "sample preparation station" may be a pre-analytical module coupled to one or more analytical devices or units within an analytical device, designed to perform a series of sample processing steps aimed at removing or at least reducing interfering matrix components in the sample and / or concentrating the analyte of interest in the sample. Such processing steps may include one or more of the following processing operations performed sequentially, in parallel, or in a staggered manner on a sample or multiple samples: pipetting (aspiration and / or delivery) of fluids, pumping of fluids, mixing with reagents, incubation at a specific temperature, heating or cooling, centrifugation, separation, filtering, sieving, drying, washing, resuspension, aliquoting, transport, storage, etc.

[0264] A liquid chromatography (LC) separation station is an analytical device or module or unit within an analytical device designed to subject a prepared sample to chromatographic separation, e.g., to separate analytes of interest from matrix components, e.g., residual matrix components after sample preparation that may still interfere with subsequent detection, e.g., mass spectrometric detection, and / or to separate analytes of interest from each other to enable individual detection. According to one embodiment, the LC separation station is an intermediate analytical device or module or unit within an analytical device designed to prepare samples for mass spectrometry and / or transfer prepared samples to a mass spectrometer. In particular, the LC separation station is a multi-channel LC station comprising multiple LC channels.

[0265] The clinical diagnostic system, e.g., a sample preparation station, may also include a buffer unit for receiving multiple samples before a new sample preparation initiation sequence is initiated, and the samples may be individually randomly accessible, and their individual preparation may be initiated according to the sample preparation initiation sequence.

[0266] Clinical diagnostic systems utilize LC coupled to mass spectrometry, which is more convenient and reliable and therefore suitable for clinical diagnostics. In particular, high throughput, e.g., up to 100 samples / hour or more, can be achieved using random-access sample preparation and LC separation while enabling online connection to mass spectrometry. Furthermore, the process can be fully automated, increasing walk-away time and reducing the level of skill required.

[0267] In further embodiments, the present invention relates to the following aspects:

[0268] 1. Formula I for mass spectrometric determination of analytes: [ka] wherein one of the substituents B1, B2, B3, B4, B5 is a coupling group Q capable of forming a covalent bond with the analyte; the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, arylyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, arylyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotopes or derivatives thereof; 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, or Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, and unsubstituted heteroaromatic. Compound. 2. The coupling group Q has the following formula II: [ka] wherein K is a reactive unit capable of forming a covalent bond with the analyte; n is 0, 1, 2, 3, 4 or 5; X is the bonding carbon atom of the pyridinium cation of formula I 2. The compound of embodiment 1, wherein X is attached according to

[0269] 3. The compound of embodiment 2, wherein K is capable of reacting with a carbonyl group, a phenol group, an amine, a hydroxyl group, or a diene group of the analyte.

[0270] 4. The compound according to aspect 2 or 3, wherein K is selected from the group consisting of hydrazide, hydrazine, hydroxylamine, Br, F, 4-substituted 1,2,4-triazoline-3,5-diones (TADs), and reactive carbonyls.

[0271] 5. The compound of any one of aspects 1-4, wherein Q is selected from methyl hydrazide, methyl hydrazine, and methyl hydroxylamine.

[0272] 6. The compound of any one of the preceding aspects 2-5, wherein n=0 and K is Br, or n=0 and K is F.

[0273] 7. The compound according to any one of aspects 1 to 6, wherein the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 that do not form the coupling group Q are each independently selected from hydrogen, methyl, ethyl, propyl, cyclopropyl, isopropyl, butyl, tert-butyl, N,N-dimethylamine, or methoxy.

[0274] 8. A compound according to any one of aspects 1-7, wherein B1 is Q, or B2 is Q, or B3 is Q.

[0275] 9. The compound according to any one of aspects 1 to 9, wherein Q is acetohydrazide or carbonylhydrazide.

[0276] 10. The compound according to any one of aspects 1-9, wherein Q is a 4-substituted 1,2,4-triazoline-3,5-dione (TAD).

[0277] 11. The compound according to any one of aspects 1-10, wherein Q is F and n=0.

[0278] 12. A compound according to any one of aspects 1-11, wherein A1 is H or methoxy.

[0279] 13. A compound according to any one of aspects 1-12, wherein A2 is H, tert-butyl or methoxy.

[0280] 14. A compound according to any one of aspects 1-13, wherein A3 is H or methoxy.

[0281] 15. A compound according to any one of aspects 1-14, wherein A4 is H, tert-butyl, or methoxy.

[0282] 16. A compound according to any one of aspects 1-15, wherein A5 is H or methoxy.

[0283] 17. The compound according to any one of aspects 1 to 16, wherein Y1 is H or forms a fused aromatic or heteroaromatic ring structure with Y2.

[0284] 18. The compound according to any one of aspects 1 to 17, wherein Y2 is H or forms a fused aromatic or heteroaromatic ring structure with Y1.

[0285] 19. The compound according to any one of aspects 1-18, wherein B3 is H or N,N dimethylamine.

[0286] 20. The compound according to any one of aspects 1-20, wherein B4 is H.

[0287] 21. A compound according to any one of aspects 1-20, wherein B5 is H.

[0288] 22. The compound according to any one of aspects 1-21, wherein B4 or B5 is N,N dimethylamine.

[0289] 23. A compound according to any one of aspects 1-22, wherein A1 is H.

[0290] 24. The compound of any of the preceding embodiments, wherein A2 is H, tert-butyl, or methoxy.

[0291] 25. The compound of any of the preceding embodiments, wherein A3 is H or methoxy.

[0292] 26. The compound of any of the preceding embodiments, wherein A4 is H, tert-butyl, or methoxy.

[0293] 27. A compound according to any one of aspects 1-26, wherein A5 is H.

[0294] 28. The compound of any of the preceding embodiments, wherein Y1 is H or forms a fused aromatic or heteroaromatic ring structure with Y2.

[0295] 29. The compound of any of the preceding embodiments, wherein Y2 is H or forms a fused aromatic or heteroaromatic ring structure with Y1.

[0296] 30. The compound according to any one of aspects 1-29, wherein B3 is H.

[0297] 31. A compound according to any one of aspects 1-20, wherein B4 is H.

[0298] 32. The compound of any of the preceding embodiments, wherein B5 is H.

[0299] 33. The compound according to any one of aspects 1-32, which is permanently positively charged.

[0300] 34. Contains a counter ion for forming a salt, the counter ion preferably being from the following group: Cl - , Br - , F - , formate, trifluoroacetate, PF6 - 34. The compound according to any one of aspects 1 to 33, wherein the hydroxyl group is selected from the group consisting of hydroxyl groups, ...

[0301] 35. A composition comprising a compound according to any one of aspects 1 to 34.

[0302] 36. A kit comprising a compound according to any one of aspects 1 to 34, or a composition according to aspect 35.

[0303] 37. A complex for detecting an analyte using mass spectrometry determination, comprising a binding analyte and a binding compound covalently bound to each other, in particular, the complex is formed by chemical reaction of the analyte with a compound according to any one of aspects 1 to 34.

[0304] 38. The binding compound has the formula III and IV: [ka] [ka] wherein one of the substituents B1, B2, B3, B4, and B5 is a coupling group Q that forms a covalent bond with the analyte. * and Other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, thioacyl, arylyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, arylyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, isotopes or derivatives thereof; Y1 and Y2 are each independently selected from hydrogen, methyl, ethyl, methoxy, and amine, or Y1 and Y2 form a ring system 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 bound analyte is K * This is true for pyridinium-hydrazides and pyridinium-1,2,4-triazoline-3,5-dione, but for fluoro-pyridiniums, K * =X * and n * =0, and X * is the bonded carbon atom of the pyridinium cation of formula III 38. The conjugate of embodiment 37, comprising:

[0305] 39.K * is selected from the group consisting of hydrazides, hydrazines, hydroxylamines, Br, F, 4-substituted 1,2,4-triazoline-3,5-diones (TADs), and reactive carbonyls.

