Reagents for mass spectrometry

Abstract

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

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to compounds suitable for use in mass spectrometry, compositions comprising said compounds, kits comprising said compositions and / or compounds and conjugates. Furthermore, the present invention relates to methods for the mass spectrometric determination of analytes using said compounds. [Background technology]

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

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

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

[0005] Several derivatization reagents (compounds) aimed at improving the sensitivity of the measurement of these analytes are known in the art. In particular, reagents containing a charged unit and a neutral loss unit combined in a single functional unit (e.g., WO 2011 / 091436) are known. Other reagents containing separate units are relatively bulky in structure and affect the general workflow of sample preparation and MS measurement (Rahimoff et al. (2017) J. Am. Chem. Soc. 139(30), pp. 10359-10364). Known derivatization reagents include, for example, dansyl chloride, RapiFluor-MS (RFMS), Cookson-type reagents, Amplifex Diene, Amplifex Keto, Girard T, Girard P, and pyridylamine (Hong and Wang, Anal Chem., 2007, 79(1):322-326; Frey et al., Steroids, 2016 December, 116:60-66; Francis et al., Journal of Pharmaceutical and Biomedical Analysis, 2005, 39(3-4), 411-417; Alley William, 28th International Carbohydrate Symposium, New Orleans, LA, United States, July 17-21 (2016), ICS-209). Others have described methods for placing a permanent charge (positive or negative) on the analyte of interest, allowing the analyte to be already ionized, thus avoiding the ionization step in the ion source (where most analyte losses occur). All of these often have disadvantages due to poor labeling efficiency, generation of structural isomers due to coupling chemistry, non-optimal ionization efficiency, disadvantages for post-coupling chromatographic separation, non-optimal fragmentation behavior due to multiple fragmentation pathways, and the need for high collision energy.

[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 sensitive detection of analytes from complex biological matrices. This is particularly important in random access, high-throughput MS setups where several different analytes with different chemical properties must be measured in a short time.

[0007] The present invention relates to novel reagents / compounds that enable sensitive quantification of analyte molecules such as steroids, proteins, and other types of analytes in biological samples. The reagents are designed in a modular fashion to allow for individual adaptation for specific needs arising from the measurement of specific analytes or for specific workflow adaptations.

[0008] An object of the present invention is to provide compounds, kits and compositions each containing the compound, for efficient detection of analytes by mass spectrometry. Furthermore, an object of the present invention is to provide complexes and methods for determination of analytes by mass spectrometry.

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

Summary of the Invention

[0010] Summary of the Invention In the following, the present invention relates to the following aspects: In a first aspect, the present invention relates to a compound for quantitative detection of an analyte using mass spectrometry-based determination, the compound contains a permanent charge, particularly a permanent net positive charge, and is capable of covalently bonding to an analyte, the compound has a mass m1 and a net charge z1, the compound is capable of forming at least one daughter ion having a mass m2 < m1 and a net charge z2 < z1 after fragmentation by mass spectrometry-based determination, m1 / z1 < m2 / z2.

[0011] In a second aspect, the present invention relates to a composition comprising a compound of the first aspect of the present invention.

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

[0013] In a fourth aspect, the present invention relates to a complex for the quantitative detection of an analyte using mass spectrometry-based determination, The complex is formed by an analyte and a compound covalently bonded to each other, and the complex contains a permanent charge, particularly a permanent net positive charge, The complex has a mass m3 and a net charge z3, The complex is capable of forming at least one daughter ion having a mass m4 < m3 and a net charge z4 < z3 after fragmentation by mass spectrometry-based determination, m3 / z3 < m4 / z4.

[0014] In a fifth aspect, the present invention relates to the use of a compound of the first aspect of the present invention for the mass spectrometry-based determination of an analyte.

