Reagents for mass spectrometry

Abstract

The present invention relates to compounds suitable for use in mass spectrometry, as well as 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 quantification 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 highly sensitive and specific method, even capable of analyzing complex biological samples, such as environmental or clinical samples. However, measurement sensitivity remains an issue for some analytes, 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 molecules of interest (analytes) being analyzed are separated by chromatography and individually subjected to mass spectrometry (Higashi et al. (2016) J. of Pharmaceutical and Biomedical Analysis 130 pp. 181-190).

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

[0005] In the art, there are known several derivatization reagents (compounds) that aim to improve the sensitivity of the measurement of these analytes. Among them, there are reagents in which a charged unit and a neutral loss unit are combined into one functional unit (for example, International Publication No. 2011 / 091436). Other reagents that contain separate units are relatively large in structure, which affects the general workflow of sample preparation and MS measurement (Rahimoff et al. (2017) J.Am.Chem.Soc.139(30), p.10359-10364). Known derivatization reagents include, for example, dansyl chloride, RapiFluor-MS (RFMS), Cookson-type reagents, Amplifex Diene, Amplifex Keto, Girard T, Girard P, and pyridylamine (Hong and Wang, Anal Chem., 2007, 79(1):322-326; Frey et al., Steroids, 2016 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, suboptimal ionization efficiency, disadvantages to post-coupling chromatographic separation, suboptimal fragmentation behavior due to the many fragmentation pathways, and the need for high collision energy. Sensitivity and selectivity are insufficient for very small amounts of analyte.

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

[0009] This object is solved by the subject matter of the independent claims. Further embodiments are set out in the dependent claims. Summary of the Invention

[0010] Summary of the Invention In the following, the present invention relates to the following items:

[0011] In a first aspect, the present invention provides a method for the mass spectrometric determination of an analyte, comprising: [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; the other substituents A1, A2, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, modified alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, aryloyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, sulfur, isotopes or derivatives thereof; A3 comprises ammonium, pyridinium, phosphonium or a derivative thereof; When A3 is ammonium and B1 or B5 is a coupling group Q, the coupling group Q contains a C atom separated from a C atom of the CA1A2A3 substituent by four single or double bonds, and the coupling group Q contains a C atom separated from a C atom of the CA1A2A3 substituent by five single or double bonds, preferably A3 is ammonium, B1 or B5 is Q, and Q does not contain at least one atom selected from O, N, S, and Br. 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 item of the present invention.

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

[0014] In a fourth aspect, the present invention relates to 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 of the second item of the present invention.

[0015] In a fifth aspect, the present invention relates to the use of a compound of the first item 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 as defined in any one of embodiments 1 to 32, thereby forming a complex as defined in any one of embodiments 35 to 38; (b) subjecting the complex from step (a) to mass spectrometry. The present invention relates to a method, comprising:

[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 an analyte; the other substituents A1, A2, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, modified alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, arylolyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, sulfur, isotopes or derivatives thereof; A3 comprises ammonium, pyridinium, phosphonium or a derivative thereof; When A3 is ammonium and B1 or B5 is a coupling group Q, the coupling group Q contains a C atom separated by four single or double bonds from a C atom of the CA1A2A3 substituent, and the coupling group Q contains a C atom separated by five single or double bonds from a C atom of the CA1A2A3 substituent. Preferably, A3 is ammonium, B1 or B5 is a coupling group Q, and Q does not contain at least one atom selected from O, N, S, and Br.

[0018] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 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] Figure 2 shows a schematic diagram of the workflow for determining the enhancement factor. DETAILED DESCRIPTION OF THE INVENTION

[0020] 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 will be limited only by the 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.

[0021] Several documents are cited herein. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. In the event of a 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.

[0022] 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 indicates otherwise.

[0023] definition The word "comprise", and variations such as "comprises" and "comprising", will be understood to mean the inclusion of a specified 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. 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 ​​explicitly recited as limiting that range, but also all or subranges of the individual numerical values ​​subsumed within that range, as if each numerical value and subrange were explicitly 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 described characteristics.

[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 may perform various functions, or two or more reactive groups may perform the same function. Reactive groups include, but are not limited to, reactive units, charged units, and neutral loss units. The compound may be designated as a label. In the context of the present invention, the term "binding compound" refers to the compound bound to an analyte. In principle, the compound and the binding compound may be identical. The compound and the binding compound may be substantially identical. Substantially identical may mean that both compounds have the same chemical structure, except that the structure of the reactive unit K and / or the structure of the coupling group Q are different from each other. Preferably, the compound is capable of forming a bond to the analyte but is not yet bound to the analyte. The binding compound binds to the analyte.

[0027] The term "C atom of the CA1A2A3 substituent" refers to the C atom to which the substituents A1, A2 and A3 are bonded. In other words, the "C" in the term "CA1A2A3 substituent" refers to the C atom to which A1, A2 and A3 are bonded.

[0028] The term "when A3 is ammonium and B1 or B5 is a coupling group Q, the coupling group Q comprises a C atom separated by four single or double bonds from a C atom of the CA1A2A3 substituent, and the coupling group Q comprises a C atom separated by five single or double bonds from a C atom of the CA1A2A3 substituent" can mean that when A3 is ammonium and B1 is a coupling group Q, the coupling group Q comprises a first C atom separated by four single or double bonds from another C atom that is a C atom of the CA1A2A3 substituent. The coupling group Q comprises a second C atom separated by five single or double bonds from another C atom that is a C atom of the CA1A2A3 substituent.

[0029] The term "when A3 is ammonium and B1 or B5 is a coupling group Q, the coupling group Q comprises a C atom separated by four single or double bonds from the C atom of the CA1A2A3 substituent, and the coupling group Q comprises a C atom separated by five single or double bonds from the C atom of the CA1A2A3 substituent" can mean that when A3 is ammonium and B5 is a coupling group Q, the coupling group Q comprises a first C atom separated by four single or double bonds from another C atom, which is a C atom of the CA1A2A3 substituent. The coupling group Q comprises a second C atom separated by five single or double bonds from another C atom, which is a C atom of the CA1A2A3 substituent. The terms "first" and "second" are used herein to distinguish two C atoms from each other.

[0030] The phrase "when A3 is ammonium and B1 or B5 is a coupling group Q, the coupling group Q comprises a C atom separated by four single or double bonds from the C atom of the CA1A2A3 substituent, and the coupling group Q comprises a C atom separated by five single or double bonds from the C atom of the CA1A2A3 substituent" can mean that when A3 is ammonium, B1 or B5 requires the coupling group Q. The coupling group Q comprises a first C atom separated by four single or double bonds from another C atom that is a C atom of the CA1A2A3 substituent, and the coupling group Q comprises a C atom separated by five single or double bonds from the C atom of the CA1A2A3 substituent. Preferably, A3 is ammonium, B1 or B5 is Q, and Q does not comprise at least one atom selected from O, N, S, or Br.

[0031] "A3 is ammonium, and B1 or B5 is a coupling group Q * When the coupling group Q * contains a C atom separated by four single or double bonds from the C atom of the CA1A2A3 substituent, and a coupling group Q *The term "contains a C atom separated by five single or double bonds from a C atom of the CA1A2A3 substituent" means that A3 is ammonium and B1 is a coupling group Q * When the coupling group Q * may mean that the coupling group Q comprises a first C atom separated by four single or double bonds from another C atom which is a C atom of a CA1A2A3 substituent. * contains a second C atom separated by five single or double bonds from another C atom, which is a C atom of a CA1A2A3 substituent. "A3 is ammonium and B1 or B5 is a coupling group Q * When the coupling group Q * contains a C atom separated by four single or double bonds from the C atom of the CA1A2A3 substituent, and a coupling group Q * The term "contains a C atom separated by five single or double bonds from a C atom of the CA1A2A3 substituent" means that A3 is ammonium and B5 is a coupling group Q * When the coupling group Q * may mean that the coupling group Q comprises a first C atom separated by four single or double bonds from another C atom which is a C atom of a CA1A2A3 substituent. * comprises a second C atom separated by five single or double bonds from another C atom, which is a C atom of a CA1A2A3 substituent. The terms "first" or "second" serve herein to distinguish the two C atoms from each other.

[0032] 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 with 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.

[0033] "Tandem mass spectrometry" or "MS / MS" involves multiple steps of selective mass analysis, with analyte fragmentation occurring between stages. 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).

[0034] Mass spectrometers separate and detect ions with slightly different masses, so they can easily distinguish different isotopes of a given element.Therefore, mass spectrometry is an important method for accurate mass measurement 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 amino acid sequence.

[0035] Most sample workflows in MS further include a sample preparation and / or enrichment process, where, for example, the analyte of interest is separated from the matrix using gas or liquid chromatography. Typically, for mass spectrometric determination, the following three steps are performed:

[0036] 1. A sample containing the 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).

[0037] 2. The ions are sorted and separated according to their mass and charge. Field Asymmetric Waveform Ion Mobility Spectroscopy (FAIMS) may be used as an ion filter.

[0038] 3. The separated ions are detected, for example, in multiple reaction mode (MRM), and the results are displayed on a chart.

[0039] 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 releases ions and neutral molecules.

[0040] 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 within 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 series of differentially excited skimmer stages. A counterflow of dry and preheated nitrogen gas can be used to improve solvent removal. Gas-phase ionization in APCI can be more effective than ESI for analyzing less polar elements.

[0041] "High Field Asymmetric Waveform Ion Mobility Spectroscopy (FAIMS)" is an atmospheric pressure ion mobility technique that separates gas phase ions according to their behavior in strong and weak electric fields.

[0042] "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.

[0043] 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.

[0044] In the context of this disclosure, the terms "analyte," "analyte molecule," or "analyte of interest" are used interchangeably to refer to a 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 characteristic of 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 an organism (e.g., therapeutic drugs, drugs of abuse, toxins, etc.), or metabolites of such substances. Such analytes may serve as biomarkers. In the context of the present invention, the term "biomarker" refers to a substance in a biological system 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 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 they differ from each other by the structure of functional groups. Preferably, the analyte can form a bond to the compound but is not also bound to the compound. The bound analyte is bound to the compound.

[0045] The term "permanently positively charged" is used in the context of this disclosure to indicate that the positive charge of the pyridinium or ammonium or phosphonium 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-permanent positive charge), which may be the result of, for example, an electrostatic interaction.

[0046] 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.

[0047] The analyte may be present in a sample of interest, for example, a biological sample or a clinical sample. The terms "sample" or "subject sample" are used interchangeably herein and refer to a portion or part of a tissue, organ, or individual, typically smaller than the tissue, organ, or individual intended to represent the entire tissue, organ, or individual. Upon analysis, the sample provides information about the tissue condition, 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.

[0048] 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).

[0049] Prior to analysis by mass spectrometry, samples may be pretreated in a manner specific to the sample and / or analyte. In the context of this disclosure, the term "pretreatment" refers to any means necessary to enable subsequent analysis of the desired analyte by mass spectrometry. Pretreatment means 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).

[0050] The term "hemolysis reagent (HR)" refers to a reagent that lyses cells present in a sample, and in the context of the present invention, hemolysis 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 hemolysis reagent is water (HO). Further examples of hemolysis reagents include, but are not limited to, deionized water, hypertonic liquids (e.g., 8 M urea), ionic liquids, and different surfactants.

[0051] 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. For 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 a 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).

[0052] In addition to pretreatment, the sample may also be subjected to one or more enrichment processes. In the context of the present disclosure, the term "first enrichment process" or "first enrichment workflow" refers to an enrichment process 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 to the analyte of interest. The coating may vary depending on the intended application, i.e., the intended capture molecule. It is well known to those skilled in the art which coating is suitable for which analyte. The beads can be made of a variety of different materials. The beads may have various sizes and may include a porous or non-porous surface. The term

[0053] 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 enriched analytes relative to the initial sample and the sample after the first enrichment process.

[0054] The term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical elements as they flow around or over a stationary liquid or solid phase.

[0055] The term "liquid chromatography" or "LC" refers to the process of selectively retarding one or more components of a fluid solution as the fluid permeates uniformly through a column or capillary passage of finely divided material. Retardation results from the distribution of mixture components between one or more stationary phases and the bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal phase liquid chromatography (NPLC), 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).

[0056] "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 a flow rate as described above and a pressure as described above (micro LC, UHPLC). A short rapid LC protocol involves trapping / washing / elution steps using a single analytical column, achieving LC in a very short time of less than 1 minute.