[0306] 40. The conjugate of any one of the preceding aspects 37-39, wherein 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 a specific modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

[0307] 41. The conjugate of any one of aspects 37-40, wherein the binding compound is covalently bound via a carbonyl group, a hydroxyl group, or a diene group of the analyte to form the conjugate.

[0308] 42. Use of a compound according to any one of embodiments 1 to 34 for the mass spectrometric determination of an analyte.

[0309] 43. Use of a compound according to aspect 42, wherein the mass spectrometric determination comprises a tandem mass spectrometric determination, in particular a triple quadrupole mass spectrometric determination.

[0310] 44. A method for mass spectrometric determination of an analyte, comprising: (a) reacting an analyte with a compound of formula I as defined in any one of embodiments 1 to 34, whereby a complex as defined in any one of embodiments 37 to 41 is formed; (b) subjecting the complex from step (a) to mass spectrometric analysis; A method comprising:

[0311] 45. The mass spectrometric analysis step (b) (i) subjecting ions of the complex to a first stage of mass spectrometry, whereby the ions of the complex are characterized according to their mass / charge (m / z) ratio; (ii) causing fragmentation of ions of the complex, whereby a first entity, particularly a low molecular weight entity, is released and daughter ions of the complex are produced, the daughter ions of the complex having an m / z ratio different from that of the ion of the complex; (iii) subjecting daughter ions of the complex to a second stage of mass spectrometry, whereby the daughter ions of the complex are characterized according to their m / z ratio; and / or (ii) may further comprise an alternative fragmentation of the ion of the complex, whereby a second entity different from the first entity, in particular the second entity, is released and a second daughter ion of the complex is generated; and The method of embodiment 44, wherein (iii) may further comprise subjecting the first daughter ion and second daughter ion of the complex to a second stage of mass spectrometry, whereby the first daughter ion and second daughter ion of the complex are characterized according to their m / z ratios.

[0312] 46.Formula V: [ka] wherein one of the substituents B1, B2, B3, B4, B5 is a coupling group Q capable of forming a covalent bond with the analyte; the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, arylyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, arylyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotopes or derivatives thereof; 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, or Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, and unsubstituted heteroaromatic. Compound.

[0313] 47. Formula VI: [ka] Compound.

[0314] 48.Formula VII: [ka] (In the formula, Y1=H, Y2=H, Y3 is H, or [ka] and B3 is [ka] or its oxidized and reactive forms: [ka] and The other substituents A1, A2, A3, A4, A5, B1, B2, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, arylyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, arylyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotopes, or derivatives thereof. Compound. [Example]

[0315] The following examples are offered to illustrate, but not to limit, the invention claimed herein.

[0316] Example 1: Synthesis of Label 1 [ka]

[0317] Step 1: Pyridine alkylation: Synthesis of methyl 2-(1-benzylpyridin-1-ium-3-yl)acetate formate: [ka]

[0318] Methyl 3-pyridylacetate (0.87 mmol) and benzyl bromide (1.14 mmol) were dissolved in 3 mL of DMF. The reaction mixture was stirred at 115° C. for 2 hours. DMF was removed in vacuo, and the residue was purified by preparative HPLC. Pure fractions were collected and lyophilized to give 214 mg (85% yield) of the desired product as a colored oil.

[0319] HPLC purification method: The residue was dissolved in 4 mL of 25 mM aqueous NH4COOH. Column: C-18 LiChroprep (45 g); Solvent A: H2O + 0.1% HCOOH; Solvent B: CH3CN + 0.1% HCOOH; Flow rate: 26 mL / min 0~5 minutes: 95% A;5% B 5~30 minutes: 10% A;90% B 30~38 minutes: 10% A; 90% B 1 H NMR(400 MHz,DMSO-d6)δ ppm 3.67(s,2 H),4.06(s,3 H),5.84(s,2 H),7.43-7.53(m,5 H),8.12-8.17(m,1 H),8.31(s,1H),8.57(d,J=8.16 Hz,1H),9.15(d,J=6.15 Hz,1H),9.20(s,1H). HPLC-MS (m / z) [M]+ calculated value 242.1, found value 242.2

[0320] Step 2: Synthesis of [[2-(1-benzylpyridin-1-ium-3-yl)acetyl]amino]ammonium diformate: [ka]

[0321] Methyl 2-(1-benzylpyridin-1-ium-3-yl)acetate formate (0.74 mmol) was dissolved in 2 mL of water. Hydrazine monohydrate (0.74 mmol) was added, and the reaction mixture was stirred at room temperature (rt) for 2 hours. The reaction mixture was purified by preparative HPLC. Pure fractions were collected and lyophilized to give 125 mg (58% yield) of the desired product as a white solid.

[0322] HPLC purification method: The reaction mixture (2 mL) was loaded onto the column without workup. Column: C-18 LiChroprep (45 g); Solvent A: HO + 0.1% HCOOH; Solvent B: CHCN + 0.1% HCOOH; Flow rate: 26 mL / min 0~8 minutes: 95% A;5% B 8~33 minutes: 10% A; 90% B 33~38 minutes: 10% A; 90% B 1 H NMR(400 MHz,DMSO-d6)δ ppm 3.67(s,2 H),5.81(s,2 H),7.39-7.49(m,5 H),8.10(dd,J=7.97;5.96 Hz,1 H),8.42(s,1H),8.46(d,J=8.16 Hz,1H),9.07(br d,J=6.15 Hz,1H),9.16(s,1H). HPLC-MS (m / z) [M]+ calculated value 242.1, found value 242.2

[0323] Example 2: Synthesis of Label 2 [ka]

[0324] Step 1: Pyridine alkylation: Synthesis of methyl 2-[1-[(2-methoxyphenyl)methyl]pyridin-1-ium-3-yl]acetate trifluoroacetate: [ka]

[0325] Methyl 3-pyridylacetate (0.87 mmol) and 1-(bromomethyl)-2-methoxybenzene (1.14 mmol) were dissolved in 3 mL of DMF. The reaction mixture was stirred at 115 °C for 2 hours. DMF was removed in vacuo, and the residue was purified by preparative HPLC. Pure fractions were collected and lyophilized to give 167 mg (94% yield) of the desired product as a colored oil.

[0326] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~60 min: 50% H2O 0.1% TFA,50% CH3CN 0.1% TFA; 60~64 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 64~80 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 80~83 minutes: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 83~89 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 89~90 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1 H NMR(400 MHz,methanol-d4)δ ppm 3.73(s,3 H)3.81(s,3 H)3.99(s,2 H)5.76(s,2 H)7.04-7.08(m,2 H)7.45-7.50(m,1 H)7.55(dd,J=7.65,1.5 Hz,1 H)8.00(dd,J=7.78,6.65 Hz,1 H)8.48(d,J=8.03 Hz,1 H)8.89(d,J=6.15 Hz,1 H)9.02(s,1 H). 13 C NMR(101 MHz, methanol-d4)δ ppm 36.12(1 C)51.61(1 C)54.66(1 C)60.93(1 C)111.14(1 C)120.84(1 C)120.93(1 C)127.10(1 C)131.20(1 C)131.95(1 C)136.00(1 C)142.89(1 C)145.34(1 C)146.72(1 C)158.07(1 C)169.96(1 C). HPLC-MS (m / z) [M]+ calculated value 272.13, found value 272.32.

[0327] Step 2: Synthesis of [2-[1-[(2-methoxyphenyl)methyl]pyridin-1-ium-3-yl]acetohydrazide bistrifluoroacetate: [ka]

[0328] 2-[1-[(2-Methoxyphenyl)methyl]pyridin-1-ium-3-yl]acetate trifluoroacetate (0.14 mmol) was dissolved in 1 mL of MeOH. Hydrazine monohydrate (1.40 mmol) was added, and the reaction mixture was stirred at 50 °C for 3 hours. The reaction mixture was purified by preparative HPLC. Pure fractions were collected and lyophilized to give 33 mg (46% yield) of the desired product as a yellow oil.