[0015] In a sixth aspect, the present invention relates to a method for the mass spectrometry-based determination of an analyte, (a) reacting the analyte with a compound of the first aspect of the present invention, whereby a complex of the fourth aspect of the present invention is formed; (b) subjecting the complex from step (a) to mass spectrometry and comprising. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] [Figure 1]Schematic diagrams of MS spectra (intensity (%) vs. m / z) are shown, describing the fragmentation behavior of compounds or complexes of the present invention, particularly complexes containing doubly charged labeled analytes relative to precursors and fragmentation of singly (singly) charged analytes. The multiple charges can be or are each the resulting net charge. [Figure 2] Schematic representation of MS spectra (intensity (%) vs m / z) showing the fragmentation behavior of a singly charged comparative compound in a + / - 0.5 Dalton window (the window is represented between two dashed lines). [Figure 3] Schematic representation of MS spectra (intensity (%) vs m / z): Describing the fragmentation behavior of doubly charged compounds or complexes of the invention in a + / - 0.5 Dalton window (the window is represented between two dashed lines). [Figure 4-1] Figures 4A-4E show the lower limits of quantitation (LLOQ) of complexes containing labeled, doubly charged estradiol. Figure 4A shows the structure of the complex (estradiol conjugated with Label 6). Figure 4B shows the MS blank chromatogram at each mass transition (intensity (%) vs. retention time). Figure 4C shows the chromatogram at each mass transition (intensity (%) vs. retention time) of estradiol conjugated with Label 6 (relative to 0.5 pg / ml of estradiol). Figure 4D shows the calibration curve of estradiol conjugated with Label 6 in the concentration range of 0-0.01 ng / ml relative to estradiol. Figure 4E shows the calibration curve data for estradiol conjugated with Label 6 in the range of 0 ng / ml-0.05 ng / ml relative to estradiol. Absolute areas of three individual injections are shown, and the respective detection limits according to DIN 32645 are reported. NG means limit of detection, EG means limit of detection with 95% accuracy, and BG means limit of quantitation (LOQ). The LLOQ for native estradiol for the best mode / setting is approximately 5 ng / ml. [Figure 4-2]Figures 4A-4E show the lower limits of quantitation (LLOQ) of complexes containing labeled, doubly charged estradiol. Figure 4A shows the structure of the complex (estradiol conjugated with Label 6). Figure 4B shows the MS blank chromatogram at each mass transition (intensity (%) vs. retention time). Figure 4C shows the chromatogram at each mass transition (intensity (%) vs. retention time) of estradiol conjugated with Label 6 (relative to 0.5 pg / ml of estradiol). Figure 4D shows the calibration curve of estradiol conjugated with Label 6 in the concentration range of 0-0.01 ng / ml relative to estradiol. Figure 4E shows the calibration curve data for estradiol conjugated with Label 6 in the range of 0 ng / ml-0.05 ng / ml relative to estradiol. Absolute areas of three individual injections are shown, and the respective detection limits according to DIN 32645 are reported. NG means limit of detection, EG means limit of detection with 95% accuracy, and BG means limit of quantitation (LOQ). The LLOQ for native estradiol for the best mode / setting is approximately 5 ng / ml. [Figure 4-3]Figures 4A-4E show the lower limits of quantitation (LLOQ) of complexes containing labeled, doubly charged estradiol. Figure 4A shows the structure of the complex (estradiol conjugated with Label 6). Figure 4B shows the MS blank chromatogram at each mass transition (intensity (%) vs. retention time). Figure 4C shows the chromatogram at each mass transition (intensity (%) vs. retention time) of estradiol conjugated with Label 6 (relative to 0.5 pg / ml of estradiol). Figure 4D shows the calibration curve of estradiol conjugated with Label 6 in the concentration range of 0-0.01 ng / ml relative to estradiol. Figure 4E shows the calibration curve data for estradiol conjugated with Label 6 in the range of 0 ng / ml-0.05 ng / ml relative to estradiol. Absolute areas of three individual injections are shown, and the respective detection limits according to DIN 32645 are reported. NG means limit of detection, EG means limit of detection with 95% accuracy, and BG means limit of quantitation (LOQ). The LLOQ for native estradiol for the best mode / setting is approximately 5 ng / ml. [Figure 5] 5a-5c show the MS chromatogram of a blank injection and the plots of the mass transitions of RHA139F2, RHA171F2 and estradiol conjugated with label 6 (intensity (%) vs. retention time for the three embodiments), respectively. [Figure 6] FIG. 1 shows the MS spectrum (intensity (%) vs. m / z) of a doubly positively charged complex comprising estradiol or a fragment thereof as the binding analyte and estradiol or a fragment thereof conjugated with label 6 as the binding compound according to the present invention. [Figure 7] 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 8] FIG. 1 shows a schematic of the workflow for determining the enhancement factor compared to underivatized analyte. [Figure 9]9A and 9B show the signal quenching effect of TFA on doubly charged compounds / derivatives or their complexes. [Figure 10-1] 10A-10D show MS spectra (intensity (%) vs. m / z) of the fragmentation patterns of compounds and / or complexes. [Figure 10-2] 10A-10D show MS spectra (intensity (%) vs. m / z) of the fragmentation patterns of compounds and / or complexes. [Figure 10-3] 10A-10D show MS spectra (intensity (%) vs. m / z) of the fragmentation patterns of compounds and / or complexes. [Figure 10-4] 10A-10D show MS spectra (intensity (%) vs. m / z) of the fragmentation patterns of compounds and / or complexes. [Figure 11] FIG. 11 shows the MS spectra (intensity (%) vs. m / z) of the fragmentation patterns of the compounds and / or complexes. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Invention Before describing the present invention in detail below, it is to be understood that the present invention is not limited to the specific embodiments and examples described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0018] 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.

[0019] Each element of the present invention is described below. While these elements are listed with specific embodiments, it is understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application unless the context dictates otherwise.

[0020] definition It will be understood that the word "comprise", and variations such as "comprises" and "comprising", imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0021] 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.

[0022] Ratios, concentrations, amounts, and other numerical data may be expressed or presented herein in the form of a "range." It is understood that such range format is used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values ​​expressly recited as boundaries of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly recited. By way of example, a numerical range of "4% to 20%" should be interpreted not only to include the explicitly recited value 4% to 20%, but also to include each individual value and subrange within the stated range. Thus, this numerical range includes individual values ​​such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20%, etc., and subranges such as 4-10%, 5-15%, 10-20%, etc. This same principle applies to ranges reciting minimum or maximum values. Moreover, such interpretation should apply regardless of the breadth of the range or the properties described.

[0023] 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.

[0024] In the context of the present invention, the term "compound" refers to a chemical substance having a specific chemical structure. The compound may contain one or more reactive groups. Each reactive group can perform a different function, and two or more reactive groups can perform the same function. Reactive groups include, but are not limited to, reactive units, charged units, and neutral loss units. In the context of the present invention, the term "binding compound" refers to the compound that is bound to an analyte. In principle, the compound and the binding compound can be identical. The compound and the binding compound can be substantially identical. Substantially identical may mean that both compounds have the same chemical structure, except that the structure of the reactive unit K and / or the structure of the coupling group Q are different from each other. Preferably, the compound is capable of forming a bond to the analyte but is not yet bound to the analyte. The binding compound is bound to the analyte.