[0057] Additionally, hydrophilic interaction chromatography (HILIC), size-exclusion LC, ion-exchange LC, and affinity LC are well known.

[0058] LC separations can be single-channel LC or multi-channel LC, which includes multiple LC channels arranged in parallel. In LC, analytes can be separated according to their polarity or log P value, size, or affinity, as commonly known to those skilled in the art.

[0059] 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 destruction point 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 destruction points 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, those skilled in the art can determine based on the overall structure of the molecule whether the pyridinium unit fragments to release a benzyl element or is completely released from the parent molecule. That is, the resulting daughter molecules are either benzyl molecules or parent molecules lacking benzyl. Fragmentation can occur via collision-induced dissociation (CID), electron-capture dissociation (ECD), electron-transfer dissociation (ETD), negative electron-transfer dissociation (NETD), electron-detachment dissociation (EDD), photodissociation, particularly infrared multiphoton dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD), surface-induced dissociation (SID), high-energy C-trap dissociation (HCD), and charge-remote fragmentation.

[0060] The term "reactive unit" refers to a unit that can react with another molecule, i.e., 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 forming a covalent bond between a reactive group and a functional group of the analyte, an atom is lost during this chemical reaction.

[0061] 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 covalent reaction product formed by the reaction of a compound with an analyte molecule.

[0062] 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 may further comprise 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 may further comprise one or more other reagents, including, but not limited to, a reaction catalyst. The kit may further comprise 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. A label may be displayed on the container to indicate that the composition is to be used for a particular application and may also indicate instructions for either in vivo or in vitro use. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., 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.

[0063] Embodiment In a first aspect, the present invention provides a compound 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; the other substituents A1, A2, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, modified alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, arylolyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, sulfur, isotopes or derivatives thereof; A3 comprises ammonium, pyridinium, phosphonium or a derivative thereof; When A3 is ammonium and B1 or B5 is a coupling group Q, the coupling group Q contains a C atom separated by four single or double bonds from a C atom of the CA1A2A3 substituent, and the coupling group Q contains a C atom separated by five single or double bonds from a C atom of the CA1A2A3 substituent. Preferably, A3 is ammonium, B1 or B5 is Q, and Q does not contain at least one atom selected from O, N, S, and Br.

[0064] 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 problems can be solved by modifying them with charge-containing moieties (e.g., pyridinium, quaternary ammonium, imidazolium, triphenylphosphonium, stable metal complexes, sulfonic acid, borates, aryltrifluoroborates, sulfates, phosphates, 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, potentially enhancing sensitivity in MS / MS mode. Regardless of the above, further MS experiments can be performed at higher energies to explore additional fragmentation pathways.

[0065] 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.

[0066] In an embodiment of the first aspect of the present invention, the alkyl is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, and cyclohexyl. The alkyl may include a modified alkyl. The modified alkyl includes the structural unit alkyl-O-alkyl, such as -CH2-O-CH3, -CH2-O-CH2-CH3.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] In an embodiment of the first aspect of the present invention, acyl is selected from the group consisting of formyl, acetyl and propionyl.

[0074] 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.

[0075] In an embodiment of the first aspect of the present invention, arylolyl is selected from the group consisting of benzoyl, naphthoyl and anthracenoyl.

[0076] In an embodiment of the first aspect of the present invention, acyloxy is selected from the group consisting of formyloxy, acetyloxy and propionyloxy.

[0077] In an embodiment of the first aspect of the present invention, aryloyloxy is selected from the group consisting of benzoyloxy, naphthoyloxy and anthracenoyloxy.

[0078] In an embodiment of the first aspect of the present invention, cycloalkyl is selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] In an embodiment of the first aspect of the present invention, the ammonium or its derivative is -NR1R2R3, where R1, R2, and R3 can independently be alkyl or aryl. The alkyl can be 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. The modified alkyl includes the structural unit alkyl-O-alkyl, such as -CH2-O-CH3, -CH2-O-CH2-CH3. The aryl is phenyl or modified phenyl, particularly methoxy-substituted phenyl.

[0084] In an embodiment of the first aspect of the present invention, the pyridinium or a derivative thereof is selected from the group consisting of bromopyridine, chloropyridine and methylpyridine.

[0085] In an embodiment of the first aspect of the present invention, the phosphonium or derivative thereof is -PR1R2R3, where R1, R2, and R3 can independently be alkyl or aryl. The alkyl can be 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. The aryl is phenyl or modified phenyl, particularly methoxy-substituted phenyl.

[0086] In an embodiment of the first aspect of the present invention, the compound of formula I, when A3 is ammonium and B1 or B5 is a coupling group Q, comprises the following structure, wherein the coupling group Q comprises a C atom separated from a C atom of the CA1A2A3 substituent by four single or double bonds and a C atom separated from a C atom of the CA1A2A3 substituent by five single or double bonds: [ka] [ka] [ka] (wherein R1, R2, and R3 are independently selected from alkyl or aryl). Alkyl may be selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, and cyclohexyl. Alkyl may include modified alkyl. Modified alkyl includes the structural unit alkyl-O-alkyl, such as -CH2-O-CH3, -CH2-O-CH2-CH3. Aryl is phenyl or modified phenyl, particularly methoxy-substituted phenyl. R4 is (CH2)nK, where n=0, 1, or 2, and K is selected from the group consisting of hydrazide, hydrazine, hydroxylamine, Br, F aromatics, 4-substituted 1,2,4-triazoline-3,5-diones (TADs), active esters, sulfonyl chlorides, and reactive carbonyls. B1, B2, B3, and B4 have the same meanings as defined above or below and / or in claim 1.

[0087] 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, B2 or B3 or B4 is Q.

[0088] In an embodiment of the first aspect of the present invention, A3 is ammonium, B1 or B5 is Q, and Q does not contain at least one atom selected from O, N, S, and Br. The term "Q does not contain at least one atom selected from O, N, S, and Br" means herein, in the context of the present disclosure, that Q does not contain O, N, S, or Br, or a combination thereof. In particular, Q does not contain O, N, S, or Br. In particular, when A3 is ammonium and B1 is Q, Q does not contain O, N, S, or Br. Alternatively, when A3 is ammonium and B5 is Q, Q does not contain O, N, S, or Br.

[0089] 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 an analyte; n is 0, 1, 2, 3, 4 or 5; X is a carbon atom of the phenyl group of formula I is bonded to X by

[0090] 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.

[0091] In an embodiment of the first aspect of the present invention, Q is linked to X via a covalent bond.

[0092] In an embodiment of the first aspect of the present invention, Q is selected from the group consisting of methyl hydrazide, methyl hydrazine, methylhydroxylamine, oxyamine, 4-methyl-oxy-1,2,4-triazoline-3,5-dione (CH2-O-TAD), 2,4-dinitro-5-fluoroaniline derivatives, chlorosulfonyl and methanesulfonyl chloride.

[0093] In an embodiment of the first aspect of the invention, carbon atom X of the phenyl group of formula I is a binding partner for B1, B2, B3, B4 or B5.

[0094] In an embodiment of the first aspect of the invention, the compound is selected from the following groups I-1, I-2, I-3, I-4 and I-5. [ka] [ka] [ka] [ka] [ka] (wherein A1, A2, A3, B1, B2, B3, B4, B5, Q and X have the above meanings.) Preferably, the compound comprises formula I-2, I-3 or I-4.

[0095] 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.

[0096] In an embodiment of the first aspect of the present invention, the reactive unit K forms a covalent bond with a 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 in the analyte molecule.

[0097] In an embodiment of the first aspect of the present invention, K may react with a carbonyl group, a phenol group, an amine, a hydroxyl group or a diene group of the analyte.

[0098] In an embodiment of the first aspect of the present invention, K is selected from the group consisting of hydrazide, hydrazine, hydroxylamine, Br, F aromatic, 4-substituted-1,2,4 triazoline-3,5-diones (TAD), active esters, sulfonyl chlorides and reactive carbonyls.

[0099] In an embodiment of the first aspect of the present invention, K is directly bonded to X (n=0).

[0100] In an embodiment of the first aspect of the present invention, K is linked to X via a methylene group (n=1).

[0101] In an embodiment of the first aspect of the present invention, K is linked to X via an ethylene group (n=2).

[0102] In an embodiment of the first aspect of the present invention, K is linked to X via a propylene group (n=3).

[0103] In an embodiment of the first aspect of the present invention, K is linked to X via a butylene group (n=4).

[0104] In an embodiment of the first aspect of the present invention, K is linked to X via a pentylene group (n=5).

[0105] 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 within the scope of common knowledge to determine which reactive unit K is suitable for binding to the functional group of the analyte of interest.

[0106] 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 contemplated 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 a reactive unit K of a compound of formula I.

[0107] 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.

[0108] 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.

[0109] In an embodiment of the first aspect of the present invention, the carbonyl group is an amide group, and those skilled in the art are familiar with the fact that, although an amide group itself is a stable group, the amide group can be hydrolyzed to a carboxylic acid group and an amino group. The 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 A.

[0110] 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 may be transferred to an intermediate imine group 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, 16-alpha-hydroxyestrone, 2-hydroxyestrone-3-methyl ether, prednisone, prednisolone, pregnenolone, progesterone, dehydroepiandrosterone (DHEA), 17-hydroxypregnenolone, 17-hydroxyprogesterone, androsterone, epiandrosterone, Δ4-androstenedione, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, allopregnanolone, and aldosterone.

[0111] In an embodiment of the first aspect of the present invention, the carbonyl group is a carboxy group. In an embodiment of the first aspect of the present invention, the carboxy group is reacted directly with a compound of formula I or converted to an activated ester group before reacting with a compound of formula I. In an embodiment of the first aspect of the present invention, the analyte molecule containing 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.

[0112] 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 may be transferred to an intermediate imine group 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 aldehyde groups is selected from the group consisting of pyridoxal, N-acetyl-D-glucosamine, alcaftadine, streptomycin, and josamycin.

[0113] 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, amyloxate, amylocaine, anileridine, alnidipine artesunate, and pethidine.

[0114] 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.

[0115] 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 containing one or more diene groups is selected from the group consisting of vitamin A, tretinoin, isotretinoin, alitretinoin, natamycin, sirolimus, amphotericin B, nystatin, everolimus, temsirolimus, and fidaxomicin.

[0116] 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 certain 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, alpha-hydroxyalprazolam, alpha-hydroxytriazolam, lorazepam, oxazepam, temazepam, ethyl glucuronide, ethylmorphine, morphine, morphine-3-glucuronide, buprenorphine, codeine, dihydrocodeine, p-hydroxypropoxyphene, O-desmethyltramadol, desmetadol, dihydroquinidine and quinidine.In an embodiment of the first aspect of the invention, wherein the analyte molecule comprises more than one hydroxyl group, the analyte is selected from the group consisting of vitamin C, glucosamine, mannitol, tetrahydropterin, cytarabine, azacytidine, ribavirin, floxuridine, gemcitabine, streptozotocin, adenosine, vidarabine, cladribine, estriol, trifluridine, clofarabine, nadolol, zanamivir, lactulose, adenosine monophosphate, idoxuridine, regadonosone, lincomycin, clindamycin, canaglyph Selected from the group consisting of rhodin, 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.

[0117] 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 succimer.

[0118] 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 pezinesatide. The selenium sulfide can be selenium disulfide (SeS2) or selenium hexasulfide (Se2S6).

[0119] 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.

[0120] 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 2The steroid or steroid-like compound has 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, estrogenic compounds, estrone (E1), estradiol (E2), 17a-estradiol, 17b-estradiol, estriol (E3), 16-epiestriol, 17-epiestriol, and 16,17-epiestriol and / or their metabolites. 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 The cannabinol may be selected from the group consisting of 4-hydroxyestradiol (4-OHE1), 2-hydroxyestradiol (2-OHE2), estrone (E1), estrone sulfate (E1), 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, and α and a can be used interchangeably.