[0329] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~30 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 30~34 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 34~50 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 50~53 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 53~59 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 59~60 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1 H NMR(400 MHz, methanol-d4)δ ppm 3.81(s,3 H)3.91(s,2 H)5.77(s,2 H)7.03-7.07(m,2 H)7.44-7.48(m,1 H)7.55(dd,J=7.53,1.63 Hz,1 H)8.01(dd,J=7.91,6.27 Hz,1 H)8.46-8.49(m,1 H)8.90(dd,J=6.15,1.13 Hz,1 H)9.03(s,1 H). 13C NMR(101 MHz, methanol-d4)δ ppm 35.58(1 C)54.70(1 C)60.93(1 C)111.14(1 C)120.84(1 C)120.91(1 C)127.15(1 C)131.23(1 C)131.95(1 C)135.77(1 C)143.07(1 C)145.17(1 C)146.53(1 C)158.08(1 C)168.03(1 C). HPLC-MS (m / z) [M]+ calculated value 272.14, found value 272.06.

[0330] Example 3: Synthesis of Label 3 [ka]

[0331] Step 1: Pyridine alkylation: Synthesis of methyl 2-[1-[(3,5-dimethoxyphenyl)methyl]pyridin-1-ium-3-yl]acetate trifluoroacetate: [ka]

[0332] Methyl 3-pyridyl acetate (0.53 mmol) and 3,5-dimethoxybenzyl bromide (0.64 mmol) were dissolved in 3 mL of DMF. The reaction mixture was stirred at 70° C. for 6 hours. DMF was removed in vacuo, and the residue was purified by preparative HPLC. Pure fractions were collected and lyophilized to give 212 mg (96% yield) of the desired product as an oil.

[0333] HPLC method C-18 column: 0 min: 98% H2O 0.1% TFA,2% CH3CN 0.1% TFA; 0~10 min: 98% H2O 0.1% TFA,2% CH3CN 0.1% TFA; 10~60 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 60~70 min: 10% H2O 0.1% TFA; 90% CH3CN 0.1% TFA. 1 H NMR(400 MHz,methanol-d4)δ ppm 3.73(s,3 H)3.78(s,6 H)4.01(s,2 H)5.72(s,2 H)6.55(t,J=2.26 Hz,1 H)6.62(d,J=2.26 Hz,2 H)8.06(dd,J=7.91,6.27 Hz,1 H)8.54(d,J=8.03 Hz,1 H)8.95(d,J=6.02 Hz,1 H)9.07(s,1 H). 13 C NMR(101 MHz,methanol-d4)δ ppm 36.18(1 C)46.92(1 C)51.65(2 C)54.56(1 C)64.38(1 C)100.86(1 C)106.42(1 C)106.48(2 C)127.61(1 C)134.92(1 C)136.56(1 C)147.05(1 C)161.86(2 C)169.94(1 C). HPLC-MS (m / z) [M]+ calculated value 302.14, found value 302.33.

[0334] Step 2: Hydrazide formation: Synthesis of 2-[1-[(3,5-dimethoxyphenyl)methyl]pyridin-1-ium-3-yl]acetohydrazide bistrifluoroacetate: [ka]

[0335] Methyl 2-[1-[(3,5-dimethoxyphenyl)methyl]pyridin-1-ium-3-yl]acetate trifluoroacetate (0.24 mmol) was dissolved in 2 mL of MeOH. Hydrazine monohydrate (2.40 mmol) was added, and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was purified by preparative HPLC. Pure fractions were collected and lyophilized to give 100 mg (79% yield) of the desired product as an oil.

[0336] HPLC method C-18 column: 0 min: 98% H2O 0.1% TFA,2% CH3CN 0.1% TFA; 0~10 min: 98% H2O 0.1% TFA,2% CH3CN 0.1% TFA; 10~60 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 60~70 min: 10% H2O 0.1% TFA; 90% CH3CN 0.1% TFA. 1 H NMR(400 MHz,methanol-d4)δ ppm 3.77(s,6 H)3.92(s,2 H)5.71(s,2 H)6.53(t,J=2.20 Hz,1 H)6.62(d,J=2.10 Hz,2 H)8.06(dd,J=7.40,6.53 Hz,1 H)8.52(d,J=7.78 Hz,1 H)8.96(d,J=6.00 Hz,1 H)9.05(s,1 H). 13 C NMR(101 MHz,methanol-d4)δ ppm 35.90(1 C)54.56(2 C)64.43(1 C)100.84(1 C)106.55(2 C)127.63(1 C)134.87(1 C)136.83(s,1 C)142.85(1 C)144.95(1 C)146.84(1 C)161.84(2 C)170.42(1 C). HPLC-MS (m / z) [M]+ calculated value 302.15, found value 302.35.

[0337] Example 4: Synthesis of Label 4 [ka] Step 1: Synthesis of tert-butyl N-[[2-(3-pyridyl)acetyl]amino]carbamate: [ka]

[0338] 3-Pyridineacetic acid (500 mg, 3.65 mmol), tert-butyl carbazate (531 mg, 4.02 mmol), and EDC·HCl (796 mg, 4.15 mmol) were dissolved in dry DCM (3 mL) and stirred at room temperature (rt) for 1 day. Saturated NaHCO3 (aq) was added and washed five times with DCM. The combined organic phase was washed with saturated NaCl (aq) and dried over Na2SO4. The solvent was removed in vacuo, and the mixture was purified by flash chromatography (eluent EtOAc). The product was obtained as a white solid (852 mg, 93% yield). 1 H NMR(400 MHz, methanol-d4)δ ppm 1.44(s,9 H)3.58(s,2 H)7.38(ddd,J=7.87,4.93,0.75 Hz,1 H)7.71-7.92(m,1 H)8.41(dd,J=4.89,1.51 Hz,1 H)8.49(d,J=1.63 Hz,1 H). 13 C NMR (101 MHz, methanol-d4) δ ppm 27.08 (3 C) 36.90 (1 C) 80.45 (1 C) 123.70 (1 C) 131.63 (1 C) 137.68 (1 C) 147.09 (1 C) 149.20 (1 C) 156.26 (1 C) 170.94 (1 C). HPLC-MS (m / z) [M+H]+ calculated 252.13, found 252.57.

[0339] Step 2: Synthesis of tert-butyl N-[[2-[1-[(3,5-ditert-butylphenyl)methyl]pyridin-1-ium-3-yl]acetyl]amino]carbamate bromide: [ka]

[0340] tert-Butyl N-[[2-(3-pyridyl)acetyl]amino]carbamate (0.40 mmol) and 3,5-ditert-butylbenzyl bromide (0.48 mmol) were dissolved in dry CH3CN. The solution was then stirred at 70 °C overnight. The solvent was removed under vacuum, and the crude product was purified by flash chromatography (gradient CHCl2 → CHCl2 / MeOH 80:20). 190 mg of pure compound was obtained as a white solid (yield 89%). 1 H NMR(400 MHz,methanol-d4)δ ppm 1.33(m,18 H)1.45(s,9 H)3.87(s,2 H)5.80(s,2 H)7.38(d,J=1.70 Hz,2 H)7.53(t,J=1.69 Hz,1 H)8.06(dd,J=7.80,6.40 Hz,1 H)8.53(d,J=8.00 Hz,1 H)8.95(d,J=6.15 Hz,1 H)9.15(s,1 H). 13 C NMR(101 MHz,methanol-d4)δ ppm 27.10(3 C)30.31(6 C)34.48(2 C)65.02(1 C)80.69(1 C)122.97(2 C)123.72(1 C)127.59(1 C)132.39(1 C)136.89(1 C)142.47(1 C)144.92(1 C)146.69(1 C)152.37(2 C)156.45(1C)173.59(1 C). HPLC-MS (m / z) [M]+ calculated 454.31, found 454.40.