[0025] The term "mass spectrometry" ("Mass Spec" or "MS") or "mass spectrometric determination" or "mass spectrometry" refers to an analytical technique used to identify compounds by their mass. MS is a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m / z." MS techniques generally involve (1) ionizing compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating their mass-to-charge ratio. Compounds can be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. Generally, one or more molecules of interest are ionized, and the ions are then introduced into a mass spectrometry instrument, where a combination of magnetic and electric fields causes the ions to follow a path in space depending on their mass ("m") and charge ("z"). The term "ionization" or "ionizing" refers to the process of generating ions of an analyte having a net charge equal to one or more units. Anions have a net negative charge of one or more units, while cations have a net positive charge of one or more units. MS methods can be performed in either "negative ion mode," in which negative ions are generated and detected, or "positive ion mode," in which positive ions are generated and detected. "Following fragmentation as determined by mass spectrometry" can mean, for example, that a compound, composition, or complex has passed through a mass spectrometer and been fragmented.

[0026] "Tandem mass spectrometry" or "MS / MS" involves multiple steps of selective mass analysis, with analyte fragmentation occurring between steps. In a tandem mass spectrometer, ions are generated in an ion source and separated by mass-to-charge ratio in the first stage of mass analysis (MS1). Ions of specific mass-to-charge ratios (precursor ions or parent ions) are selected, and fragment ions (daughter ions) are generated by collision-induced dissociation, ion-molecule reactions, or photodissociation. The resulting ions are then separated and detected in the second stage of mass analysis (MS2).

[0027] Mass spectrometers separate and detect ions of slightly different masses, easily distinguishing between different isotopes of a given element. Mass spectrometry is therefore an important method for accurate mass determination and characterization of analytes, including, but not limited to, low molecular weight analytes, peptides, polypeptides, or proteins. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits, and functional interactions, and the total measurement of proteins in proteomics. De novo sequencing of peptides or proteins by mass spectrometry can usually be performed without prior knowledge of the amino acid sequence. Most sample workflows in MS further include a sample preparation and / or enrichment step, where, for example, the analyte of interest is separated from the matrix using gas or liquid chromatography. Typically, for a mass spectrometry measurement, the following three steps are performed:

[0028] 1. A sample containing an analyte of interest is ionized, usually by complexation with a cation, often by protonation to a cation. Ionization sources include, but are not limited to, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).

[0029] 2. The ions are sorted and separated according to their mass and charge. High field asymmetric waveform ion mobility spectrometry (FAIMS) can be used as an ion filter.

[0030] 3. The separated ions are detected, for example, in multiple reaction mode (MRM), and the results are displayed on a chart.

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

[0032] 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 sustained by an electrical discharge between the spray capillary and a counter electrode. The ions are then typically extracted into a mass analyzer using a set of differentially pumped skimmer stages. A counterflow of dry, preheated Ni gas may be used to improve solvent removal. Gas-phase ionization in APCI can be more effective than ESI for analyzing less polar entities.

[0033] "High-field asymmetric waveform ion mobility spectrometry (FAIMS)" is an atmospheric pressure ion mobility technique that separates gas-phase ions according to their behavior in strong and weak electric fields.

[0034] "Multiple reaction mode" or "MRM" is a detection mode of an MS instrument in which a precursor ion and one or more fragment ions are selectively detected.

[0035] 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.

[0036] In the context of this disclosure, the terms "analyte," "analyte molecule," or "analyte of interest" are used interchangeably to refer to chemical species analyzed by mass spectrometry. Chemical species, i.e., analytes, suitable for analysis by mass spectrometry can be any type of molecule present in a living organism, including, but not limited to, nucleic acids (e.g., DNA, mRNA, miRNA, rRNA, etc.), amino acids, peptides, proteins (e.g., cell surface receptors, cytoplasmic proteins, etc.), metabolites or hormones (e.g., testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (e.g., vitamin D), molecules characterized by a particular modification of another molecule (e.g., a sugar moiety or phosphoryl residue on a protein, a methyl residue on genomic DNA), or substances internalized by the organism (e.g., therapeutic drugs, drugs of abuse, toxins, etc.), or metabolites of such substances. Such analytes may serve as biomarkers. In the context of the present invention, the term "biomarker" refers to a substance in a biological system that is used as an indicator of the biological state of said system. In the context of the present invention, the term "bound analyte" refers to said analyte that is bound to a compound to form a complex. In principle, the analyte and the bound analyte can be identical. The analyte and the bound analyte can be substantially identical. Substantially identical may mean that both analytes have the same chemical structure, except that the structures of the functional groups differ from each other. Preferably, the analyte is capable of forming a bond to a compound but is not yet bound to the compound. The bound analyte is bound to the compound.

[0037] The term "permanent charge" or "permanently charged" is used in the context of this disclosure to mean that the charge of a unit, e.g., a positive or negative charge, is not readily reversible, e.g., by washing, dilution, filtration, etc. In particular, in context, this means that the permanent charges are not in equilibrium with their non-permanent charges. Permanent charges are formed by covalent bond formation, which is stable both in high pH environments and low pH environments (e.g., positive charge pH > 12 and negative charge pH < 1). Thus, each permanently charged molecule is either a strong base or a strong acid. In the case of a positive charge z = 1, the permanent charge exhibits a pKs value of pKs > 12, and in the case of a negative charge z = -1, pKs < 1. Permanent charges can be, for example, the result of covalent bonds. Reversible charges (non-permanent charges) can be, for example, the result of electrostatic interactions in contrast to permanent charges. The compounds of the present invention have a net charge z1, particularly prior to fragmentation. After fragmentation, the compound can be split or cleaved into at least one daughter ion. The daughter ion has a net charge z2, which is smaller than the net charge z1 (z2 < z1). The complexes of the present invention have a net charge z3, particularly prior to fragmentation. After fragmentation, the complex can be split or cleaved into at least one daughter ion having a net charge z4 (z4 < z3) that is smaller than the net charge z3. At least one daughter ion can, in this context, mean that more than one daughter ion is formed after fragmentation. One daughter ion and another daughter ion are distinguishable from each other at least by their mass, charge, or structure. In the context of this disclosure, by comparing the net charge z2 of the daughter ion with the net charge z1 of the compound, the absolute value of the net charge is exact. For example, if the compound has a double negative net charge z1 (z1 = -2) and the daughter ion has a single negative net charge z2 (z2 = -1), the absolute value of the net charge z1 (z1 = 2) is greater than the absolute value of the net charge z2 (z2 = 1), so the net charge z2 is smaller than the net charge z1 (z2 < z1). By comparing the net charges z1 and z2, the absolute value of the net charge is compared instead of the total value.