[0121] 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-fluoromethcathinone, 3-fluoromethcathinone, 3-methyl ... Methionone, 4-methoxymethcathinone, 4-methylethotcathinone, 4-methylmethylmethcathinone, amfepramone, butyrone, etcathinone, elefehedron, methcathinone, methylone, methylenedioxypyrovalerone, benzoylecgonine, dehydronorketamine, ketamine, norketamine, methadone, normethadone, 6-acetylmorphine, diacetylmorphine, morphine, norhydrocodone, oxycodone, o Selected from the group consisting of ximorphone, phencyclidine, norpropoxyphene, amitriptyline, clomipramine, dothiepin, doxepin, imipramine, nortriptyline, trimipramine, fentanyl, glycylxylidide, lidocaine, monoethylglycylxylidide, N-acetylprocainamide, procainamide, pregabalin, 2-methylamino-1-(3,4-methylenedioxyphenyl)butane, N-methyl-1,3-benzodioxolylbutanamine, 2-amino-1-(3,4-methylenedioxyphenyl)butane, 1,3-benzodioxolylbutanamine, normeperidine, O-destramadol, desmetamadol, tramadol, lamotrigine, theophylline, amikacin, gentamicin, tobramycin, vancomycin, methotrexate, gabapentin, sisomicin, and 5-methylcytosine.

[0122] 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.

[0123] 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.

[0124] 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 is 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.

[0125] 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.

[0126] In an embodiment of the first aspect of the present invention, the reactive unit K is a carbonyl-reactive unit and can react with any type 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.

[0127] In an embodiment of the first aspect of the present invention, the carbonyl-reactive unit is selected from the group: (i) a hydrazine unit, e.g., 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, particularly 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, particularly 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.

[0128] In embodiments of the first 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 embodiments of the first 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.

[0129] In an embodiment of the first aspect of the present invention, the analyte molecule comprises a ketone or aldehyde group, K is a carbonyl reactive unit and is selected from the group: (i) a hydrazine unit, (ii) a hydrazide unit, (iii) a hydroxylamino unit, and (iv) Dithiol unit is selected from.

[0130] In an embodiment of the first aspect of the present invention, the reactive unit K is a diene-reactive unit capable of reacting with an analyte comprising a diene group.

[0131] In an embodiment of the first aspect of the present invention, the diene reactive unit is selected from the group consisting of Cockson-type reagents that can act as dienophiles, such as 1,2,4-triazoline-3,5-dione.

[0132] In an embodiment of the first aspect of the present invention, the reactive unit K is a hydroxyl-reactive unit capable of reacting with an analyte containing 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 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 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.

[0133] 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.

[0134] 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.

[0135] 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 active ester unit, such as 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 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), via a reaction (H. Ban et al., J. Am. Chem. Soc., 2010, 132(5), pp. 1523-1525), or by diazotization, or by ortho-nitration followed by reduction to an amine, which can then be reacted with an amine-reactive reagent. In an embodiment of the first aspect of the present invention, the phenol-reactive unit is fluoro-1-pyridinium.

[0136] In an embodiment of the first aspect of the present invention, the reactive unit K is an epoxide-reactive unit capable of reacting 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 NH-N / O molecule.

[0137] In an embodiment of the first aspect of the present invention, the epoxide reactive unit is selected from the group: (i) a hydrazine unit, e.g., 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, particularly 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, particularly 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.

[0138] In an embodiment of the first aspect of the present invention, the reactive unit K is a disulfide-reactive unit capable of reacting 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.

[0139] 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.

[0140] 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]

[0141] In an embodiment of the first aspect of the present invention, A3 is ammonium, B1 or B5 is K, and n is 3, 4 or 5.

[0142] In an embodiment of the first aspect of the present invention, A3 is ammonium, B1 or B5 is K, and K does not contain at least one atom selected from O, N, S, and Br. The term "K does not contain at least one atom selected from O, N, S, and Br" means, in the context of the present disclosure, that K does not contain O, N, S, Br, or a combination thereof. In particular, K does not contain O, N, S, or Br.

[0143] In an embodiment of the first aspect of the present invention, A3 is selected from the group consisting of pyridinium, trimethylammonium, phosphonium, triethylammonium, tripropylammonium, tributylammonium, dimethylethylammonium, methyldiethylammonium and trialkylammonium.

[0144] In an embodiment of the first aspect of the present invention, A3 is NR1R2R3 or PR4R5R6, where R1, R2, R3, R4, R5, and R6 are each independently selected from methyl, ethyl, propyl, substituted phenyl, unsubstituted phenyl, alkyl, modified alkyl, and short chain alkyl.

[0145] In particular, a short chain alkyl comprises 1, 2, 3, 4, 5 or 6 C atoms in the alkyl chain.

[0146] In an embodiment of the first aspect of the present invention, B1, B2, B3, B4, and B5 are each independently selected from OR7, NR8R9, H, and aliphatic, wherein R7, R8, and R9 are each independently selected from methyl, ethyl, propyl, substituted phenyl, unsubstituted phenyl, alkyl, modified alkyl, and short chain alkyl.

[0147] In an embodiment of the first aspect of the present invention, A1 and A2 are each independently selected from H, an aliphatic group, and an aromatic group.

[0148] In an embodiment of the first aspect of the present invention, A1 is H or CH3.

[0149] In an embodiment of the first aspect of the present invention, A2 is H or CH3.

[0150] In an embodiment of the first aspect of the present invention, A1 is H, A2 is H and A3 is pyridinium.

[0151] In an embodiment of the first aspect of the present invention, B3 is Q.

[0152] In an embodiment of the first aspect of the present invention, B2 is Q.

[0153] In an embodiment of the first aspect of the present invention, B4 is Q.

[0154] In an embodiment of the first aspect of the present invention, B1 is H or methoxy.

[0155] In an embodiment of the first aspect of the present invention, B2 is H or methoxy

[0156] In an embodiment of the first aspect of the present invention, B4 is H or methoxy.

[0157] In an embodiment of the first aspect of the present invention, B5 is H or methoxy.

[0158] In an embodiment of the first aspect of the present invention, Q comprises F.

[0159] In an embodiment of the first aspect of the present invention, n is 0.

[0160] In an embodiment of the first aspect of the present invention, n is 1.

[0161] In an embodiment of the first aspect of the present invention, n is 0 or 1 and K is hydrazide.

[0162] In an embodiment of the first aspect of the present invention, B3 is Q. Q is selected from the following group: hydrazine unit, methylhydrazine unit, ethylhydrazine unit, hydrazide unit, methylhydrazide unit, ethylhydrazide unit, hydroxylamino unit. Other substituents that are not Q are A1=H, A2=H, A3=methylpyridinium, B1=H, B2=H, B4=H and B5=H.

[0163] In an embodiment of the first aspect of the present invention, B3 is Q. Q is selected from the following group: hydrazine unit, methylhydrazine unit, ethylhydrazine unit, hydrazide unit, methylhydrazide unit, ethylhydrazide unit, hydroxylamino unit. Other substituents that are not Q are A1=H, A2=H, A3=methylpyridinium, B1=H, B2=H, B4=H and B5=methoxy.

[0164] In an embodiment of the first aspect of the present invention, B3 is Q. Q is selected from the following group: hydrazine unit, methylhydrazine unit, ethylhydrazine unit, hydrazide unit, methylhydrazide unit, ethylhydrazide unit, hydroxylamino unit. Other substituents that are not Q are A1=H, A2=H, A3=methylpyridinium, B1=methoxy, B2=H, B4=H and B5=H.

[0165] In an embodiment of the first aspect of the present invention, B3 is Q. Q is selected from the following group: hydrazine unit, methylhydrazine unit, ethylhydrazine unit, hydrazide unit, methylhydrazide unit, ethylhydrazide unit, hydroxylamino unit. Other substituents that are not Q are A1=H, A2=H, A3=methylpyridinium, B1=H, B2=methoxy, B4=H and B5=H.

[0166] In an embodiment of the first aspect of the present invention, B3 is Q. Q is selected from the following group: hydrazine unit, methylhydrazine unit, ethylhydrazine unit, hydrazide unit, methylhydrazide unit, ethylhydrazide unit, hydroxylamino unit. Other substituents that are not Q are A1=H, A2=H, A3=methylpyridinium, B1=methoxy, B2=H, B4=H and B5=methoxy.

[0167] In an embodiment of the first aspect of the present invention, B3 is Q. Q is selected from the following group: hydrazine unit, methylhydrazine unit, ethylhydrazine unit, hydrazide unit, methylhydrazide unit, ethylhydrazide unit, hydroxylamino unit. Other substituents that are not Q are A1 = H, A2 = H, A3 = methylpyridinium, B1 = methoxy, B2 = H, B4 = methoxy and B5 = methoxy.

[0168] In an embodiment of the first aspect of the present invention, B3 is Q. Q is selected from the following group: hydrazine unit, methylhydrazine unit, ethylhydrazine unit, hydrazide unit, methylhydrazide unit, ethylhydrazide unit, hydroxylamino unit. Other substituents that are not Q are A1=H, A2=H, A3=ammonium, B1=H, B2=H, B4=H and B5=H.

[0169] In an embodiment of the first aspect of the present invention, B3 is Q. Q is selected from the following group: hydrazine unit, methylhydrazine unit, ethylhydrazine unit, hydrazide unit, methylhydrazide unit, ethylhydrazide unit, hydroxylamino unit. Other substituents that are not Q are A1=H, A2=H, A3=ammonium, B1=H, B2=H, B4=H and B5=methoxy.

[0170] In an embodiment of the first aspect of the present invention, B4 is Q. Q is selected from the following group: hydrazine unit, methylhydrazine unit, ethylhydrazine unit, hydrazide unit, methylhydrazide unit, ethylhydrazide unit, hydroxylamino unit. Other substituents that are not Q are A1=H, A2=H, A3=ammonium, B1=methoxy, B2=H, B3=H and B5=H.

[0171] In an embodiment of the first aspect of the present invention, B4 is Q. Q is selected from the following group: hydrazine unit, methylhydrazine unit, ethylhydrazine unit, hydrazide unit, methylhydrazide unit, ethylhydrazide unit, hydroxylamino unit. Other substituents that are not Q are A1=H, A2=H, A3=ammonium, B1=H, B2=methoxy, B3=H and B5=H.

[0172] In an embodiment of the first aspect of the present invention, B3 is Q. Q is selected from the following group: hydrazine unit, methylhydrazine unit, ethylhydrazine unit, hydrazide unit, methylhydrazide unit, ethylhydrazide unit, hydroxylamino unit. Other substituents that are not Q are A1=H, A2=H, A3=ammonium, B1=methoxy, B2=H, B4=H and B5=methoxy.

[0173] In an embodiment of the first aspect of the present invention, B3 is Q. Q is CH2-Br, TAD or SO2Cl. Other substituents that are not Q are A1=H, A2=H, A3=methylpyridinium or ammonium, B1=H, B2=H, B4=H and B5=H.

[0174] In an embodiment of the first aspect of the present invention, the other substituents 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, ethoxy or methoxy.

[0175] In an embodiment of the first aspect of the invention, the compound is permanently positively charged.

[0176] In an embodiment of the first aspect of the invention, the compound is simply permanently positively charged.

[0177] In an embodiment of the first aspect of the invention, the compound is doubly or triply permanently positively charged.

[0178] In an embodiment of the first aspect of the present invention, the compound comprises a counterion for forming a salt, the counterion being preferably selected from the group consisting of Cl - , Br - , F - , formate, trifluoroacetate, PF6 - , sulfonates, phosphates, acetates.

[0179] In an embodiment of the first aspect of the invention, the compound is The following formula: [ka] Includes.

[0180] In an embodiment of the first aspect of the invention, the compound of formula I is selected from the following group (see Table 2): [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0181] Each of the above labels 1-25 can form a compound alone or in combination with a counterion.

[0182] Stabilization of the carbocation after neutral loss fragmentation at the benzylic position allows for low-energy neutral loss, providing a highly favorable fragmentation pathway that allows for high signal enhancement. In some embodiments, favorable LC elution characteristics can be observed, particularly with meta-substitution patterns (B2 or B4 is Q). Either one of the two E / Z stereoisomers that can form with hydrazides or oximes (e.g., testosterone labeling) is predominantly produced, or both isomers co-elute. This improves the signal-to-noise ratio, thereby improving sensitivity. Electron-donating substituents B1-B5 (e.g., OMe) allow for lower-energy fragmentation. Particularly high signal enhancement can be achieved with A1, A2 = H, B1-B5 = H, n = 1, and K = hydrazide (1). Aliphatic (e.g., methyl) and aromatic (e.g., phenyl or substituted analogs) modifications A1 and A2 at carbon atoms that bear a positive charge after neutral loss fragmentation allow fragmentation at low energies (e.g., 1 or 2: R1, R2 = Me, Ph).