[0341] Step 3: Hydrazide formation: Synthesis of 2-[1-[(3,5-ditert-butylphenyl)methyl]pyridin-1-ium-3-yl]acetohydrazide bistrifluoroacetate: [ka]

[0342] tert-Butyl N-[[2-[1-[(3,5-ditert-butylphenyl)methyl]pyridin-1-ium-3-yl]acetyl]amino]carbamate bromide (0.19 mmol) was treated with 50% TFA in CHCl (2 mL). The reaction mixture was stirred at room temperature for 1 hour. The solvent was removed in vacuo, and the crude product was suspended in water and lyophilized. 108 mg of pure compound was obtained as a white solid, TFA salt (quantitative yield). 1 H NMR(400 MHz,methanol-d4)δ ppm 1.30(s,18 H)3.97(s,2 H)5.81(s,2 H)7.37(d,J=1.76 Hz,2 H)7.53(t,J=1.76 Hz,1 H)8.08(dd,J=7.91,6.27 Hz,1 H)8.53(d,J=8.03 Hz,1 H)8.97(d,J=6.15 Hz,1 H)9.15(s,1 H). 13 C NMR(101 MHz, methanol-d4)δ ppm 30.29(6 C)34.48(2 C)35.56(s,1 C)65.01(1 C)122.92(2 C)123.73(1 C)127.67(1 C)132.39(1 C)136.12(1 C)142.79(1 C)145.05(1 C)146.81(1 C)152.39(2 C)167.95(1 C). HPLC-MS (m / z) [M]+ calculated value 354.25, found value 354.43

[0343] Scheme 5: Synthesis of Labeled 5 [ka]

[0344] Step 1: Synthesis of tert-butyl N-[[2-(1-benzhydrylpyridin-1-ium-3-yl)acetyl]amino]carbamate bromide: [ka]

[0345] tert-Butyl N-[[2-(3-pyridyl)acetyl]amino]carbamate [synthesized similarly to Example 4, Step 1] (0.40 mmol) and bromodiphenylmethane (0.48 mmol) were dissolved in dry CH3CN. The solution was then stirred at 70 °C overnight. The solvent was removed under vacuum, and the crude product was purified by flash chromatography (gradient CHCl2 → CHCl2 / MeOH 80:20). 70 mg (44% yield) of the pure compound was obtained as a white solid. 1 H NMR(400 MHz,methanol-d4)δ ppm 1.45(s,9 H)3.34(s,2 H)7.27-7.30(m,4 H)7.47-7.50(m,6 H)7.54(s,1H)8.09(dd,J=7.90,6.30 Hz 1 H)8.58(d,J=8.10 Hz,1 H)8.84(d,J=6.22 Hz,1 H)8.97(s,1 H). 13 C NMR(101 MHz,methanol-d4)δ ppm 27.07(3 C)77.79(1 C)80.70(1 C)127.73(1 C)128.60(4 C)129.28(4 C)129.59(2 C)135.37(2 C)137.10(1 C)142.68(1 C)144.74(1 C)147.50(1 C)161.94(s,1 C)171.19(1 C). HPLC-MS (m / z) [M]+ calculated value 418.21, found value 418.30.

[0346] Step 2: Synthesis of 2-(1-benzhydrylpyridin-1-ium-3-yl)acetohydrazide bistrifluoroacetate: [ka]

[0347] tert-Butyl N-[[2-(1-benzhydrylpyridin-1-ium-3-yl)acetyl]amino]carbamate bromide (0.19 mmol) was treated with 50% TFA in CHCl (2 mL). The reaction mixture was stirred at room temperature for 1 hour. The solvent was removed in vacuo, and the crude product was suspended in water and lyophilized. 24 mg of the pure compound (34% yield) was obtained as an oil.

[0348] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~60 min: 40% H2O 0.1% TFA;60% CH3CN 0.1% TFA; 60~65 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 65~80 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA. 1 H NMR(400 MHz, methanol-d4)δ ppm 3.94(s,2 H)7.17-7.36(m,4 H)7.41-7.57(m,6 H)8.10(dd,J=7.84,6.46 Hz,1 H)8.59(d,J=7.91 Hz,1 H)8.86(d,J=6.02 Hz,1 H)8.98(s,1 H). 13 C NMR (101 MHz, methanol-d4) δ ppm 38.99 (1 C) 77.83 (1 C) 127.80 (2 C) 128.55 (4 C) 129.31 (4 C) 129.65 (1 C) 135.32 (2 C) 136.31 (1 C) 142.91 (1 C) 144.89 (1 C) 147.63 (1 C) 167.97 (1 C). HPLC-MS (m / z) [M]+ calculated value 318.2, found value 318.3

[0349] Example 6: Synthesis of Label 6 [ka]

[0350] Step 1: Synthesis of tert-butyl N-[[2-[1-[(3,4,5-trimethoxyphenyl)methyl]pyridin-1-ium-3-ylacetyl]amino]carbamate bromide: [ka]

[0351] tert-Butyl N-[[2-(3-pyridyl)acetyl]amino]carbamate [synthesized similarly to Example 4, Step 1] (0.40 mmol) and 3,4,5-trimethoxybenzyl bromide (0.48 mmol) were dissolved in dry CH3CN. The solution was then stirred at 70 °C overnight. The solvent was removed in vacuo, and the crude product was purified by flash chromatography (gradient CHCl2 → CHCl2 / MeOH 80:20). 55 mg, 34% yield, of the pure compound was obtained as a brownish oil. 1 H NMR(400 MHz,methanol-d4)δ ppm 1.46(s,9 H)3.74(s,3 H)3.84(s,6 H)5.74(s,2 H)6.89(s,2 H)8.05(dd,J=7.78,6.27 Hz,1 H)8.52(d,J=8.16 Hz,1 H)8.98(d,J=5.90 Hz,1 H)9.15(s,1 H). 13 C NMR(101 MHz, methanol-d4)δ ppm 27.07(3 C)36.03(1 C)55.46(2 C)59.62(1 ​​C)64.57(1 C)80.69(1 C)106.36(2 C)127.59(1 C)128.51(1 C)136.87(1 C)138.92(1 C)142.53(1 C)144.89(s,1 C)146.70(s,1 C)153.93(2 C)166.93(1 C)168.98(1 C). HPLC-MS (m / z) [M]+ calculated value 432.21, found value 432.29.

[0352] Step 2: Synthesis of 2-[1-[(3,4,5-trimethoxyphenyl)methyl]pyridin-1-ium-3-yl]acetohydrazide bistrifluoroacetate: [ka]

[0353] tert-Butyl N-[[2-[1-[(3,4,5-trimethoxyphenyl)methyl]pyridin-1-ium-3-yl]acetyl]amino]carbamate bromide (0.19 mmol) was treated with 50% TFA in CHCl (2 mL). The reaction mixture was stirred at room temperature for 1 hour. The solvent was removed in vacuo, and the crude product was suspended in water and lyophilized. 52 mg of pure compound was obtained as a brown oil (quantitative yield). 1 H NMR(400 MHz,methanol-d4)δ ppm 3.74(s,3 H)3.85(s,6 H)4.00(s,2 H)5.77(s,2 H)6.91(s,2 H)8.07(dd,J=7.91,6.27 Hz,1 H)8.54(d,J=8.03 Hz,1 H)9.02(d,J=6.15 Hz,1 H)9.20(s,1 H). 13 C NMR(101 MHz,methanol-d4)δ ppm 35.60(1 C)55.50(2 C)59.64(1 C)64.53(1 C)106.43(2 C)127.64(1 C)128.55(1 C)136.03(1 C)138.87(1 C)142.82(1 C)145.04(1 C)146.81(1 C)153.90(2 C)168.06(1 C). HPLC-MS (m / z) [M]+ calculated value 332.2, found value 332.4.