[0038] In the context of this disclosure, the term "permanent charge" or "permanent net charge" refers to pseudo-molecular ions, e.g., [M+ H] + or [M - H] - or [M+Na + ] + or [M+Cl - ] - Does not include etc.

[0039] The term "permanent net charge" or "net charge" is used in the context of this disclosure where permanent net charge is the total permanent charge possessed by an ion or molecule. Permanent net charge can be calculated as follows: Number of protons - Number of electrons = Permanent net charge. Permanent net charge can be viewed as the covalent combination of atoms that form charged moieties within a molecule through bond rearrangements (e.g., quaternary nitrogen, tetramethylammonium), and net charge also exists through the addition or abstraction of atoms, such as hydrogen, to form [M + H] + or [M - H] - -. For example, if a compound has two permanent positive charges and one permanent negative charge, the permanent net charge is +1 (2 * (+1)+(-1)=(+1)).

[0040] The term "the compound is capable of covalently binding to an analyte" means that the compound is suitable for binding to the analyte. The bond between the compound and the analyte is covalent.

[0041] The term "quantitative detection of an analyte using mass spectrometry determination" means that the amount of an analyte of interest is measured or determined by mass spectrometry.

[0042] The term "mass", e.g., m1, m2, m3, m4, or mx (x>4), refers to atomic mass, specifically unified atomic mass. The unit of unified atomic mass is u. In the biomedical field, the Dalton [Da] can be used instead of unified atomic mass [u]. The Dalton is not an SI unit. The Dalton is equivalent to unified atomic mass in that there is no conversion factor between these units. A "mass spectrum" is a two-dimensional representation of signal intensity (ordinate) versus m / z (abscissa). The position of the peak, usually called the signal, reflects the m / z of the ion generated from the compound, analyte, or combination thereof (complex) in the ion source. The intensity of this peak correlates with the abundance of that ion. Often, but not always, the peak with the highest m / z represents the intact ionized molecule, the molecular ion, M + The molecular ion peak usually results from the detection of a molecular ion. The molecular ion peak is usually accompanied by several peaks of lower or higher m / z caused by fragmentation of the compound, analyte, or complex to produce fragment ions. Thus, each peak in the mass spectrum can be referred to as a fragment ion peak or a daughter ion peak. m / z is by definition dimensionless.

[0043] The term "fragmentation" may refer to the dissociation of a compound, analyte, and / or complex into ions, e.g., at least one daughter ion, by passing the compound, analyte, and / or complex through the ionization chamber of a mass spectrometer. The fragments cause a unique pattern in a mass spectrum. The term "fragmentation" may refer to the dissociation of a single molecule into two or more separate molecules. As used herein, the term fragmentation refers to a specific fragmentation event in which the point of destruction of the parent molecule at which the fragmentation event occurs is well defined and the multiple daughter molecules resulting from the fragmentation event are well characterized. Methods for determining the points of destruction of the parent molecule and the resulting multiple daughter molecules are well known to those skilled in the art. The resulting daughter molecules may be stable or may dissociate during subsequent fragmentation events. To illustrate, if the parent molecule undergoing fragmentation contains an N-benzylpyridinium unit, one skilled in the art can determine, based on the overall structure of the molecule, whether the pyridinium unit will fragment to release a benzyl entity or be released entirely from the parent molecule, i.e., the resulting daughter molecule will be either a benzyl molecule or the parent molecule lacking the benzyl.Fragmentation can occur via collision-induced dissociation (CID), electron-capture dissociation (ECD), electron-transfer dissociation (ETD), negative electron-transfer dissociation (NETD), electron-detachment dissociation (EDD), photodissociation, particularly infrared multiphoton dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD), surface-induced dissociation (SID), higher-energy C-trap dissociation (HCD), and charge-remote fragmentation.

[0044] The term "m1 / z1 < m2 / z2" means that the mass-to-charge ratio (m1 / z1) of a compound is less than the mass-to-charge ratio (m2 / z2) of at least one or exactly one daughter ion of the compound.

[0045] The term "m3 / z3 < m4 / z4" means that the mass-to-charge ratio (m3 / z3) of a complex is less than the mass-to-charge ratio (m4 / z4) of at least one or exactly one daughter ion of the complex.

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

[0048] The analyte may be present in a sample of interest, e.g., a biological sample or a clinical sample. The terms "sample" or "sample of interest" are used interchangeably herein and refer to a portion or piece of a tissue, organ, or individual, and are usually smaller than such tissue, organ, or individual, which is intended to represent the entire tissue, organ, or individual. Upon analysis, the sample yields information regarding the state of the tissue, or the health or disease state of the organ or individual. Examples of samples include, but are not limited to, liquid samples such as blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva, and lymphatic fluid, or solid samples such as dried blood spots and tissue extracts. A further example of a sample is a cell culture or tissue culture.

[0049] A "covalent bond" or "covalently linked" or "covalently bonded" is at least one chemical bond involving the sharing of an electron pair between atoms or molecules, e.g., between a compound and an analyte.

[0050] The terms "unit" and "moiety" can be used interchangeably, for example, McLafferty fragmentation moiety and McLafferty fragmentation unit can be used interchangeably.

[0051] The numerical value of the charge, for example, 1, 2, 3, 4, 5, or 6, such as z1, z2, z3, z4, or zx (x>4), is the absolute value of the charge. For example, a net charge z1=2 can mean that the net charge z1 is +2, or that the net charge is -2. Preferably, the charge in this case is a positive numerical value, for example, 2=+2.