[0183] In an embodiment of the first aspect of the present invention, each of A1 and A2, independently of one another, is H, aliphatic (e.g., Me) or aromatic (e.g., phenyl); A3 is NR'3 (R', e.g., Me), pyridine or PR''3 (R'', e.g., Me, Ph); and each of B1, B2, B3, B4, and B5, independently of one another, is OR', NR'2, H or aliphatic (e.g., Me). K is a conjugable group, e.g., hydrazide, hydrazine, hydroxylamine, bromo, PTAD, or TAD.

[0184] In an embodiment of the first aspect of the present invention, more than one of the substituents B1, B2, B3, B4, and B5 is a coupling group Q, for example, two, three, or four of the substituents B1, B2, B3, B4, and B5 are coupling groups Q. In other words, the compound of Formula I contains more than one Q, for example, a first Q, a second Q, or a third Q, and the Qs may be structurally different from each other or structurally identical to each other. The first, second, third, fourth, or fifth Q may be independently selected from the embodiments described above for Q.

[0185] 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.

[0186] In a third aspect, the present invention relates to a kit comprising a compound of formula I as disclosed in detail hereinbefore with respect to the first aspect of the invention, or a composition of the second aspect of the invention as disclosed in detail hereinbefore. 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.

[0187] 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 the compound of the first aspect of the invention. 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 characteristic of specific modifications of other molecules, substances internalized by living organisms, metabolites of such substances, and combinations thereof. All embodiments described for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention also apply to the fourth aspect of the invention, and vice versa.

[0188] In an embodiment of the fourth aspect of the present invention, the binding complex of formula III results from the formation of a covalent bond between 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.

[0189] In an embodiment of the fourth aspect of the invention, the binding compound has formula III [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 the other substituents A1, A2, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, modified alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, arylolyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, sulfur, isotopes or derivatives thereof; A3 comprises ammonium, pyridinium, phosphonium or a derivative thereof; Coupling group Q * comprises formula IV: [ka] (In the formula, n * is 0, 1, 2, 3, 4, or 5; The bound analyte is K * It is covalently bonded via X * is a carbon atom of the phenyl group of formula III, A3 is ammonium and B1 or B5 is a coupling group Q *When the coupling group Q * contains a C atom separated by four single or double bonds from the C atom of the CA1A2A3 substituent, and a coupling group Q * contains a C atom separated from the C atom of the CA1A2A3 substituent by five single or double bonds) Includes.

[0190] In an embodiment of the fourth aspect of the present invention, A3 is ammonium and B1 or B5 is Q * and Q * does not contain at least one atom selected from O, N, S, and Br. * The term "does not contain at least one atom selected from O, N, S, Br" is used herein to mean that Q * does not contain O or N or S or Br or combinations thereof.

[0191] In an embodiment of the fourth aspect of the present invention, each of A1, A2, A3, B1, B2, B3, B4, B5 of formula III has the same meaning as described above for the first aspect of the present invention (A1, A2, A3, B1, B2, B3, B4, B5 of formula I).

[0192] 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.

[0193] In an embodiment of the fourth aspect of the present invention, K * is due to 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.

[0194] Furthermore, it is also contemplated within the scope of the present invention that a functional group present on the analyte molecule is first converted into another readily available group by reaction with a reactive unit K of the compound of formula I.

[0195] In an embodiment of the fourth aspect of the present invention, the analyte is selected from the group consisting of a nucleic acid, an amino acid, a peptide, a protein, a metabolite, a hormone, a fatty acid, a lipid, a carbohydrate, a steroid, a ketosteroid, a secosteroid, a molecule characteristic of a particular modification of another molecule, a substance internalized by an organism, a metabolite of such a substance, and combinations thereof.

[0196] In an embodiment of the fourth aspect of the 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 epoxy 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.

[0197] 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.

[0198] All embodiments of the analyte mentioned for the first aspect of the invention apply to the fourth aspect of the invention and vice versa.

[0199] 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 said complex.

[0200] 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 described for 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 apply to the fifth aspect of the present invention, and vice versa.

[0201] 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.

[0202] 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 detection levels, particularly lower quantification levels.

[0203] In an embodiment of the fifth 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 molecule. The reactive unit K is capable of reacting with the 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 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 in the analyte molecule.

[0204] 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 within the scope of common knowledge to determine which reactive unit K is suitable for binding to the functional group of the analyte of interest.

[0205] What has been said above for analytes applies to analytes in the context of the fifth aspect of the invention.

[0206] 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 is 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.

[0207] In a sixth aspect, the present invention provides a method for mass spectrometric determination of an analyte, comprising: reacting the analyte with a compound of formula I as disclosed herein above with respect to the first aspect of the invention, thereby forming a complex as disclosed herein above with respect to the fourth aspect of the invention; (b) subjecting the complex from step (a) to mass spectrometry. The present invention relates to a method comprising:

[0208] In an embodiment of the fifth aspect of the present invention, the mass spectrometry step (b) comprises: (i) subjecting ions of the complex to a first stage of mass spectrometry, thereby characterizing the ions of the complex according to their mass / charge (m / z) ratio; (ii) causing fragmentation of the complex ion, thereby releasing a first component, particularly a first neutral component, particularly a low molecular weight neutral component, and generating daughter ions of the complex, the daughter ions having a different m / z ratio than the complex ion; (iii) subjecting the daughter ions of the complex to a second stage of mass spectrometry, thereby characterizing the daughter ions of the complex according to their m / z ratios. and / or (ii) may further comprise alternative fragmentation of the complex ion, whereby a second element, in particular a second neutral element different from the first element, is released and a second daughter ion of the complex is produced; and (iii) may further include subjecting the first daughter ion and the second daughter ion of the complex to a second stage of mass spectrometry, thereby characterizing the first and second daughter ions of the complex according to their m / z ratios.

[0209] In an embodiment of the sixth aspect of the present invention, the first entity and / or the second entity is selected from the group consisting of trimethylamine, pyridine, phosphine, trimethylamine, tripropylamine, tributylamine, dimethylethylamine, methyldiethylamine and trialkylamine.

[0210] 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 be familiar with which pretreatment method is suitable for which sample type. Those skilled in the art will also be familiar with which enrichment method is suitable for which analyte of interest.

[0211] 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.

[0212] 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, particularly in an amount of 0.5:1 to 20:1 mL of water / mL of sample, particularly 1:1 to 10:1 mL of water / mL of sample, particularly 2:1 to 5:1 mL of water / mL of sample.

[0213] 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 sample PT workflows, each of which involves the addition of an internal standard and an enzyme reagent, followed by a predetermined incubation period; the difference between the two workflows is the order in which the internal standard and enzyme reagent are added. The enzyme reagent is typically a reagent used for glucuronide cleavage or protein cleavage, or any pretreatment of the analyte or matrix. 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.

[0214] 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-preotease. 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.

[0215] In an embodiment of the sixth aspect of the invention, the sample is plasma or serum and is assigned to another predetermined PT workflow, which only involves the addition of an internal standard (ISTD) prior to a predetermined incubation time.

[0216] As described hereinabove or below, the incubation time and temperature to be selected for a contemplated sample treatment, chemical reaction, or method are well known to those skilled in the art. In particular, those skilled in the art will recognize 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, particularly 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.

[0217] Thus, step a) of this method embodiment is carried out following any of the sample pretreatment processes disclosed above.

[0218] 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 of 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 spectrometry in step b).

[0219] 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).

[0220] 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 target analyte(s) 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 using 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) involves 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.

[0221] In an embodiment of the sixth aspect of the present invention, the first enrichment workflow includes adding magnetic beads carrying a matrix-selective group to the pretreated sample. In an embodiment of the sixth aspect of the present invention, the addition of the magnetic beads includes 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.

[0222] 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.

[0223] 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.

[0224] 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 hereinbefore or hereinafter 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).

[0225] 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 directly to an LC station or after a dilution step by adding a diluent.

[0226] In an embodiment of the sixth aspect of the invention, matrix-specific magnetic beads are used and a compound of formula I as disclosed hereinbefore or hereinafter 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 directly to an LC station or after a dilution step by adding a diluent.

[0227] Therefore, in an embodiment of the sixth aspect of the present invention, in which the reaction between the compound of formula I and the analyte molecule in step a) is carried out after the first enrichment process, 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, is completed. Thus, the sample is first pretreated as described hereinabove, and then subjected to the 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 the second enrichment process before being subjected to mass spectrometry in step b).

[0228] In another embodiment of the sixth aspect of the present 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 present 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 present invention, the liquid chromatography is selected from the group consisting of HPLC, fast LC, micro LC, flow injection, and trap and elute.

[0229] In an embodiment of the sixth aspect of the present invention, step (a) of the method is carried out simultaneously with or after chromatographic separation. In an embodiment of the sixth aspect of the present invention, the compound of formula I is added to the column with the elution buffer. In another embodiment, the compound of formula I is added post-column.

[0230] 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.

[0231] Therefore, in an embodiment of the sixth aspect of the present invention, in which the reaction between the compound of formula I and the analyte molecule in step a) is carried out after the second enrichment process, the compound of formula I is added to the target sample after the second enrichment process using chromatography, particularly liquid chromatography, is completed.Therefore, in this case, the sample is first pretreated as described hereinabove, then subjected to the 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 spectrometry in step b).

[0232] 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 an analyte; the other substituents A1, A2, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, modified alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, arylolyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, sulfur, isotopes or derivatives thereof; A3 comprises ammonium, pyridinium, phosphonium or a derivative thereof; When A3 is ammonium and B1 or B5 is a coupling group Q, the coupling group Q contains a C atom separated by four single or double bonds from a C atom of the CA1A2A3 substituent, and the coupling group Q contains a C atom separated by five single or double bonds from a C atom of the CA1A2A3 substituent. Preferably, A3 is ammonium, B1 or B5 is a coupling group Q, and Q does not contain at least one atom selected from O, N, S, and Br.

[0233] 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, B1, B2, B3, B4 and B5, apply to compounds of formula V. In particular, compounds of formula V may be used for mass spectrometry determination. Alternatively, or in addition, compounds of formula V may be used in other technical fields, such as chemical labeling and drug delivery.

[0234] In an embodiment of the present invention, a clinical diagnostic system comprises a compound according to the first aspect of the present invention and / or a composition according to the second aspect of the present invention and / or a kit according to the third aspect of the present invention and / or a complex according to the fourth aspect of the present invention. Additionally or optionally, the compound according to the first aspect of the present invention is used for mass spectrometric determination of an analyte, and the clinical diagnostic system comprises mass spectrometric determination. Additionally or optionally, a method for mass spectrometric determination of an analyte according to the sixth aspect of the present invention is performed by the clinical diagnostic system.

[0235] 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 coupling 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 may be an analytical function, a pre-analytical or post-analytical function, or a function supporting either a pre-analytical, analytical, or post-analytical function. In particular, a module may be configured to cooperate with one or more other modules to perform a dedicated task of a sample processing workflow, e.g., by performing one or more pre-analytical and / or analytical and / or post-analytical steps. In particular, a clinical diagnostic system may comprise one or more analytical devices, each designed to perform a respective workflow optimized for a specific type of analysis, such as clinical chemistry, immunochemistry, coagulation, hematology, liquid chromatography separation, mass spectrometry, etc. Thus, a clinical diagnostic system may include one analytical device or any combination of such analytical devices with their respective workflows, and pre-analytical and / or post-analytical modules may be coupled to individual analytical devices or shared by multiple analytical devices. Alternatively, pre-analytical and / or post-analytical functions may be performed by units integrated into the analytical devices. Clinical diagnostic systems may include functional units such as liquid handling units 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. Clinical diagnostic systems may include a sample preparation station for automatically preparing samples containing analytes of interest, a liquid chromatography (LC) separation station including multiple LC channels, and / or a sample preparation / LC interface for inputting prepared samples into any 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 sequence 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. As used herein, the terms "automatically" and "automated" 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 special or customized meaning. The terms may specifically refer to, but are not limited to, processes performed entirely by at least one computer and / or computer network and / or machine, particularly without the need for manual action and / or user interaction.