[0354] Example 7: Synthesis of Label 7 Synthesis of 4-[1-[(2-methoxyphenyl)methyl]pyridin-1-ium-4-yl]-1,2,4-triazolidine-3,5-dione trifluoroacetate: [ka]

[0355] 4-(Pyridin-4-yl)-1,2,4-triazolidine-3,5-dione (0.28 mmol) and 1-(bromomethyl)-2-methoxybenzene (0.29 mmol) were dissolved in 1 mL of dry DMF. The reaction mixture was stirred at 70 °C overnight. The solvent was removed in vacuo, and the residue was purified by preparative HPLC. Pure fractions were collected and concentrated in vacuo to give 71 mg (61% yield) of the desired product as a pale yellow solid.

[0356] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~60 min: 40% H2O 0.1% TFA,60% CH3CN 0.1% TFA; 60~64 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 64~74 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 74~79 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 79~90 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1 H NMR(400 MHz, methanol-d4)δ ppm 3.83(s,3 H)5.70(s,2 H)7.04-7.10(m,2 H)7.44-7.51(m,1 H)7.54(dd,J=7.84,1.69 Hz,1 H)8.73-8.83(m,2 H)8.92-8.99(m,2 H). HPLC-MS(m / z)[M] + Calculated value 299.11, detected value 299.33.

[0357] Label 9 can be synthesized in a similar manner.

[0358] Example 8: Synthesis of Label 8: Synthesis of 4-(1-methylpyridin-1-ium-4-yl)-1,2,4-triazolidine-3,5-dione iodide: [ka]

[0359] 4-(Pyridin-4-yl)-1,2,4-triazolidine-3,5-dione (0.34 mmol) was dissolved in dry DMF. Then, MeI (0.40 mmol) was added, and the solution was stirred at room temperature overnight. The solvent was removed under vacuum to give a yellow solid (quantitative yield) that was not further purified. 1 H NMR(400 MHz,DMSO-d6)δ ppm 4.27(s,5 H)8.47-8.57(m,4 H)8.90-8.98(m,4 H)11.37(br s,4 H). HPLC-MS(m / z)[M] + Calculated value 193.07, Found value 193.27.

[0360] Example 9: Synthesis of Label 10: Synthesis of 2-fluoro-1-[(2-methoxyphenyl)methyl]-N,N-dimethyl-pyridin-1-ium-4-amine bromide [ka]

[0361] 2-Fluoro-N,N-dimethylpyridin-4-amine (0.35 mmol) and 1-(bromomethyl)-2-methoxybenzene (0.39 mmol) were dissolved in 1 mL of dry DMF. The reaction mixture was stirred at 70 °C overnight. The solvent was removed in vacuo, and the residue was purified by silica gel chromatography (eluent CHCl / MeOH 100:0 to 95:5). Pure fractions were collected and concentrated in vacuo to give 100 mg (82% yield) of the desired product as a colorless oil. 1H NMR(400 MHz,chloroform-d)δ ppm 3.24(s,3 H)3.35(s,3 H)3.85(s,3 H)5.38(d,J=1.76 Hz,2 H)6.47(dd,J=8.66,2.89 Hz,1 H)6.88(d,J=7.78 Hz,1 H)6.97(td,J=7.47,1.00 Hz,1 H)7.23(d,J=2.89 Hz,1 H)7.33-7.40(m,1 H)7.44(dt,J=7.40,1.51 Hz,1 H)8.44(dd,J=7.78,6.15 Hz,1 H). 19 F NMR (376 MHz, chloroform-d) δ ppm -87.92 (s, 1 F). HPLC-MS (m / z) [M]+ calculated value 261.14, found value 261.36.

[0362] Example 10: Synthesis of Label 11: Synthesis of 2-fluoro-N,N-dimethyl-1-[(3,4,5-trimethoxyphenyl)methyl]pyridin-1-ium-4-amine bromide: [ka]

[0363] 2-Fluoro-N,N-dimethylpyridin-4-amine (0.35 mmol) and 3,4,5-trimethoxybenzyl bromide (0.39 mmol) were dissolved in 1 mL of dry DMF. The reaction mixture was stirred at 70 °C overnight. The solvent was removed in vacuo, and the residue was purified by silica gel chromatography (eluent: CHCl / MeOH 100:0 to 95:5). Pure fractions were collected and concentrated in vacuo to give 73 mg (51% yield) of the desired product as a brown oil. 1H NMR(400 MHz,chloroform-d)δ ppm 3.22(s,3 H)3.28(s,3 H)3.46(s,3 H)3.80(s,3 H)3.88(s,3 H)5.52(d,J=1.76 Hz,2 H)6.44(dd,J=8.53,2.76 Hz,1 H)6.85(s,2 H)6.91-7.07(m,1 H)9.30(br t,J=6.90 Hz,1 H). 19 F NMR (376 MHz, chloroform-d) δ ppm -88.40 (s, 1 F). HPLC-MS (m / z) [M]+ calculated value 321.16, found value 321.31.

[0364] Example 11: General synthetic procedure: Example 11.1: Labels 1-3 Labels 1-3 are synthesized in two steps: pyridine alkylation and hydrazide formation.

[0365] Step 1: General protocol for pyridine alkylation: [ka]

[0366] The pyridine derivative (0.87 mmol) and the benzyl bromide derivative (1.14 mmol) were dissolved in 3 mL of DMF. The reaction mixture was stirred at 115 °C for 2 h. DMF was removed in vacuo, and the residue was purified by preparative HPLC. Pure fractions were collected and lyophilized to give the product (85-96% yield) as a colored oil. FG in this context refers to a functional group.

[0367] Step 2: General protocol for hydrazide formation: [ka]

[0368] The pyridine-methyl-acetate derivative (0.74 mmol) was dissolved in 2 mL of water or MeOH. Hydrazine monohydrate (0.74 mmol) was added, and the reaction mixture was stirred at room temperature or 100 °C for 30 min to 2 h. The reaction mixture was purified by preparative HPLC. Pure fractions were collected and lyophilized to give the product (yields 46–79%).

[0369] Example 11.2: Labels 4-6 Labels 4 to 6 involve tert-butyl N-[[2-(3-pyridyl)acetyl]amino]carbamate alkylation and Boc deprotection. The tert-butyloxycarbonyl protecting group or tert-butoxycarbonyl protecting group can be abbreviated as Boc.

[0370] A general protocol for the alkylation of tert-butyl N-[[2-(3-pyridyl)acetyl]amino]carbamate is as follows. [ka]

[0371] tert-Butyl N-[[2-(3-pyridyl)acetyl]amino]carbamate (0.40 mmol) and the benzyl bromide derivative (0.48 mmol) were dissolved in dry CH3CN. The solution was then stirred at 70 °C overnight. The solvent was removed in vacuo, and the crude product was purified by flash chromatography (gradient CHCl2 → CHCl2 / MeOH 80:20). The pure compound was obtained as a white solid (yields 34-89%).

[0372] The general protocol for Boc deprotection is as follows: [ka]

[0373] The Boc-protected pyridinium salt (0.19 mmol) was treated with 50% TFA in CHCl (2 mL). The reaction mixture was stirred at room temperature for 1 hour. The solvent was removed under vacuum, and the crude product was suspended in water and lyophilized. The pure compound was obtained as a white solid, TFA salt (quantitative yield).

[0374] Example 11.3: Labels 7-9 Labels 7 and 9 are pyridine-urazole derivatives. A general protocol for pyridine-urazole alkylation is as follows: [ka]

[0375] 4-(Pyridin-4-yl)-1,2,4-triazolidine-3,5-dione (0.28 mmol) and the benzyl bromide derivative (0.29 mmol) were dissolved in 1 mL of dry DMF. The reaction mixture was stirred at 70° C. overnight. The solvent was removed under vacuum, and the residue was purified by preparative HPLC. The pure fractions were collected and concentrated under vacuum to give the product (61% yield) as a solid.