[0052] The terms "compound" and "label" can be used interchangeably.

[0053] The term "fragments" in the phrase "the compound can form further daughter ions, each of which comprises a fragment of the compound or fragments of the compound" means that the compound comprises one fragment or more than one fragments that are or can be different from each other. In particular, the further daughter ions are distinguished from each other by at least their mass, charge, or structure.

[0054] 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).

[0055] Prior to analysis by mass spectrometry, samples may be pretreated in a manner specific to the sample and / or analyte. In the context of the present disclosure, the term "pretreatment" refers to any measure necessary to enable subsequent analysis of the desired analyte by mass spectrometry. Pretreatment measures typically include, but are not limited to, elution of solid samples (e.g., elution of dried blood spots), addition of a hemolyzing reagent (HR) to whole blood samples, and addition of an enzyme reagent to urine samples. Also contemplated as sample pretreatment is the addition of an internal standard (ISTD).

[0056] The term "hemolytic reagent (HR)" refers to a reagent that lyses cells present in a sample, and in the context of the present invention, hemolytic reagent specifically refers to a reagent that lyses cells present in a blood sample, including but not limited to red blood cells present in a whole blood sample. A well-known hemolytic reagent is water (HO). Further examples of hemolytic reagents include, but are not limited to, deionized water, hypertonic liquids (e.g., 8M urea), ionic liquids, and different surfactants.

[0057] Typically, an internal standard (ISTD) is a known quantity of a substance that exhibits similar properties to the analyte of interest when subjected to a mass spectrometric detection workflow (i.e., including any pretreatment, enrichment, and actual detection steps). The ISTD exhibits similar properties to the analyte of interest, but is clearly distinguishable from it. By way of example, during a chromatographic separation, such as gas or liquid chromatography, the ISTD has approximately the same retention time as the analyte of interest from the sample. Thus, both the analyte and the ISTD enter the mass spectrometer simultaneously. The ISTD, however, exhibits a different molecular weight than the analyte of interest from the sample. This allows ions from the ISTD and the analyte to be distinguished in mass spectrometry using their different mass-to-charge (m / z) ratios. Both are subjected to fragmentation to produce daughter ions. These daughter ions can be distinguished by their m / z ratios and their respective parent ions. As a result, the signals from the ISTD and the analyte can be determined and quantified separately. Because the ISTD is added in a known amount, the signal intensity of an analyte from the sample can be attributed to a specific quantitative amount of the analyte. Thus, the addition of the ISTD allows for relative comparison of the amount of analyte detected, allowing for unambiguous identification and quantification of the analyte of interest present in the sample when the analyte(s) reach the mass spectrometer. Typically, although not necessarily, the ISTD is an isotopically labeled variant of the analyte of interest (e.g., 2 H, 13 C, or 15 (including labels such as N).

[0058] In addition to pretreatment, the sample may also be subjected to one or more enrichment steps. In the context of the present disclosure, the term "first enrichment process" or "first enrichment workflow" refers to an enrichment step that occurs following sample pretreatment and provides a sample containing enriched analytes compared to the initial sample. The first enrichment workflow may include chemical precipitation (e.g., using acetonitrile) or the use of a solid phase. Suitable solid phases include, but are not limited to, solid phase extraction (SPE) cartridges and beads. The beads may be nonmagnetic, magnetic, or paramagnetic. The beads may be coated differently to be specific for the analyte of interest. The coating may vary depending on the intended application, i.e., the intended capture molecule. Those skilled in the art will be familiar with which coating is suitable for which analyte. The beads can be made of a variety of different materials. The beads may have various sizes and may include porous or non-porous surfaces.

[0059] In the context of the present disclosure, the term "second enrichment process" or "second enrichment workflow" refers to an enrichment process that occurs following sample pretreatment and a first enrichment process and provides a sample containing an analyte that is enriched relative to the initial sample and the sample after the first enrichment process.

[0060] The term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of the differential distribution of the chemical components as they flow around or over a stationary liquid or solid phase.

[0061] The terms "liquid chromatography" or "LC" refer to the process of selectively retarding one or more components of a fluid solution as the fluid permeates uniformly through a column or capillary passage of finely divided material. Retardation results from the distribution of mixture components between one or more stationary phase(s) and the bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal phase liquid chromatography (NPLC), while methods in which the stationary phase is less polar than the mobile phase (e.g., a water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) are called reversed phase liquid chromatography (RPLC).

[0062] "High-performance liquid chromatography" or "HPLC" refers to a method of liquid chromatography in which the degree of separation is increased by passing a mobile phase under pressure through a stationary phase, typically a densely packed column. Typically, the column is packed with a stationary phase composed of irregular or spherical particles, a porous monolithic layer, or a porous membrane. HPLC has historically been divided into two distinct subclasses based on the polarity of the mobile and stationary phases. Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal-phase liquid chromatography (NPLC), while the opposite (e.g., water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) is called reverse-phase liquid chromatography (RPLC). Micro-LC refers to HPLC methods using columns with narrow internal diameters, typically less than 1 mm, e.g., about 0.5 mm. "Ultra-high performance liquid chromatography" or "UHPLC" refers to an HPLC method using a pressure of 120 MPa (17,405 lbf / in2), or approximately 1200 atmospheres. Rapid LC refers to an LC method using a short column with an internal diameter as described above and a length of less than 2 cm, e.g., 1 cm, at the above flow rate and pressure (micro LC, UHPLC). A short rapid LC protocol involves trapping / washing / elution steps using a single analytical column, achieving LC in a very short time of less than 1 minute.