[0236] A "sample preparation station" is a pre-analytical module coupled to one or more analytical devices or units within an analytical device, designed to perform a series of sample processing steps aimed at removing or at least reducing interfering matrix components in the sample and / or concentrating analytes 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, and storage.

[0237] A "liquid chromatography (LC) separation station" is an analytical device or module or unit within an analytical device designed to chromatographically separate a prepared sample, e.g., to separate analytes of interest from matrix components, e.g., remaining matrix components after sample preparation that may still interfere with subsequent detection, e.g., mass spectrometry detection, and / or to separate analytes of interest from each other to enable their individual detection. In one embodiment, the LC separation station is an intermediate analytical device or module or unit within an analytical device designed to prepare the sample for mass spectrometry analysis and / or to transfer the prepared sample to a mass spectrometer. In particular, the LC separation station is a multi-channel LC station containing multiple LC channels.

[0238] 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 order.

[0239] Clinical diagnostic systems make LC coupled to mass spectrometry more convenient and reliable, and therefore more suitable for clinical diagnostics. Random-access sample preparation and LC separation, combined with online coupling to mass spectrometry, enable high throughput (e.g., up to 100 samples per hour or more). Furthermore, the process can be fully automated, improving yield and reducing the required skill level.

[0240] In further embodiments, the present invention relates to the following aspects:

[0241] 1. For mass spectrometric determination of analytes, a compound 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; the other substituents A1, A2, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, modified alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, arylolyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, sulfur, isotopes or derivatives thereof; A3 comprises ammonium, pyridinium, phosphonium or a derivative thereof; When A3 is ammonium and B1 or B5 is a coupling group Q, the coupling group Q contains a C atom separated by four single or double bonds from a C atom of the CA1A2A3 substituent, and the coupling group Q contains a C atom separated by five single or double bonds from a C atom of the CA1A2A3 substituent. Compound.

[0242] 2. The coupling group Q has the following formula II: [ka] wherein K is a reactive unit capable of forming a covalent bond with an analyte; n is 0, 1, 2, 3, 4 or 5; X is a carbon atom of the phenyl group of formula I 2. The compound of embodiment 1, wherein X is attached to X by

[0243] 3. The compound of aspect 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.

[0244] 4. The compound of aspect 2 or 3, wherein K is selected from the group consisting of hydrazides, hydrazines, hydroxylamines, Br, F aromatics, 4-substituted 1,2,4-triazoline-3,5-diones (TADs), active esters, sulfonyl chlorides, and reactive carbonyls.

[0245] 5. The compound according to any one of aspects 1 to 4, wherein A3 is ammonium, B1 or B5 is Q, and Q does not contain at least one atom selected from O, N, S, and Br, and in particular Q does not contain O, N, S, or Br.

[0246] 6. The compound according to any one of aspects 1 to 5, wherein A3 is ammonium, B1 or B5 is K, and n is 3, 4, or 5.

[0247] 7. The compound according to any one of aspects 1 to 6, wherein A3 is ammonium, B1 or B5 is K, and K does not contain at least one atom selected from O, N, S, Br, and in particular K does not contain O, N, S or Br.

[0248] 8. The compound of any one of aspects 1-7, wherein Q is selected from the group consisting of methyl hydrazide, methyl hydrazine, methylhydroxylamine, oxyamine, 4-methyl-oxy-1,2,4-triazoline-3,5-dione (CH2-O-TAD), 2,4-dinitro-5-fluoroaniline derivatives, chlorosulfonyl, and methanesulfonyl chloride.

[0249] 9. The compound according to any one of aspects 1-8, wherein A3 is selected from the group consisting of pyridinium, trimethylammonium, phosphonium, triethylammonium, tripropylammonium, tributylammonium, dimethylethylammonium, methyldiethylammonium, and trialkylammonium.

[0250] 10.A3 is NR1R2R3 or PR4R5R6 and The compound of any one of aspects 1-9, wherein R1, R2, R3, R4, R5, and R6 are each independently selected from methyl, ethyl, propyl, substituted phenyl, unsubstituted phenyl, alkyl, modified alkyl, and short chain alkyl.

[0251] 11. B1, B2, B3, B4, and B5 are each independently selected from OR7, NR8R9, H, and aliphatic; The compound of any one of aspects 1-10, wherein R7, R8, and R9 are each independently selected from methyl, ethyl, propyl, substituted phenyl, unsubstituted phenyl, alkyl, modified alkyl, and short chain alkyl.

[0252] 12. The compound according to any one of aspects 1 to 11, wherein A1 and A2 are each independently selected from H, an aliphatic group, and an aromatic group.

[0253] 13. A compound according to any one of aspects 1-12, wherein A1 is H or CH3.

[0254] 14. A compound according to any one of aspects 1-13, wherein A2 is H or CH3.

[0255] 15. The compound according to any one of aspects 1-14, wherein A1 is H, A2 is H, and A3 is pyridinium.

[0256] 16. A compound according to any one of aspects 1-15, wherein B3 is Q.

[0257] 17. A compound according to any one of aspects 1-16, wherein B2 is Q.

[0258] 18. A compound according to any one of aspects 1-17, wherein B4 is Q.

[0259] 19. A compound according to any one of aspects 1-18, wherein B1 is H or methoxy.

[0260] 20. A compound according to any one of the preceding aspects, wherein B2 is H or methoxy.

[0261] 21. A compound according to any one of aspects 1-20, wherein B4 is H or methoxy.

[0262] 22. A compound according to any one of aspects 1-21, wherein B5 is H or methoxy.

[0263] 23. The compound according to any one of aspects 1-22, wherein Q comprises F.

[0264] 24. The compound according to any one of aspects 1 to 23, wherein n is 0.

[0265] 25. A compound according to any one of aspects 1 to 24, wherein n is 1.

[0266] 26. A compound according to any one of aspects 1 to 25, wherein n is 0 or 1 and K is hydrazide.

[0267] 27. The compound according to any one of aspects 1 to 26, wherein the other substituents 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, ethoxy, or methoxy.

[0268] 28. The compound according to any one of aspects 1-27, wherein the compound is permanently positively charged.

[0269] 29. The compound according to any one of aspects 1-28, wherein the compound is simply permanently positively charged.

[0270] 30. The compound according to any one of aspects 1-29, wherein the compound is doubly or triply permanently positively charged.

[0271] 31. The compound according to any one of aspects 1 to 30, wherein the compound comprises a counterion for forming a salt, the counterion being preferably selected from the group consisting of Cl - , Br - , F - , formate, trifluoroacetate, PF6 - , sulfonates, phosphates, acetates.

[0272] 32. The following formula [ka] 32. The compound of any one of aspects 1-31, comprising:

[0273] 33. A composition comprising a compound according to any one of aspects 1 to 32.

[0274] 34. A kit comprising a compound according to any one of aspects 1 to 32 or a composition according to aspect 33.

[0275] 35. 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 32.

[0276] 36. The binding compound has the formula III [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 the other substituents A1, A2, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, modified alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, arylolyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, sulfur, isotopes or derivatives thereof; A3 comprises ammonium, pyridinium, phosphonium or a derivative thereof; Coupling group Q * comprises formula IV: [ka] (In the formula, n * is 0, 1, 2, 3, 4, or 5; The bound analyte is K * It is covalently bonded via X * is a carbon atom of the phenyl group of formula III, A3 is ammonium and B1 or B5 is a coupling group Q * When the coupling group Q * contains a C atom separated by four single or double bonds from the C atom of the CA1A2A3 substituent, and a coupling group Q * contains a C atom separated from the C atom of the CA1A2A3 substituent by five single or double bonds) 36. The conjugate of embodiment 35, comprising:

[0277] 37. The conjugate according to aspect 35 or 36, 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 characteristic of a particular modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

[0278] 38. The conjugate of any one of aspects 35-37, wherein the binding compound is covalently bound via a carbonyl group, a hydroxyl group, or a diene group of the analyte to form said conjugate.

[0279] 39. Use of a compound according to any one of aspects 1 to 32 for the mass spectrometric determination of an analyte.

[0280] 40. Use of a compound according to aspect 39, wherein the mass spectrometric determination comprises a tandem mass spectrometric determination, in particular a triple quadrupole mass spectrometric determination.

[0281] 41. 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 32, thereby forming a complex as defined in any one of embodiments 35 to 38; (b) subjecting the complex from step (a) to mass spectrometry. A method comprising:

[0282] 42.The mass spectrometry step (b) is (i) subjecting ions of the complex to a first stage of mass spectrometry, thereby characterizing the ions of the complex according to their mass / charge (m / z) ratio; (ii) causing fragmentation of the complex ion, thereby releasing a first component, particularly a first neutral component, particularly a low molecular weight neutral component, and generating daughter ions of the complex, the daughter ions having a different m / z ratio than the complex ion; (iii) subjecting the daughter ions of the complex to a second stage of mass spectrometry, thereby characterizing the daughter ions of the complex according to their m / z ratios. and / or (ii) may further comprise alternative fragmentation of the complex ion, whereby a second element, in particular a second neutral element different from the first neutral element, is released and a second daughter ion of the complex is produced; and 42. The method of embodiment 41, wherein (iii) can further comprise subjecting the first daughter ion and the second daughter ion of the complex to a second stage of mass spectrometry, thereby characterizing the first and second daughter ions of the complex according to their m / z ratios.

[0283] 43. The method of embodiment 41 or 42, wherein the first entity and / or the second entity is selected from the group consisting of trimethylamine, pyridine, phosphine, trimethylamine, tripropylamine, tributylamine, dimethylethylamine, methyldiethylamine, and trialkylamine.

[0284] 44. 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 an analyte; the other substituents A1, A2, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, modified alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, arylolyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, sulfur, isotopes or derivatives thereof; A3 comprises ammonium, pyridinium, phosphonium or a derivative thereof; When A3 is ammonium and B1 or B5 is a coupling group Q, the coupling group Q contains a C atom separated by four single or double bonds from a C atom of the CA1A2A3 substituent, and the coupling group Q contains a C atom separated by five single or double bonds from a C atom of the CA1A2A3 substituent. Compound.

[0285] Example The following examples are offered to illustrate, but not to limit, the invention claimed herein.

[0286] Example 1: Synthesis of Labels 1 to 5 Label 1: Step 1: Synthesis of hydrazide derivatives containing a pyridinium group [ka] 587 mg (2 mmol) of methyl 4-(bromomethyl)phenylacetate, 10 ml of ethanol, and 324 ml (4 mmol) of pyridine were mixed and refluxed for 3 hours, and the mixture was concentrated under reduced pressure to give an oil.

[0287] Step 2: [ka] The oil from step 1 was dissolved in 8 ml of water, 0.9 ml of hydrazine hydrate (80% in HO) was added, and the reaction was refluxed for 70 minutes. The reaction mixture was concentrated under reduced pressure and purified by preparative RP HPLC (water to acetonitrile). The calculated LC-ESI+ m / z is 242.3. The experimentally determined m / z for Label 1 is 242.2. RP HPLC (reverse phase HPLC) is known to those skilled in the art and will therefore not be described in detail.

[0288] Labels 2-5 are synthesized accordingly as described for label 1. The calculated LC-ESI+ m / z values ​​and experimentally determined m / z values ​​for labels 2-5 are shown in Table 3. [Table 3]

[0289] Example 2: Synthesis of Labels 6-13 Label 6: Step 1: Synthesis of hydrazide derivatives bearing a trimethylammonium group [ka] 587 mg (2 mmol) of methyl 4-(bromomethyl)phenylacetate, 10 ml of ethanol, and 0.95 ml of trimethylamine (25% in H2O) were mixed and refluxed for 3 hours, and the mixture was concentrated under reduced pressure to give an oil.

[0290] Step 2: [ka] The oil from step 1 was dissolved in 8 ml of water, 0.9 ml of hydrazine hydrate (80% in HO) was added, and the reaction was refluxed for 70 minutes. The reaction mixture was concentrated under reduced pressure and purified by preparative RPHPLC (water to acetonitrile). LC-ESI+ m / z calculated: 222.3. The experimentally determined m / z for label 6 is 222.3.