[0376] Example 11.4: Labels 10-11 Labels 10 and 11 are synthesized by the following general steps: General protocol for fluoropyridine alkylation: [ka]

[0377] 2-Fluoro-N,N-dimethylpyridin-4-amine (0.35 mmol) and the benzyl bromide derivative (0.39 mmol) were dissolved in 1 mL of dry DMF. The reaction mixture was stirred at 70 °C overnight. The solvent was removed in vacuo, and the residue was purified by silica gel chromatography (eluent CHCl / MeOH 100:0 to 95:5). Pure fractions were collected and concentrated in vacuo to give the product (51-82% yield) as an oil.

[0378] Example 12: General protocol for the reaction of testosterone with hydrazide derivatives: [ka]

[0379] The 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 or 50° C. The solvent was removed in vacuo and the crude mixture was subjected to purification by preparative HPLC to give the desired product as a white solid.

[0380] Example 12.1: Preparation of Labeled 3-Testosterone Derivatives and Their Analysis by MS [ka] HPLC-MS(m / z)[M] + Calculated value 572.35, Found value 572.39.

[0381] Example 13: General protocol for in situ activation of urazole for TAD-containing labels and reaction with vitamin D: [ka]

[0382] 25-Hydroxyvitamin D monohydrate (0.024 mmol) and the urazole derivative (0.029 mmol) were dissolved in MeOH (500 μL). A solution of iodobenzene diacetate (0.033 mmol) in MeOH was added to the first solution. The reaction mixture was stirred at room temperature for 15 minutes. Complete conversion of vitamin D to the corresponding product was observed. The solvent was removed in vacuo, and the residue was purified by preparative HPLC. The pure product was obtained as a white solid.

[0383] Example 13.1: Preparation of labeled 7-vitamin D derivatives and their analysis by MS [ka]

[0384] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~20 min: 5% H2O 0.1% TFA,95% CH3CN 0.1% TFA; 20~40 min: 5% H2O 0.1% TFA; 95% CH3CN 0.1% TFA; 40~45 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 45~50 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 50~55 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 55~65 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1 H NMR(400 MHz, methanol-d4)δ ppm 0.43(s,2 H)0.59(s,1 H)0.87-0.91(m,4 H)1.12-1.20(m,6 H)1.25-1.48(m,10 H)1.56-2.29(m,12 H)2.37-2.46(m,1 H)2.87-2.95(m,1 H)3.82(s,3 H)3.87-4.05(m,2 H)4.14-4.25(m,1 H)4.74-4.82(m,1 H)5.06-5.16(m,1 H)5.70(s,2 H)7.00-7.11(m,2 H)7.43-7.51(m,1 H)7.54(dt,J=7.72,1.85 Hz,1 H)8.69-8.79(m,2 H)8.91-9.04(m,2 H). HPLC-MS(m / z)[M] + Calculated value 697.43, Found value 697.56.

[0385] Example 13.2: Preparation of Labeled 8-Vitamin D Derivatives and Their Analysis by MS [ka]

[0386] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~20 min: 5% H2O 0.1% TFA,95% CH3CN 0.1% TFA; 20~40 min: 5% H2O 0.1% TFA; 95% CH3CN 0.1% TFA; 40~45 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 45~50 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 50~55 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 55~65 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1 H NMR(400 MHz, methanol-d4)δ ppm 0.45(s,2 H)0.60(s,1 H)0.87-1.02(m,4 H)1.14(s,3 H)1.15(s,3 H)1.25-1.50(m,10 H)1.61-2.29(m,12 H)2.37-2.46(m,1 H)2.89-2.87(m,1 H)3.87-4.05(m,2 H)4.17-4.26(m,1 H)4.34(s,3 H)4.76-4.82(m,1 H)5.10-5.17(m,1 H)8.72-8.81(m,2 H)8.88(t,J=6.74 Hz,2 H). HPLC-MS(m / z)[M] + Calculated value 591.39, Found value 591.50.

[0387] Example 14: General protocol for the reaction of estradiol with fluoropyridine derivatives: [ka]

[0388] Estradiol (0.09 mmol) and the fluoropyridine derivative (0.11 mmol) were dissolved in CH3CN (1 mL). DIPEA (0.14 mmol) was then added, and the reaction mixture was stirred at room temperature for 30 min. The solvent was removed in vacuo, and the residue was purified by preparative HPLC. Pure fractions were collected and lyophilized to give the product as a solid (yields 21-45%). Example 14.1: Preparation of labeled 10-estradiol derivatives and their analysis by MS [ka]

[0389] HPLC method C-18 column: 0 min: 90% H2O 0.1% TFA,10% CH3CN 0.1% TFA; 0~60 min: 30% H2O 0.1% TFA,70% CH3CN 0.1% TFA; 60~64 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 64~74 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 74~79 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 79~90 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1H NMR(400 MHz, methanol-d4)δ ppm 0.78(s,3 H)1.11-1.60(m,7 H)1.63-1.78(m,1 H)1.84-2.11(m,3 H)2.19-2.29(m,1 H)2.36(dq,J=13.40,3.52 Hz,1 H)2.76-2.87(m,2 H)2.92(br s,3 H)3.19(br s,3 H)3.66(t,J=8.60 Hz,1 H)3.85(s,3 H)5.40(s,2 H)5.80(d,J=2.76 Hz,1 H)6.73-6.79(m,2 H)6.81(dd,J=8.47,2.70 Hz,1 H)6.91-7.00(m,1 H)7.05(d,J=8.16 Hz,1 H)7.30(dd,J=7.47,1.57 Hz,1 H)7.41(br d,J=8.53 Hz,2 H)8.09(d,J=7.78 Hz,1 H). HPLC-MS (m / z) [M]+ calculated value 513.31, found value 513.41.

[0390] Example 14.2: Preparation of labeled 11-estradiol derivatives and their analysis by MS [ka]

[0391] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~60 min: 40% H2O 0.1% TFA,60% CH3CN 0.1% TFA; 60~64 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 64~74 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 74~79 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 79~90 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1H NMR(400 MHz, methanol-d4)δ ppm 0.78(s,3 H)1.12-1.59(m,7 H)1.64-1.79(m,1 H)1.82-2.10(m,3 H)2.18-2.30(m,1 H)2.31-2.42(m,1 H)2.81-2.89(m,2 H)2.95(br s,3 H)3.21(br s,3 H)3.66(t,J=8.66 Hz,1 H)3.74(s,3 H)3.76(s,6 H)5.37(s,2 H)5.88(d,J=2.76 Hz,1 H)6.70(s,2 H)6.79(d,J=2.76 Hz,1 H)6.84(dd,J=7.72,2.82 Hz,1 H)6.88(dd,J=8.60,2.70 Hz,1 H)7.43(d,J=8.66 Hz,1 H)8.18(d,J=7.78 Hz,1 H). HPLC-MS (m / z) [M]+ calculated value 573.33, found value 573.49.

[0392] Example 14.3: Preparation of labeled 12-estradiol derivatives and their analysis by MS [ka]

[0393] Estradiol (0.50 mmol), fluoropyridine (0.52 mmol), and CsCO (0.59 mmol) were dissolved in dry DMF (1 mL). The reaction mixture was stirred at 70 °C for 7 days. The solvent was removed under vacuum, and the residue was purified by preparative HPLC. The pure fractions were collected and lyophilized to give 81 mg (35% yield) of the product as a colorless solid. The obtained product (0.09 mmol) was further reacted with the benzyl bromide derivative (0.11 mmol) in dry DMF (1 mL) at 70 °C for 2 days. The solvent was removed under vacuum, and the residue was purified by preparative HPLC. The pure fractions were collected and lyophilized to give 18 mg (36% yield) of the product as a colorless solid.