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

[0064] LC separations may be single-channel LC or multi-channel LC, comprising multiple LC channels arranged in parallel, in which analytes may be separated according to their polarity or log P value, size, or affinity, as commonly known to those skilled in the art.

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

[0066] In the context of the present disclosure, the term "complex" refers to a product produced by the reaction of a compound with an analyte molecule. This reaction results in the formation of a covalent bond between the compound and the analyte. Thus, the term complex refers to a covalently linked reaction product formed by the reaction of a compound with an analyte molecule.

[0067] A "kit" is any product (e.g., a package or container) that includes 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 performing the method of the present invention. Typically, the kit can further include carrier means compartmentalized to closely enclose and receive one or more container means, such as vials, tubes, etc. In particular, each of the container means comprises one of the distinct elements used in the method of the first aspect. The kit can further include one or more other reagents including, but not limited to, a reaction catalyst. The kit may further include one or more other containers containing additional materials including, but not limited to, buffers, diluents, filters, needles, syringes, and an accompanying document with instructions for use. A label can be presented on the container to indicate that the composition is for a particular use and can also indicate instructions regarding either in vivo use or in vitro use. Computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disc), or directly to a computer or data processing device. Additionally, the kit can include a standard amount of a biomarker as described elsewhere herein for calibration purposes.

[0068] Embodiment In a first aspect, the present invention relates to a compound or at least one compound for quantitatively detecting an analyte using a mass spectrometry determination, the compound comprising a permanent charge, particularly a permanent net positive charge, capable of covalently binding to the analyte, having a mass m1 and a net charge z1, and capable of forming at least one daughter ion having a mass m2 < m1 and a net charge z2 < z1 after fragmentation by a mass spectrometry determination, where m1 / z1 < m2 / z2.

[0069] The inventors have surprisingly found that the reagents (compounds) described herein that are capable of depositing multiple permanent charges (either x positive charges, y negative charges, or (xy) net positive and negative charges) exhibit fragmentation behavior into one or more fragments that carry information about the portion of the label and the portion of the analyte molecular ion after fragmentation. Thus, MS signal enhancement of an analyte can be important, for example, for low-abundance analytes.

[0070] For example, a doubly permanently charged molecule / compound has half the m / z value of a singly permanently charged ion of the same analyte (the analyte molecule before labeling). When this multiply permanently charged molecule, such as a doubly permanently charged compound, is fragmented into differently charged molecules, the fragmentation pathway of the label information proceeds from the precursor with a higher m / z value to the label information with a lower m / z value, and from the precursor with a lower m / z value to the analyte ion with a higher m / z value (see Figures 1 and 6).

[0071] By attaching a permanently charged label, the fragmentation behavior of the molecule alternates compared to the native molecule. After fragmentation of the analyte-labeled molecule in the MSMS process, the permanent charge remains on either the analyte molecule or the label. Therefore, the quantitation ions measured at high intensity can carry either molecular or label information after the fragmentation process. To obtain molecular information of the labeled structure, a further ion isolation process of the resulting fragments is required, which requires an MS3 instrument and further reduces the overall sensitivity of each scan. Since the resulting ions after the fragmentation process can be either label information ions or analyte information-containing ions, the concept of modifier and quantitator ions described herein cannot be implemented if only one permanent charge is attached to the complex or compound.

[0072] In an embodiment of the first aspect of the present invention, a compound for quantitatively detecting an analyte using mass spectrometry determination includes a permanent charge, particularly a permanent net positive charge, can covalently bond to the analyte, has a mass m1 and a net charge z1, and forms at least one daughter ion having a mass m2 < m1 and a net charge z2 < z1 after fragmentation by mass spectrometry determination, where m1 / z1 < m2 / z2.

[0073] In an embodiment of the first aspect and / or the sixth aspect of the present invention, the fragmentation is a one-step process. This may mean that the compound is directly fragmented into at least one daughter ion without an intermediate step of rearrangement or pseudomolecular ion species formation.

[0074] In an embodiment of the first aspect of the present invention, the compound includes at least two permanent charges, particularly at least two permanent net positive charges.

[0075] In an embodiment of the first aspect of the present invention, m1 / z1 is at least 60 or more than that. More than 60 means, in the case of C7H₂ON₂ 2+ it can mean 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75, for example 66. Instead of this, or in addition to this, m1 / z1 is at least 60, m2 / z2 is at least 70 or more than that, for example 74 in the case of C4H 12 N + Here it is 74. More than 70 can mean 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80. Further, each or both of m1 / z1 and m2 / z2 can be at most 1500, for example 1300, 1350, 1400, 1450 or 1500. In other words, m1 / z1 and / or m2 / z2 can be in the range of 60 to 1500 (including the boundaries).

[0076] In an embodiment of the first aspect of the present invention, the compound does not contain a sulfoxide unit (SO). In particular, the compound does not contain a sulfoxide unit (SO) and inhibits neutral loss, thereby enabling a preferred fragmentation method from a doubly charged derivative to a singly charged ion. Thus, for example, the fragmentation pathway from a doubly charged compound to a singly charged compound is easier for a compound without an SO unit compared to a compound having an SO unit.

[0077] In an embodiment of the first aspect of the present invention, z1 is an integer and is 2 or greater than 2. In particular, z1 is a positive integer. A positive integer can mean an integer rather than a fraction, for example, +2, +3, +4, +5. Alternatively, z1 is a negative integer, for example, -2, -3, -4, -5. Positive integers, for example, +2, +3, +4, +5, in this context, mean that when the compound is +2, it has a double positive net charge z1, and when it is +3, it has a triple positive net charge z1, etc. Negative integers, for example, -2, -3, -4, -5, in this context, mean that when the compound is -2, it has a double negative net charge z1, and when it is -3, it has a triple negative net charge z1, etc.