[0291] Labels 7–13 are synthesized accordingly as described for label 6. The calculated LC-ESI+ m / z and experimentally determined m / z values ​​for labels 7–13 are shown in Table 4. [Table 4]

[0292] Example 3: Synthesis of Label 14: Synthesis of a hydroxylamine derivative having a trimethylammonium group Step 1: [ka] 1 g of 4-bromobenzyl bromide (4 mmol) was dissolved in 50 ml of THF and 1.5 equivalents of 25% aqueous trimethylamine was added. The mixture was stirred for 16 hours, and the resulting precipitate was collected by filtration and dried. LC-ESI+ m / z calculated: 229.1. Experimentally determined m / z: 230.2.

[0293] Step 2: [ka] 7.5 mg (11 mg) of allylpalladium(II) chloride dimer (20 μmol), 40 mg of tBuBrettPhos (2-(di-tert-butylphosphino)-2',4',6'-triisopropyl-3,6-dimethoxy-1,1'-biphenyl, 80 μmol), 490 mg of cesium carbonate (1.5 mmol), and 310 mg of the solid from step 1 (1 mmol) were dissolved in 2 ml of anhydrous toluene in a dry microwave vial under argon. 125 μl of ethyl acetohydroxamate (1.25 mmol) was added, and the reaction mixture was stirred at 65 °C in a sealed vial. The reaction mixture was then poured into 25 ml of methanol, filtered, and concentrated under reduced pressure. The resulting solid was dissolved in water / methanol and purified by RP HPLC (water to acetonitrile, reverse-phase HPLC). LC-ESI+ m / z calculated: 251.3. The experimentally determined m / z value is 251.3.

[0294] Step 3: [ka] 109 mg (329 μmol) of the solid from step 2 was dissolved in 2 ml of dioxane, 400 μl (2.35 mmol) of 6 M HCl in water was added, and the mixture was stirred at room temperature for 80 min. 9 ml of water was added, and the mixture was purified by RP HPLC (water to acetonitrile). LC-ESI+ m / z calculated: 181.3. Experimentally determined m / z: 181.3.

[0295] Example 4: Synthesis of Label 15 Step 1: Synthesis of [4-(3,5-dioxo-1,2,4 triazolidin-4-yl)phenyl]methyl-trimethyl-ammonium trifluoroacetate [ka] 4-[4-(hydroxymethyl)phenyl]-1,2,4-triazolidine-3,5-dione (0.39 mmol) (synthesized as previously reported in J. Chem. Soc., Chem. Commun. 1990, 191, 1416-1417) was dissolved in anhydrous DMF, followed by the addition of PPh (0.54 mmol) and CBr (0.58 mmol). After stirring at room temperature (rt) for 2 h, TMA (1 M solution in THF, 0.46 mmol) was added, and the solution was stirred at room temperature for 2 h. The solvent was removed in vacuo, and the residue was suspended in water and extracted with CHCl. ​​The aqueous phase was dried, and the crude product was purified by preparative HPLC. The pure product was obtained as a white solid (75 mg, 54% yield).

[0296] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA, 0% CH3CN 0.1% TFA; 0~40 min: 80% H2O 0.1% TFA, 20% CH3CN 0.1% TFA; 40~60 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 60~74 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 3.12 (s, 9 H) 4.56 (s, 2 H) 7.65-7.72 (m, 4 H). 13 C NMR (101 MHz, methanol-d4) δ ppm 51.68 (3 C) 68.41 (1 C) 126.24 (2 C) 127.23 (1 C) 133.25 (2 C) 134.14 (1 C) 153.66 (2 C). HPLC-MS (m / z) [M]+ calculated value 249.13460, found value 249.39.

[0297] Step 2: Synthesis of [4-(3,5-dioxo-1,2,4-triazol-4-yl)phenyl]methyl-trimethyl-ammonium trifluoroacetate [ka] 4-(3,5-Dioxo-1,2,4-triazolidin-4-yl)phenyl]methyl-trimethyl-ammonium trifluoroacetate (0.041 mmol) was dissolved in anhydrous CH3CN (500 μL) under argon. DBH (0.041 mmol) was added, and the red solution was stirred at room temperature in the dark for 10 minutes. The solvent was then removed in vacuo, and the residue was suspended in anhydrous CHCl2. The red solid was washed again with CHCl2 and dried. The solid was used without further purification and stored at -20 °C in the dark.

[0298] Example 5: Synthesis of Label 16: Synthesis of N-[4-[(dimethylamino)methyl]phenyl]-5-fluoro-2,4-dinitroaniline [ka] 4-[(Dimethylamino)methyl]aniline (0.58 mmol) was added to a solution of 1,5-difluoro-2,4-dinitrobenzene (0.35 mmol) in CHCN (1 mL). The resulting solution was stirred at room temperature overnight. The solvent was removed under vacuum, and the crude product was purified by chromatography (eluent: CHCl / MeOH 100:0 to 95:5). The product was obtained as a yellow oil (97 mg, 83% yield). 1 H NMR (400 MHz, chloroform-d) δ ppm 2.50 (s, 6 H), 3.77 (s, 2 H), 6.85 (d, J = 13.30 Hz, 1 H), 7.26-7.31 (m, 2 H), 7.52 (d, J = 8.03 Hz, 2 H), 9.17 (br d, J = 7.65 Hz, 1 H), 9.94 (br s, 1 H). HPLC-MS (m / z) [M+H]+ calculated value 335.11501, found value 335.36

[0299] The dimethylamino residue of the resulting product can be methylated to form label 16. Methylation can occur before or after complex formation with the analyte.

[0300] Example 6: Synthesis of Label 17: Synthesis of [4-(bromomethyl)phenyl]methanesulfonyl chloride [ka] 4-Methylbenzylsulfonyl chloride (2.59 mmol), DBH (1.81 mmol), and AIBN (0.26 mmol) were dissolved in CHCN. The solution was stirred at 60 °C for 18 h. The solvent was removed in vacuo, and the crude compound was purified by chromatography (eluent: hexane / EtOAc 100:0 to 90:10). The product was obtained as a white solid (50 mg). 1 H NMR (400 MHz, chloroform-d) δ ppm 4.49 (s, 2 H), 4.85 (s, 2 H), 7.38-7.55 (m, 4 H). 13C NMR (101 MHz, chloroform-d) δ ppm 32.11 (1 C) 70.36 (1 C) 126.15 (1 C) 129.82 (2 C) 131.76 (2 C) 140.09 (1 C).

[0301] Example 7: Synthesis of Label 18: Synthesis of Bromo Derivative with Pyridinium Group [ka] 1.6 g of alpha,alpha'-dibromo-p-xylol was dissolved in ethanol and pyridine was added at reflux for 2 hours. The mixture was then stirred for 30 minutes and the solvent was removed in vacuo. The crude material was digested twice with ether, and the remaining solid was dissolved in water and purified by RP HPLC (water to acetonitrile). LC-ESI+ m / z calculated: 263.2. Experimentally determined m / z: 263.1.

[0302] Example 8: Synthesis of Label 19: Synthesis of bromo derivative bearing trimethylammonium group [ka] 1.6 g of alpha,alpha'-dibromo-p-xylol was dissolved in 100 mL of THF and trimethylamine was added. The mixture was stirred at room temperature for 16 h, and the resulting precipitate was filtered and washed with ether. The crude material was purified by RP HPLC (water to acetonitrile). LC-ESI+ m / z calculated: 243.1. Experimentally determined m / z: 244.1.

[0303] Example 9: Synthesis of Label 20: Step 1: [ka] Under an argon atmosphere, α,α'-dibromo-p-xylene (2.27 mmol) was added to a solution of N-hydroxyphthalimide (2.06 mmol) in 10 mL of anhydrous THF. DIPEA (4.54 mmol) was then added dropwise, and the reaction mixture was refluxed for 24 hours. After cooling to room temperature, the reaction mixture was diluted with water and extracted with CHCl. ​​The organic phase was collected and dried under vacuum. The crude product was purified by chromatography (eluent: hexane / EtOAc 100:0 to 80:20). The product was obtained as a white solid (264 mg, 37% yield). 1 H NMR (400 MHz, chloroform-d) δ ppm 4.47 (s, 2 H), 5.19 (s, 2 H), 7.40 (d, J = 8.03 Hz, 2 H), 7.51 (d, J = 8.16 Hz, 2 H), 7.70-7.77 (m, 2 H), 7.77-7.84 (m, 2 H). 13 C NMR(101 MHz,chloroform-d)δ ppm 32.85(1 C)77.19(1 C)79.30(2 C)123.53(2 C)128.82(1 C)129.22(2 C)130.17(2 C)133.92(2 C)134.47(2 C)138.86(2 C)163.45(2 C). HPLC-MS (m / z) [M+H]+ calculated value 346.00733, found value 346.19

[0304] Step 2: Synthesis of [4-[(1,3-dioxoisoindolin-2-yl)oxymethyl]phenyl]methyl-trimethyl-ammonium bromide (Label 16) [ka] N-[[4-(bromomethyl)phenyl]methoxy]phthalimide (0.37 mmol) was dissolved in anhydrous CHCl (2.5 mL) and a TMA solution (1 M in THF, 0.45 mmol) was added. The reaction mixture was stirred at room temperature for 4 hours. The white solid was collected by filtration and washed with CHCl. ​​The product was obtained as a white solid (130 mg, 85% yield). 1H NMR(400 MHz,acetonitrile-d3)δ ppm 3.08(s,9 H)4.59(s,2 H)5.24(s,2 H)7.56-7.65(m,2 H)7.68(br d,J=8.03 Hz,2 H)7.83(s,4 H). 1 3 C NMR(101 MHz,acetonitrile-d3)δ ppm 52.29(3 C)68.33(2 C)78.83(2 C)123.15(2 C)128.71(2 C)128.95(2 C)130.28(2 C)133.12(2 C)134.76(2 C)136.96(2 C)163.45(2 C). HPLC-MS (m / z) [M]+ calculated 325.15467, found 325.35.

[0305] Step 3: Synthesis of [4-(aminooxymethyl)phenyl]methyl-trimethyl-ammonium chloride (Label 20) [ka] [4-[(1,3-Dioxoisoindolin-2-yl)oxymethyl]phenyl]methyl-trimethyl-ammonium bromide (0.30 mmol) was dissolved in CH3CN (2 mL). Aqueous KOH (0.33 mmol, 1 mL) was added, and the solution was refluxed for 4 h. Concentrated HCl (200 μL) was then added, and the solution was stirred at room temperature overnight. The solvent was removed in vacuo, and the crude compound was purified by preparative HPLC. The compound was obtained as a white solid (chloride salt after anion exchange, 23 mg, 34% yield).

[0306] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA, 0% CH3CN 0.1% TFA; 0~20 min: 100% H2O 0.1% TFA; 0% CH3CN 0.1% TFA. 20~24 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 24~34 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 34~40 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 40~50 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1H NMR (400 MHz, methanol-d4) δ ppm 3.11 (s, 9 H), 4.55 (s, 2 H), 5.09 (s, 2 H), 7.57-7.66 (m, 4 H). 1 3 C NMR (101 MHz, methanol-d4) δ ppm 53.27 (3 C) 69.95 (1 C) 77.29 (1 C) 130.42 (1 C) 131.07 (2 C) 134.62 (2 C) 137.33 (1 C). HPLC-MS (m / z) [M]+ calculated value 195.14919, found value 195.35.

[0307] Example 10: Synthesis of Label 21: [ka] Dibromo-p-xylol (1 mmol) and triphenylphosphine (0.5 mmol) were mixed in anhydrous toluene under an argon atmosphere and stirred for 90 min at 80° C. After cooling to room temperature, the product was filtered, washed with toluene and purified by RP HPLC (acetonitrile, water, RP = reverse phase). LC-ESI+ m / z (calculated) = 445.1; m / z (observed) = 445.2

[0308] Example 11: Synthesis of Label 22 [ka] Dibromo-p-xylol (1 mmol) and 500 μL of 0.1 M trimethylphosphine in THF were dissolved in 3 mL of toluene and stirred for 60 min at 80° C. After cooling to room temperature, the product was filtered, washed with toluene and purified by RP HPLC (acetonitrile, water). LC-ESI+ m / z (calculated) = 259.0; m / z (observed) = 259.1

[0309] Example 12: Synthesis of Label 25 [ka] Dibromo-p-xylol (1 mmol) and triphenylphosphine (0.5 mmol) were mixed in anhydrous toluene under Ar atmosphere and stirred for 90 min at 80° C. After cooling to room temperature, the product was filtered, washed with toluene and purified by RP (acetonitrile, water). LC-ESI+ m / z (calculated) = 535.1; m / z (observed) = 535.1

[0310] Preparation of label-analyte derivatives (conjugates) and their analysis by MS The labels referred to herein in this disclosure can be bound to an analyte, such as testosterone, vitamin D, estradiol, or a derivative thereof. This is exemplified as follows. The present invention is not limited to the following examples:

[0311] Example 13: Preparation of labeled testosterone derivatives (conjugates) and their analysis by MS Example 13.1: Preparation of labeled 7-testosterone derivatives [ka] 315 mg of Labeled 7 was dissolved in 13 mL of ethyl acetate and 5 mL of glacial acetic acid, testosterone was added, and the reaction was stirred at 40° C. for 30 min and then concentrated under reduced pressure at 40° C. The crude mixture was purified by RP HPLC (water to acetonitrile). LC-ESI+ m / z calculated is 478.7. Experimentally determined m / z is 278.4.