[0394] First purification HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~60 min: 20% H2O 0.1% TFA,80% CH3CN 0.1% TFA; 60~64 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 64~80 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 80~83 minutes: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 83~89 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 89~90 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. HPLC-MS (m / z) [M+H]+ calculated 350.47, found 350.50.

[0395] Second Purification HPLC Method C-18 Column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~60 min: 2% H2O 0.1% TFA,98% CH3CN 0.1% TFA; 60~64 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 64~80 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 80~83 minutes: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 83~89 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 89~90 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. HPLC-MS (m / z) [M]+ calculated value 470.27, found value 470.49.

[0396] Example 15: Analytical derivatization of testosterone with labels 1-6 A 500 ng / mL solution of testosterone (S1) was prepared in methanol. A solution (S2) containing an excess of one of the derivatization reagents, labels 1–12, diluted in methanol (molar ratio >1000) relative to solution (S1) was added, and the solution was acidified with glacial acetic acid (20% v / v). Solutions S1 and S2 were mixed to obtain solution S3, which was then kept at 65°C for 2 hours and then at room temperature for 12 hours. After 12 hours, solution S3 was diluted with methanol to obtain five independent concentration levels based on the molecular weight of testosterone: 1 / 20 equivalent of testosterone; 1 / 40 equivalent of testosterone; 1 / 100 equivalent of testosterone; and 1 / 200 equivalent of testosterone. One equivalent is selected because it is within the proportionality range of the detector, while 1 / 200 equivalent of testosterone is below the detection limit of the instrument used. As an 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 blank solution of 0 ng / mL was prepared using solution S2.

[0397] After derivatization of the analyte molecule, isomers can be introduced, which can result in multiple peaks. The chromatographic behavior of these isomers needs to be addressed in a way that quantifies their properties relatively. Illustrated in Figure 1 describes the signal distribution (%) over time when a specific chromatographic method is applied at 5% of the absolute peak height. The parameter "resolution" describes the ability of the chromatographic system to separate the isomers resulting from the derivatization reaction of the analyte molecule. The retention times measured at the centroids of the isomer peaks can describe the polarity of the resulting derivatives when reversed-phase chromatographic materials are used for the separation. The areas A1 and A2 are the signal numbers. * The signal intensity is measured in minutes and reported as the ratio of the resulting derivative isomers. If the ratio of peaks A1 and A2 is 50 / 50, the resulting isomers will be produced in equal amounts. If the labeling can affect this ratio so that it is not equal to 50 / 50, one isomer will be produced predominantly. The signal intensity will therefore be unevenly distributed, increasing the signal height relative to the unaffected background noise.

[0398] For each labeled substance, each testosterone derivative was measured by electrospray ionization mass spectrometry after liquid chromatography separation, and different fragmentation scans with collision energies of 15V, 20V, 25V, 30V, 35V, and 40V were performed.

[0399] Fragmentation was optimized using the mass signals of the molecular ion peak and the most abundant fragment ion peak (neutral loss). Regarding signal gain for charged molecules with neutral loss units, fragmentation energy is one of the most important tuning parameters. The fragmentation energy is important because the molecule must not be fragmented during source and further ion path conditions. The cleavage or scission of neutral loss fragments should only occur in the collision cell (and / or associated instrumentation) to form specific fragments of interest. If the collision energy is too high for the cleavage of neutral loss fragments, other undesired fragmentation pathways may also occur, resulting in a loss of signal intensity relative to the neutral loss fragmentation pathway. It is assumed that the collision energy is neither too small nor too large to obtain optimal fragmentation behavior. The maximum area count value at each collision energy was used for further detection limit quantification approaches.

[0400] For each labeled substance, the respective testosterone derivative was tuned on a triple quadrupole mass spectrometer by injecting it into the mass spectrometer via liquid chromatography. The tuning parameter was the collision energy that resulted in the highest signal for each chromatographic peak in five independent collision energy experiments.

[0401] Chromatography and MS parameters Polarity ES+ Calibration Static 2 Soft transmit mode disabled Capillary (kV) 3.00 3.14 Corn (V) 50.00 144.92 Source Offset (V) 30.0 Source temperature (℃) 140 140 Desolvation temperature (℃)350 350 Cone gas flow rate (L / Hr) 150 149 Desolvation gas flow rate (L / Hr) 1000 990 Collision gas flow (mL / min) 0.15 0.14 Nebulizer gas flow (Bar) 7.00 6.52 LM1 resolution 3.0 HM1 resolution 15.0 Ion 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 Entrance 1.00 Gas On MS Mode Exit 1.00 Gas On MSMS Mode Entrance 1.00 Gas On MSMS Mode Exit 1.00 Gas off MS mode entrance 30.00 Gas off MS mode exit 30.00 Gas Off MSMS Mode Entrance 30.00 Gas Off MSMS Mode Exit 30.00 ScanWave MS mode entrance 1.00 ScanWave MS mode exit 1.00 ScanWave MSMS Mode Entrance 1.00 ScanWave MSMS Mode Exit 1.00 LM2 resolution 3.0 HM2 resolution 15.0 Ion Energy2 0.2 Gain 1.00 Multiplier 513.80 Active Reservoir C Cone 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 transmit mode disabled Capillary (kV) 3.00 3.14 Corn (V) 50.00 144.92 Source Offset (V) 30.0 Source temperature (℃) 140 140 Desolvation temperature (℃)350 350 Cone gas flow rate (L / Hr) 150 149 Desolvation gas flow rate (L / Hr) 1000 990 Collision gas flow (mL / min) 0.15 0.14 Nebulizer gas flow (Bar) 7.00 6.52 LM1 resolution 3.0 HM1 resolution 15.0 Ion 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 Entrance 1.00 Gas On MS Mode Exit 1.00 Gas On MSMS Mode Entrance 1.00 Gas On MSMS Mode Exit 1.00 Gas off MS mode entrance 30.00 Gas off MS mode exit 30.00 Gas Off MSMS Mode Entrance 30.00 Gas Off MSMS Mode Exit 30.00 ScanWave MS mode entrance 1.00 ScanWave MS mode exit 1.00 ScanWave MSMS Mode Entrance 1.00 ScanWave MSMS Mode Exit 1.00 LM2 resolution 3.0 HM2 resolution 15.0 Ion Energy2 0.2 Gain 1.00 Multiplier 513.80 Active Reservoir C Cone 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 (sec) 0.003 Polarity / Mode Switch Inter-Scan Delay (sec) 0.020 Enhanced inter-scan delay (sec) 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 -----------------Method Parameter Execution------------ Waters Acquity SDS Duration: 6.50 minutes comment: Solvent Selection A:A2 Solvent Selection B: B1 Low pressure limit: 0.000 bar High pressure limit: 1034.200 bar Solvent A: Water + NH4Ac + 0.1% formic acid Solvent name B: MeOH + NH4Ac + 0.1% formic acid Switch 1: No change Switch 2: No change Switch 3: No change Seal cleaning: 5.0 minutes Chart Out 1: System Pressure Chart Out 2: %B System pressure data channel: Yes Flow Data Channel: None %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 Run event: Yes Gradient start (for injection): 0 uL 2D Iterations: None Waters Acquity PDA Duration: 6.50 minutes PDA detector type: UPLC LG 500nm Lamp: On Sampling rate: 10 points / second Filter time constant: 0.2000 seconds Exposure time: automatic msec Quadratic filter area interpolation: None Use of UV blocking filters: No 3D Channel... Range: 200-400 Resolution: 2.4nm Initial Switch 1: No change Initial Switch 2: No change Waters ACQUITY FTN AutoSampler Duration: 6.50 minutes comment: Preload: Disabled Offline Loop: Automatic min Cleaning solvent: ACN: Water Pre-injection cleaning time: 15.0 seconds Post-injection cleaning time: 15.0 seconds Purging solvent name: ACN:Water Dilution: Ineffective Dilution volume: 0uL 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 Pre-suction air gap: Automatic Post-suction air gap: Automatic Column temperature data channel: None Room temperature data channel: Yes Sample temperature data channel: Yes Specimen Organizer Temperature Data Channel: None Sample pressure data channel: None Preheater temperature data channel: None Seal Force Data Channel: None No injection mode: No Automatic Additive Mixing Stroke Cycle: Automatic Automatic addition mixing stroke volume: Automatic uL Active Preheater: Use the console configuration Run Events: None Sample run injection parameters Injection volume (ul)-5.00 Feature 1 Active scans: 738 Cycle time (seconds): Automatic Delay between scans (seconds): Automatic Inter-channel delay (seconds): Automatic Span (Da): 0.500 Start time and end time (minutes): 0.000 to 5.000 Ionization mode: ES+ Data type: SIR or MRM enhancement Function type: 1 channel MRM Chan reaction dwell (sec) Kohnbolt. Col. energy delay (sec) Compound 1:289.25 > 108.78 0.200 Tune 25.0 Automatic Testosterone Feature 2 Active scans: 737 Cycle time (seconds): Automatic Delay between scans (seconds): Automatic Inter-channel delay (seconds): Automatic Span (Da): 0.500 Start time and end time (minutes): 0.000 to 5.000 Ionization mode: ES+ Data type: SIR or MRM enhancement Function type: 1 channel MRM Chan reaction dwell (sec) Kohnbolt. Col. energy delay (sec) Compound formula | mass 1:624.40 > 203.00 0.200 Tune 40.0 Automatic DMA041 CE40 420.2 Feature 3 Active scans: 3901 Function Type: Diode Array Wavelength range (nm): 200-400