[0078] This means that in the context of the present disclosure, by comparing the net charge z2 of the daughter ion with the net charge z1 of the compound, the absolute value of the net charge is exact. For example, when the compound has a double negative net charge z1 (z1 = -2) and the daughter ion has a single negative net charge z2 (z2 = -1), the absolute value of the net charge z1 (z1 = 2) is greater than the absolute value of the net charge z2 (z2 = 1), so the net charge z2 is smaller than the net charge z1 (z2 < z1). By comparing the net charges z1 and z2, the absolute value of the net charge is compared instead of the total value.

[0079] In an embodiment of the first aspect of the invention, z1 is 2, 3, 4 or 5, preferably z1 is 2. In this context, 2 can have the meaning +2 (2=+2), 3 can have the meaning +3, 4 can have the meaning +4, and 5 can have the meaning +5. Alternatively, if the compound is negatively charged, 2 can have the meaning -2 (2=-2), 3 can have the meaning -3, 4 can have the meaning -4, and 5 can have the meaning -5.

[0080] In an embodiment of the first aspect of the present invention, z2 is smaller than z1. In particular, z2=z1-1, and preferably z2 is 1. In this context, 1 has the same meaning as +1 or -1.

[0081] In an embodiment of the first aspect of the present invention, each or both of z1 and z2 is a permanent charge, in particular a permanent net charge.

[0082] In an embodiment of the first aspect of the present invention, each or both of z1 and z2 is a permanent positive charge, in particular a permanent net charge.

[0083] In an embodiment of the first aspect of the present invention, each or both of z1 and z2 is a permanent negative charge, in particular a permanent net charge.

[0084] In an embodiment of the first aspect of the present invention, the net charge z1 is the sum of x times positive and y times negative permanent charges of the compound.

[0085] In an embodiment of the first aspect of the present invention, the net charge z2 is the sum of x times positive and y times negative permanent charges of at least one daughter ion.

[0086] In an embodiment of the first aspect of the present invention, the compound is capable of forming additional daughter ions different from the at least one daughter ion having mass m2 and net charge z2. The additional daughter ions can be one, two, three, four, five, six, or more than six daughter ions. Each of these daughter ions has a mass-to-noise m / z. The m / z values ​​of the additional daughter ions can generally be designated as mx / zx values ​​(x>4). For example, one additional daughter ion has an m5 / z5 value, two additional daughter ions have m5 / z5 and m6 / z6 values, three additional daughter ions have m5 / z5, m6 / z7, and m8 / z8 values, etc.

[0087] In an embodiment of the first aspect of the present invention, the m / z of the further daughter ion is smaller than m1 / z1. That is, the position of the peak of the further daughter ion in the mass spectrum is located to the left of the peak (parent ion) having the m1 / z1 value. In contrast, the position of the peak of at least one daughter ion having the m2 / z2 value is located to the right of the parent ion peak. The parent ion peak may also be called a base peak. Typically, the intensity of the base peak is normalized to a relative intensity of 100%. Normalization can be performed because the relative intensity is essentially independent of the absolute ion abundance recorded by the detector.

[0088] In an embodiment of the first aspect of the invention, the compound is capable of forming further daughter ions, each comprising a fragment or fragments of the compound, each having an mx / zx value x>4, and each of the mx / zx values ​​of the further daughter ions being less than the m1 / z1 value. In particular, the fragments of the compound differ from each other at least by their m / z values.

[0089] In an embodiment of the first aspect of the invention, the compound comprises at least three units Z1, Z2, Q and an optional further unit L1, which units are covalently linked to each other, Q is a reactive unit capable of forming a covalent bond with an analyte; Z1 is a charge unit comprising at least one permanently charged moiety, in particular a permanently positively charged moiety or a permanently negatively charged moiety; Z2 is a charge unit containing at least one permanently charged moiety, in particular a permanently positively charged moiety or a permanently negatively charged moiety; and L1 is a substituted or unsubstituted linker, in particular a fragmentation cleavable group, such as a McLafferty fragmentation moiety, a Retro Diels Alder fragmentation moiety, benzyl, or aliphatic; The net charge of the compound is greater than one.

[0090] In an embodiment of the first aspect of the invention, the compound comprises a reactive unit Q capable of reacting with an analyte molecule. The reactive unit Q is capable of reacting with the analyte molecule such that a covalent bond is formed between the compound and the analyte molecule. In an embodiment of the first aspect of the invention, the reactive unit Q forms a covalent bond with the compound. In particular, the covalent bond is formed between the reactive unit of the compound and a functional group present on the analyte molecule.

[0091] Depending on the functional groups present in the analyte molecule to be measured, a person skilled in the art will select an appropriate reactive unit Q for the compound. It is well known to determine which reactive unit Q is suitable for binding to the functional group of the analyte of interest.

[0092] 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 the reactive unit Q of the compound. Furthermore, it is also considered within the scope of the present invention that a functional group present on the analyte molecule is first converted into another group that is readily available for reaction with the reactive unit Q of the compound.

[0093] 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.

[0094] 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.

[0095] In an embodiment of the first aspect of the present invention, the carbonyl group is an amide group, and those skilled in the art will recognize that, although an amide group itself is a stable group, it can be hydrolyzed to a carboxylic acid group and an amino group. Hydrolysis of the amide group can be achieved via an acid / base catalysis reaction or an enzymatic process, both of which are well known to those skilled in the art. In an embodiment of the first aspect of the present invention in which the carbonyl group is a masked aldehyde group or a masked keto group, each group is either a hemiacetal group or an acetal group, particularly a cyclic hemiacetal group or an acetal group. In an embodiment of the first aspect of the present invention, the acetal group is converted to an aldehyde group or a keto group before reaction with the compound.