[0312] Accordingly, other combined complexes can be prepared, for example:

[0313] Example 13.2: Labeled 3-testosterone derivatives: [ka] LC-ESI+ m / z (calculated) = 498.7; m / z (observed = experimentally determined) = 498.3

[0314] Example 13.3: Labeled 10-Testosterone Derivatives [ka] LC-ESI+ m / z (calculated) = 508.7; m / z (observed) = 508.3

[0315] Example 13.4: Analytical derivatization of testosterone derivatives using labels 1-14 A 500 ng / ml solution of testosterone in methanol (S1) was prepared. Compared to a solution (S1) containing an excess of either derivatization reagent labeled 1-14 or 20, a solution (S2) diluted with methanol was added (molar ratio >1000) and 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 h and then at room temperature for 12 h. After 12 h, solution S3 was diluted with methanol to obtain five independent concentration levels based on the molecular weight of 1 equivalent of testosterone: 1 / 20 equivalent of testosterone; 1 / 40 equivalent of testosterone; 1 / 100 equivalent of testosterone; and 1 / 200 equivalent of testosterone. While 1 equivalent is still within the linear range of the detector, 1 / 200 equivalent of testosterone is chosen to be below the detection limit of the instrument used. For more sensitive instruments, the concentration was adjusted to match the appropriate system detection limit. As an example, the following concentrations were 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 by using solution S2.

[0316] After derivatization of the analyte molecule, isomers are introduced, which can often result in multiple peaks. The chromatographic behavior of these isomers needs to be addressed in a way that quantifies their properties relatively. The exemplary Figure 1 shows the signal distribution (%) over time applying a specific chromatographic method at 5% of the absolute peak height. The parameter "resolution" describes the ability of a chromatographic system to separate isomers resulting from the derivatization reaction of the analyte molecule. The retention time measured at the centroid of the isomer peak can represent the polarity of the resulting derivative when reversed-phase chromatographic materials are used for the separation. The areas A1 and A2 are the signal number * 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 are produced in equal amounts. If the labeling affects this ratio so that it is not equal to 50 / 50, mainly two isomers are produced. The signal intensity is therefore unevenly distributed, increasing the signal height relative to the unaffected background noise.

[0317] For each labeled substance, the respective testosterone derivatives were measured by electrospray ionization mass spectrometry after liquid chromatography separation, and various fragmentation scans with collision energies of 15V, 20V, 25V, 30V, 35V, and 40V were performed.

[0318] The mass signals of the molecular ion peak and the most abundant fragment ion peak (neutral loss or specific main fragment) were used to optimize fragmentation. Fragmentation energy is one of the most important tuning parameters when it comes to signal gain of charged molecules with neutral loss and / or fragmentation method units. The fragmentation energy is important because the molecule must not be fragmented during source and further ion path conditions. Cleavage of neutral loss fragments, or cleavage, should only occur in the collision cell (and / or related devices) to form the specific fragments of interest. If the collision energy needs to be very 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.

[0319] For all labeled substances, each testosterone derivative was tuned on a triple quadrupole mass spectrometer by injecting it into the mass spectrometer via liquid chromatography. The tuning parameters were the collision energy that produced the highest signal for each chromatographic peak in five independent collision energy experiments. The optimized MS parameters are shown in point (C) for general LC and MS parameters.

[0320] Chromatographic and MS parameters ● Polarity ES+ ● Calibration Static 2 ● Capillary (kV) 3.00 3.00 ● Corn (V) 50.00 53.36 ● Extractor (V)3.00 3.54 ● RF lens (V) 0.2 0.2 ● Sauce temperature (℃) 140 138 ● Desolvation temperature (℃)350 348 ● Cone gas flow rate (L / h) 50 49 ● Desolvation gas flow rate (L / h) 650 646 ● LM1 resolution 5.0 ● HM1 resolution 15.0 ● Ion Energy 1 1.0 ● Entrance 50-65 ● Collision 2-15 ● Exit 50-65 ● LM2 resolution 5.0 ● HM2 resolution 15.0 ● Ion Energy 2 1.0 ● Multiplier (V) 650-647 Syringe pump flow rate (uL / min) 40.0 ● Gas cell Pirani pressure (mbar) 1.87e-4 ● Device parameters-function 2: ● Polarity ES+ ● Calibration Static 2 ● Capillary (kV) 3.00 3.00 ● Corn (V) 50.00 53.36 ● Extractor (V)3.00 3.54 ● RF lens (V) 0.2 0.2 ● Sauce temperature (℃) 140 138 ● Desolvation temperature (℃)350 348 ● Cone gas flow rate (L / h) 50 49 ● Desolvation gas flow rate (L / h) 650 646 ● LM1 resolution 5.0 ● HM1 resolution 15.0 ● Ion Energy 1 1.0 ● Entrance 50-65 ● Collision 2-15 ● Exit 50-65 ● LM2 resolution 5.0 ● HM2 resolution 15.0 ● Ion Energy 2 1.0 ● Multiplier (V) 650-647 Syringe pump flow rate (uL / min) 40.0 ● Gas cell Pirani pressure (mbar) 1.87e-4 ● Equipment parameters-function 3: ● Polarity ES+ ● Calibration Static 2 ● Capillary (kV) 3.00 3.00 ● Corn (V) 50.00 53.36 ● Extractor (V)3.00 3.54 ● RF lens (V) 0.2 0.2 ● Sauce temperature (℃) 140 138 ● Desolvation temperature (℃)350 348 ● Cone gas flow rate (L / h) 50 49 ● Desolvation gas flow rate (L / h) 650 646 ● LM1 resolution 5.0 ● HM1 resolution 15.0 ● Ion Energy 1 1.0 ● Entrance 50-65 ● Collision 2-15 ● Exit 50-65 ● LM2 resolution 5.0 ● HM2 resolution 15.0 ● Ion Energy 2 1.0 ● Multiplier (V) 650-647 Syringe pump flow rate (uL / min) 40.0 ● Gas cell Pirani pressure (mbar) 1.87e-4 ● ACE experiment record ●----------------Method parameter execution----------- ● Waters Acquity SDS ● Driving time: 10.00 minutes ● Comments: ● Solvent Choice A: A1 = 0.1% formic acid in water ● Solvent option B: B1 = 0.1% formic acid in methanol ● Low pressure limit: 0.000 bar ● High pressure limit: 1034.200 bar ● Solvent name A: Water ● Solvent name B: Acetonitrile ● Switch 1: No change ● Switch 2: On ● Switch 3: No change ● Seal wash: 5.0 minutes ● Chart Out 1: System Pressure ● Chart Out 2:%B ● System pressure data channel: Yes ● Flow data channel: None ● %A Data Channel: None ● %B Data Channel: None ● 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 98.0 2.0 ● 2.7.000.40020.080.06 ● 3.7.100.4000.0100.06 ● 4.8.000.4000.0100.06 ● 5.8.100.40098.02.06 ● 6.10.000.40098.02.06 ● Event execution: Yes ● [Event Table] ● Execution time (minutes) Event operation parameters ● 1.0.10 Switch 2 on 0.00 ● 2.3.50 Switch 2 off 0.00 ● 3.9.00 Switch 2 on 0.00 ● Waters996 PDA ● Starting wavelength (nm) 210.00 ● End wavelength (nm) 400.00 ● Resolution (nm) 1.2 ● Sampling rate (spectrum / second) 2.000 ● Filter response 1 ● Exposure time (milliseconds) automatic ● Interpolate 656 available ● Acquisition stop time (min) 10.00 ● Save to disk: Yes ● Waters 996 PDA Analog Channel 1 ● Output mode off ● Waters 996 PDA Analog Channel 2 ● Output mode off ● ● Waters Acquity AutoSampler ● Driving time: 10.00 minutes ● Comments: ● Preload: Disabled ● Loop option: Partial loop with needle overfill ● LoopOffline: Disabled ● Weak cleaning solvent name: Methanol ● Weak cleaning volume: 600uL ● Strong cleaning solvent name: Methanol ● Strong cleaning volume: 200uL ● Target column temperature: 40.0℃ ● Column temperature alarm band: 20.0℃ ● Target sample temperature: 10.0℃ ● Sample temperature alarm band: 10℃ ● Full Loop Overfill Element: Automatic ● Syringe withdrawal speed: Automatic ● Needle placement: Automatic ● Pre-suction air gap: Automatic ● Air gap after suction: Automatic ● Column temperature data channel: Yes ● Ambient temperature data channel: Yes ● Sample temperature data channel: None ● Sample Organizer Temperature Data Channel: None ● Sample pressure data channel: None ● Switch 1: No change ● Switch 2: On ● Switch 3: No change ● Switch 4: No change ● Chart out: Sample pressure ● Sample Temperature Alarm: Enabled ● Column temperature alarm: Enabled ● Event execution: Yes ● [Event Table] ● Execution time (minutes) event operation ● 1.0.10 Switch 2 on ● 2.3.00 Switch 2 off ● 3.3.10 Switch 2 Toggle ● 4.8.00 Switch 2 Pulse ● 5.8.10 Switch 2 off ● 6.10.00 Switch 2 on ● Needle overfill flush: Automatic ● Sample analysis injection parameters ● Injection volume (uL)-10.00

[0321] From the linear calibration curves, the respective detection limits were obtained using the procedure described in DIN EN ISO 32645. The enhancement factor is calculated based on the detection limit (LOD) of the labeled analyte, e.g., labeled testosterone, compared to the underivatized analyte LOD, e.g., underivatized testosterone LOD. The workflow is shown in Figure 2. The results are shown in Table 5 below. [Table 5]

[0322] As can be seen from Table 5, all the presented derivatives or conjugates show signal enhancement compared to the underivatized analyte. Almost all derivatives of the new structures (except Label 2) outperform state-of-the-art derivatization agents, such as GirardT and Amplifex from Sciex.

[0323] Example 14: Preparation of labeled vitamin D derivatives (conjugates) and their analysis by MS Example 14.1: General Reaction of TADs (Triazolinediones) with Vitamin D [ka] 25-Hydroxyvitamin D monohydrate (0.024 mmol) and the TAD derivative (0.036 mmol) were dissolved in CH3CN. The solution was stirred at room temperature (rt) for 10 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 product was obtained as a solid.

[0324] Example 14.2: In situ activation of urazole to TAD reagent (Label 15) and reaction with vitamin D [ka] 25-Hydroxyvitamin D monohydrate (0.024 mmol) and [4-(3,5-dioxo-1,2,4 triazolidin-4-yl)phenyl]methyl-trimethyl-ammonium trifluoroacetate (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 under vacuum, and the residue was purified by preparative HPLC. The pure product was obtained as a white solid.

[0325] 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. 1H NMR (400 MHz, methanol-d4) δ ppm 0.52(s,3 H)0.87-1.01(m,4 H)1.15(s,6 H)1.19-1.47(m,12 H)1.65-1.78(m,4 H)1.83-1.92(m,2 H)1.95-2.08(m,4 H)2.15-2.30(m,2 H)2.86-2.94(m,1 H)3.12(s,9 H)4.06-3.86(m,2 H)4.12-4.23(m,1 H)4.56(s,2 H)5.01-5.11(m,1 H)7.62-7.74(m,4 H). The calculated LC-ESI+ m / z is 647.45308. The experimentally determined m / z is 647.49.