[0402] From the linear calibration curve, the respective detection limits were obtained using the procedure described in DIN EN ISO 32645. The enhancement factors are calculated based on the labeled analyte detection limit (LOD), here labeled testosterone LOD, compared to the underivatized analyte LOD, here underivatized testosterone LOD. The workflow is shown in Figure 2. The results are shown in Table 2 below. [Table 2]

[0403] As can be seen from Table 2, labels 1, 2, and 4-6 and their conjugates show signal enhancement compared to underivatized analytes. These labels and their conjugates show better enhancement factors than state-of-the-art derivatizing agents, such as Amplifex from Sciex.

[0404] Example 16: Analytical derivatization of estradiol with labels 10-12 A 500 pg / mL solution of estradiol (S1) was prepared in acetonitrile. Horse serum was depleted using acetonitrile (-20°C) at a ratio of 1 mL horse serum + 3 mL acetonitrile. The horse serum / acetonitrile mixture was mixed, centrifuged, and the supernatant was decanted to obtain solution (S2). Solutions S1 and S2 were mixed in a 1:5 ratio to obtain solution S3. Solution S4, which contained an excess of either the derivatization reagent, Label 10, or Label 12, diluted in methanol (molar ratio >1000) compared to solution (S3), was added. A 5 μg / mL solution of K2CO3 (S5) was prepared in acetonitrile / H2O 90 / 10 + 0.1% formic acid. Solutions S3, S4, and S5 were mixed to obtain solution S6, which was then kept at 50°C for 1 hour and then at room temperature for 12 hours. Solution S6 was diluted with acetonitrile / H2O + 0.1% formic acid to obtain an appropriate concentration level for quantification.

[0405] From the linear calibration curve, the respective detection limits were obtained using the procedure described in DIN EN ISO 32645. The enhancement factors are calculated based on the labeled estradiol limit of detection (LOD) compared to the underivatized estradiol LOD. The results are shown in Table 3 below. [Table 3]

[0406] As can be seen from Table 3, labels 10 and 11 and their conjugates show signal enhancement compared to underivatized analytes.

[0407] Example 17: Analytical derivatization of vitamin D with labels 7 and 8 A 500 pg / ml solution of 25-OH vitamin D3 (S1) was prepared in methanol. Horse serum was depleted using methanol (-20°C) at a ratio of 1 mL horse serum + 3 mL methanol. The horse serum / methanol mixture was mixed, centrifuged, and the supernatant was decanted to obtain solution (S2). Solutions S1 and S2 were mixed in a 1:5 ratio to obtain solution S3. Solution S3 was concentrated to dryness. A solution (S4) containing an excess of either derivatization reagent, Label 7 or 8, diluted in methanol (molar ratio >1000) relative to solution (S3) was added, and the resulting solution was mixed. A 2 mg / mL solution of iodobenzene diacetate in methanol (S5) was added, and the resulting solution (S6) was stirred at 40°C for 2 min. Solution S6 was diluted with methanol / HO + 0.1% formic acid to obtain the appropriate concentration level for quantification. [Table 4]

[0408] As can be seen from Table 4, labels 7 and 8 and their conjugates show signal enhancement compared to the underivatized analyte.

[0409] In addition to testosterone, estradiol, and vitamin D, other analytes can be selected as analytes of interest. Complexes with these other analytes exhibit enhanced signals compared to the underivatized analytes.

[0410] This patent application claims priority to European patent application 20175801.8, the contents of which are incorporated herein by reference.

Claims

1. Formula (I) for mass spectrometric determination of analytes: 【Chemical 1】 wherein one of the substituents B1, B2, B3, B4, B5 is a coupling group Q capable of forming a covalent bond with the analyte, wherein Q is selected from the group consisting of methyl hydrazide, methyl hydrazine, methylhydroxylamine, acetohydrazide, 4-substituted 1,2,4-triazoline-3,5-diones (TAD), and F; The other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, and B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, aryloyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, heteroaryl, heterocycloalkyl, or isotopes thereof; 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, or Y1 and Y2 form a ring structure selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, and unsubstituted heteroaromatic. Compound.

2. 2. The compound of claim 1, wherein B1 is Q, or B2 is Q, or B3 is Q.

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

4. A composition comprising a compound according to any one of claims 1 to 3.

5. A kit comprising the compound according to any one of claims 1 to 3 or the composition according to claim 4.

6. 4. A conjugate for detecting an analyte using mass spectrometry determination, comprising a binding analyte and a binding compound covalently bound to each other, said conjugate being formed by chemical reaction of said analyte with a compound according to any one of claims 1 to 3.

7. The complex described in claim 6, wherein 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 a specific modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

8. 4. Use of a compound according to any one of claims 1 to 3 for mass spectrometric determination of said analyte, wherein said mass spectrometric determination comprises tandem mass spectrometric determination or triple quadrupole mass spectrometric determination.

9. 1. A method for mass spectrometric determination of an analyte, comprising: (a) reacting the analyte with a compound of formula (I) according to any one of claims 1 to 3, whereby a complex according to claim 6 or 7 is formed; (b) subjecting the complex from step (a) to mass spectrometric analysis; A method comprising:

10. The method according to claim 9, wherein step (b) comprises: (i) subjecting ions of said complex to a first stage of mass spectrometric analysis, whereby said ions of said complex are characterized according to their mass-to-charge (m / z) ratio; (ii) causing fragmentation of the ions of the complex, whereby the first entity or low molecular weight entity is released and daughter ions of the complex are produced, the daughter ions of the complex having a different m / z ratio than the ions of the complex; (iii) subjecting the daughter ions of the complex to a second stage of mass spectrometric analysis, whereby the daughter ions of the complex are characterized according to their m / z ratio; and / or (ii) may further comprise alternative fragmentation of ions of the complex, whereby a second entity different from the first entity is released and a second daughter ion of the complex is produced; and (iii) may further comprise subjecting the first and second daughter ions of the complex to a second stage of mass spectrometry, whereby the first and second daughter ions of the complex are characterized according to their m / z ratios.

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