[0326] Example 14.3: Analytical derivatization of vitamin D using the exemplary labels above or below A 500 pg / ml solution of 25-hydroxyvitamin D3 in methanol (S1) was prepared. Horse serum was depleted with methanol (-20°C) using 1 mL of horse serum + 3 mL of 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 diluted with methanol (molar ratio >1000) was added to solution S3 containing an excess amount of derivatization reagent label (S4), 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 minutes. Solution S6 was diluted with methanol / HO + 0.1% formic acid to obtain the appropriate concentration level for quantification.

[0327] 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 limit of detection (LOD) of labeled vitamin D compared to the underivatized vitamin D LOD. The results for the conjugates are shown in Table 6 below. [Table 6]

[0328] All derivatives of vitamin D or complexes with vitamin D show significant signal enhancement. No isomers of the derivatives have been detected. Therefore, parameters of retention time, splitting, and peak width are not required and therefore are not presented in this disclosure.

[0329] In embodiments, the conjugate may be selected from the following group: [ka] [ka] [ka]

[0330] Example 15: Preparation of Labeled Estradiol Derivatives (Conjugates) and Their Analysis by MS Example 15.1: Reaction of [4-(3,5-dioxo-1,2,4 triazolidin-4-yl)phenyl]methyl-trimethyl-ammonium trifluoroacetate with estradiol (label 15 and a complex containing estradiol or a derivative thereof) [ka] [4-(3,5-Dioxo-1,2,4-triazolidin-4-yl)phenyl]methyl-trimethyl-ammonium trifluoroacetate (0.05 mmol) was dissolved in CH3CN (200 μL) and DBH (0.05 mmol) was added. The solution, which rapidly turned red, was stirred at room temperature for 10 minutes. This solution was then added dropwise to a solution of β-estradiol (0.05 mmol) in 1:1 CH3CN / phosphate buffer (1.2 mL). After stirring at room temperature for 30 minutes, the solvent was removed in vacuo. The crude product was purified by preparative HPLC, and two isomers, A and B, were isolated in a 3:1 ratio.

[0331] 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; 60~65 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 65~75 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 75~80 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 80~90 minutes: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA.

[0332] Isomer A: 1H NMR(400 MHz,methanol-d4)δ ppm 0.77(d,J=2.51 Hz,3 H)1.12-1.58(m,7 H)1.65-1.76(m,1 H)1.90-2.10(m,3 H)2.12-2.26(m,1 H)2.34(br d,J=12.92 Hz,1 H)2.71-2.84(m,1 H)2.86-2.96(m,1 H)3.13(s,9 H)3.61-3.70(m,1 H)4.57(s,2 H)6.78(d,J=8.53 Hz,1 H)7.31(d,J=8.66 Hz,1 H)7.67-7.73(m,2 H)7.73-7.79(m,2 H). HPLC-MS (m / z) [M]+ calculated value 519.29658, found value 519.45.

[0333] Isomer B: 1 H NMR(400 MHz, methanol-d4)δ ppm 0.78(s,3 H)1.15-1.58(m,7 H)1.65-1.77(m,2 H)1.86-2.09(m,2 H)2.13-2.22(m,1 H)2.25-2.33(m,1 H)2.82(br dd. HPLC-MS (m / z) [M]+ calculated value 519.29658, found value 519.33.

[0334] Example 15.2: Preparation of labeled 16-estradiol derivatives [ka] β-Estradiol (0.09 mmol), N-[4-[(dimethylamino)methyl]phenyl]-5-fluoro-2,4-dinitroaniline (0.11 mmol) and K2CO3 (0.18 mmol) were dissolved in anhydrous DMF (1 mL). After stirring at room temperature for 1 hour, MeI (0.14 mmol) was added, and the solution was stirred at room temperature overnight. The solution was acidified with 1 M HCl, and the solvent was removed under vacuum. The crude compound was purified by preparative HPLC to obtain a yellow solid. Preparative HPLC is known to those skilled in the art and therefore will not be described in detail.

[0335] HPLC method C-18 column: 0 min:80% H2O 0.1% TFA,20% 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; 74~80 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 80~90 minutes: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1H NMR(400 MHz, methanol-d4)δ ppm 0.78(s,3 H)1.17-1.45(m,6 H)1.47-1.58(m,1 H)1.63-1.78(m,1 H)1.84-1.94(m,1 H)1.94-2.11(m,2 H)2.11-2.18(m,1 H)2.23-2.38(m,1 H)2.82(br dd,J=8.41,4.14 Hz,2 H)3.05-3.12(m,9 H)3.67(t,J=8.60 Hz,1 H)4.47(s,2 H)6.56-6.64(m,1 H)6.82(d,J=2.64 Hz,1 H)6.86(dd,J=8.53,2.64 Hz,1 H)7.31(d,J=8.28 Hz,1 H)7.35-7.42(m,2 H)7.45-7.54(m,2 H)9.05(s,1 H). HPLC-MS (m / z) [M]+ calculated value 601.30206, found value 601.39.

[0336] Example 15.3: Preparation of labeled 17-estradiol derivatives [ka] N-(p-chlorosulfonylphenylmethyl)pyridinium bromide was synthesized as reported in WO2000009498.

[0337] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA, 0% 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; 74~80 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 80~90 minutes: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1 H NMR(400 MHz, methanol-d4)δ ppm 0.75(s,3 H)1.11-1.56(m,7 H)1.62-1.75(m,1 H)1.80-1.89(m,1 H)1.90-2.08(m,2 H)2.10-2.20(m,1 H)2.23-2.32(m,1 H)2.67-2.75(m,2 H)3.64(t,J=8.60 Hz,1 H)5.97(s,2 H)6.61-6.75(m,2 H)7.19(d,J=8.41 Hz,1 H)7.66(d,J=8.16 Hz,2 H)7.90(m,J=8.28 Hz,2 H)8.17(t,J=7.09 Hz,2 H)8.59-8.72(m,1 H)9.09(d,J=5.77 Hz,2 H). HPLC-MS (m / z) [M]+ calculated value 504.22030, found value 504.28.

[0338] Example 15.4: General Reaction of Estradiol with Sulfonyl Chloride Derivatization Reagents (e.g., Labels 23 and 24) [ka] β-Estradiol (0.07 mmol) and the sulfonyl chloride derivative (0.07 mmol) were dissolved in anhydrous CH3CN (2 mL). A solution of base (TMA or TEA, 0.21 mmol) was then added, and the solution was stirred overnight at room temperature. The solvent was removed under vacuum, and the crude compound was purified by preparative HPLC. The purified product was obtained as a solid.

[0339] Example 15.4.1: Preparation of Labeled 24-Estradiol Derivatives [ka]

[0340] 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; 64~74 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 74~80 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 80~90 minutes: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1 H NMR(400 MHz, methanol-d4)δ ppm 0.75(s,3 H)1.11-1.55(m,7 H)1.59-1.77(m,1 H)1.81-1.90(m,1 H)1.90-2.07(m,2 H)2.11-2.22(m,1 H)2.28(dq,J=13.32,3.59 Hz,1 H)2.70-2.78(m,2 H)3.13(s,9 H)3.64(t,J=8.66 Hz,1 H)4.63(s,2 H)6.69-6.74(m,2 H)7.22(d,J=9.16 Hz,1 H)7.80(d,J=8.41 Hz,2 H) 7.98 (d, J = 8.41 Hz, 2 H). HPLC-MS (m / z) [M]+ calculated value 484.25160, found value 484.31.

[0341] Example 15.4.2: Preparation of Labeled 23-Estradiol Derivatives [ka]

[0342] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA, 0% 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; 74~80 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 80~90 minutes: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1 H NMR(400 MHz, methanol-d4)δ ppm 0.77(s,3 H)1.15-1.58(m,8 H)1.64-1.77(m,1 H)1.85-2.10(m,4 H)2.24(br d,J=11.42 Hz,1 H)2.34(br dd,J=13.49,3.45 Hz,1 H)2.85(br dd,J=8.47,3.95 Hz,2 H)3.11(s,9 H)3.66(t,J=8.53 Hz,1 H)4.54(s,2 H)4.77(s,2 H)6.92(d,J=2.64 Hz,1 H)6.97(dd,J=8.47,2.70 Hz,1 H)7.33(d,J=8.41 Hz,1 H)7.56-7.63(m,2 H)7.63-7.69(m,2 H). HPLC-MS (m / z) [M]+ calculated value 498.26725, found value 498.42.

[0343] Example 15.5: Analytical derivatization of estradiol using labels 15, 17, 23, and 24 A 500 pg / mL solution of estradiol in acetonitrile (S1) was prepared. Horse serum was depleted with acetonitrile (-20°C) using 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. A solution containing excess derivatization reagent (labeled 10 or 12) diluted with methanol (molar ratio >1000) was added to solution S3 (S4). A 5 μg / mL solution of K2CO3 (S5) in acetonitrile / H2O 90 / 10 + 0.1% formic acid was prepared. Solutions S3, S4, and S5 were mixed to obtain solution S6, which was then stored at 50°C for 1 hour, followed by 12 hours at room temperature. Solution S6 was diluted with acetonitrile / H2O + 0.1% formic acid to obtain an appropriate concentration level for quantification.

[0344] From the linear calibration curve, the respective detection limits were obtained using the procedure described in DIN EN ISO 32645. The enhancement factors were calculated based on the labeled estradiol limit of detection (LOD) compared to the underivatized estradiol LOD. The results are shown in Table 7 below. [Table 7-1] [Table 7-2]

[0345] As can be seen from Table 7, all of the derivatives or conjugates presented show an enhanced signal compared to the underivatized analyte, in this case estradiol.

[0346] Example 16: Analysis of Labeled 20-Analyte Derivatives (Conjugates) and Analysis by MS The conjugate and label 20 are synthesized as previously described.

[0347] From the linear calibration curves, the respective detection limits were obtained using the procedure described in DIN EN ISO 32645. The enhancement factors are calculated based on the limit of detection (LOD) of the labeled analyte compared to the LOD of the underivatized analyte. The results are shown in Table 8 below. [Table 8]

[0348] As can be seen from Table 8, label 20 exhibits a 10-fold signal enhancement compared to the underivatized analyte.

[0349] The above compounds and exemplary labels can be combined with each appropriate analyte. The exemplary labels or exemplary analytes should not limit the scope of protection. Those skilled in the art know how to combine other disclosed analytes with the disclosed compounds to form the complexes of the present invention.

[0350] This patent application claims priority to European patent application 20175798.6, the contents of which are incorporated herein by reference.

Claims

1. for mass spectrometric determination of analytes, below: 【Chemistry 3-1】 【Chemistry 3-2】 A compound selected from:

2. The compound has the following formula: 【Chemistry 4】 2. The compound of claim 1, wherein the compound is labeled 6.

3. A composition comprising a compound according to claim 1 or 2.

4. A kit comprising a compound according to claim 1 or 2, or a composition according to claim 3.

5. 1. A conjugate for detecting an analyte using mass spectrometry, comprising a binding analyte and a binding compound covalently bound to each other, wherein said binding compound is: 【Chemistry 7-1】 【Chemistry 7-2】 A complex comprising a compound selected from

6. 3. Use of a compound according to claim 1 or 2 for mass spectrometric determination of said analyte, in particular said mass spectrometric determination comprising a tandem mass spectrometric determination, in particular a triple quadrupole mass spectrometric determination.

7. 1. A method for mass spectrometric determination of an analyte, comprising: (a) reacting the analyte with a compound of claim 1 or 2, thereby forming a complex of claim 5; and (b) subjecting the complex from step (a) to mass spectrometric analysis. In particular, the mass spectrometric analysis step (b) comprises (i) subjecting ions of said complex to a first stage of mass spectrometry analysis, whereby said ions of said complex are characterized according to their mass / charge (m / z) ratio; (ii) causing fragmentation of the ion of the complex, thereby releasing a first component, particularly a first neutral component, particularly a small neutral component, and generating daughter ions of the complex, the daughter ions having a different m / z ratio than the ion of the complex; and (iii) subjecting said daughter ions of said complex to a second stage of mass spectrometry, thereby characterizing said daughter ions of said complex according to their m / z ratios. and / or (ii) may further comprise alternative fragmentation of the ion of the complex, whereby a second element, in particular a second neutral element different from the first neutral element, is released and a second daughter ion of the complex is produced; (iii) may further comprise subjecting the first and second daughter ions of the complex to a second stage of mass spectrometry analysis, thereby characterizing the first and second daughter ions of the complex according to their m / z ratios. method.

Images