[0096] In an embodiment of the first aspect of the present invention, the carbonyl group is a keto group. In an embodiment of the first aspect of the present invention, the keto group can be transferred to an imine group of an intermediate before reacting with a reactive unit of a compound. In an embodiment of the first aspect of the present invention, the analyte molecule containing one or more keto groups is a ketosteroid. In certain embodiments of the first aspect of the present invention, the ketosteroid is selected from the group consisting of testosterone, epitestosterone, dihydrotestosterone (DHT), desoxymethyltestosterone (DMT), tetrahydrogestrinone (THG), aldosterone, estrone, 4-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone, 16-ketoestradiol, 16α-hydroxyestrone, 2-hydroxyestrone-3-methyl ether, prednisone, prednisolone, pregnenolone, progesterone, dehydroepiandrosterone (DHEA), 17-hydroxypregnenolone, 17-hydroxyprogesterone, androsterone, epiandrosterone, Δ4-androstenedione, 11-deoxycortisol, corticosterone, 21-deoxycortisol, 11-deoxycorticosterone, allopregnenolone, and aldosterone.

[0097] 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 carboxyl group is reacted directly with a compound or converted to an activated ester group prior to reaction with a compound. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more carboxyl groups is selected from the group consisting of Δ8-tetrahydrocannabinolic acid, benzoylecgonine, salicylic acid, 2-hydroxybenzoic acid, gabapentin, pregabalin, valproic acid, vancomycin, methotrexate, mycophenolic acid, montelukast, repaglinide, furosemide, telmisartan, gemfibrozil, diclofenac, ibuprofen, indomethacin, zomepirac, isoxepak, and penicillin. In 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.

[0098] In an embodiment of the first aspect of the present invention, the carbonyl group is an aldehyde group. In an embodiment of the first aspect of the present invention, the aldehyde group can be transferred to an imine group of the intermediate before reacting with the reactive unit of the compound. 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.

[0099] In an embodiment of the first aspect of the present invention, the carbonyl group is a carbonyl ester group. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more ester groups is selected from the group consisting of cocaine, heroin, Ritalin, aceclofenac, acetylcholine, amcinonide, amiloxate, amylocaine, anileridine, aranidipine, artesunate, and pethidine.

[0100] 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.

[0101] In an embodiment of the first aspect of the present invention, the analyte molecule comprises one or more diene groups, particularly conjugated diene groups, as functional groups. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more diene groups is a secosteroid. In an embodiment, the secosteroid is selected from the group consisting of cholecalciferol (vitamin D3), ergocalciferol (vitamin D2), calcifediol, calcitriol, tachysterol, lumisterol, and tacalcitol. In particular, the secosteroid is vitamin D, particularly vitamin D2 or D3, or a derivative thereof. In certain embodiments, the secosteroid is selected from the group consisting of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 (calcifediol), 3-epi-25-hydroxyvitamin D2, 3-epi-25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3 (calcitriol), 24,25-dihydroxyvitamin D2, and 24,25-dihydroxyvitamin D3. In embodiments of the first aspect of the invention, the analyte molecule comprising one or more diene groups is selected from the group consisting of vitamin A, tretinoin, isotretinoin, alitretinoin, natamycin, sirolimus, amphotericin B, nystatin, everolimus, temsirolimus, and fidaxomicin.

[0102] In embodiments of the first aspect of the present invention, the analyte molecule comprises one or more hydroxyl groups as functional groups. In embodiments of the first aspect of the present invention, the analyte molecule comprises a single hydroxyl group or two hydroxyl groups. In embodiments where multiple hydroxyl groups are present, the two hydroxyl groups (1,2-diol) may be located adjacent to each other or may be separated by one, two, or three C atoms (1,3-diol, 1,4-diol, 1,5-diol, respectively). In particular embodiments of the first aspect, the analyte molecule comprises a 1,2-diol group. In embodiments where only one hydroxyl group is present, the analyte is selected from the group consisting of primary alcohols, secondary alcohols, and tertiary alcohols. In embodiments of the first aspect of the invention wherein the analyte molecule comprises one or more hydroxyl groups, the analyte is selected from the group consisting of benzyl alcohol, menthol, L-carnitine, pyridoxine, metronidazole, isosorbide mononitrate, guaifenesin, clavulanic acid, miglitol, zalcitabine, isoprenaline, acyclovir, methocarbamol, tramadol, venlafaxine, atropine, clofedanol, α-hydroxyalprazolam, α-hydroxytriazolam, lorazepam, oxazepam, temazepam, ethyl glucuronide, ethylmorphine, morphine, morphine-3-glucuronide, buprenorphine, codeine, dihydrocodeine, p-hydroxypropoxyphene, O-desmethyltramadol, desmetramadol, dihydroquinidine, and quinidine. In an embodiment of the first aspect of the invention, wherein the analyte molecule comprises multiple hydroxyl groups, the analyte is selected from the group consisting of vitamin C, glucosamine, mannitol, tetrahydrobiopterin, cytarabine, azacytidine, ribavirin, floxuridine, gemcitabine, streptozotocin

[0103] , adenosine, vidarabine, cladribine, estriol, trifluridine, clofarabine, nadolol, zanamivir, lactulose, adenosine monophosphate, idoxuridine, regadenoson, lincomycin, clindamycin, canaglifodine, tobramycin, netilmicin, kanamycin, ticagrelor, epirubicin, doxorubicin, arbekacin, streptomycin, ouabain, amikacin, neomycin, framycetin, paromomycin, erythromycin, clarithromycin, azithromycin, vindesine, digitoxin, digoxin, metrizamide, acetyldigitoxin, deslanoside, fludarabine, clofarabine, gemcitabine, cytarabine, capecitabine, vidarabine, and plicamycin.

[0104] In an embodiment of the first aspect of the present invention, the analyte molecule comprises one or more thiol groups (including, but not limited to, alkylthiol and arylthiol groups) as functional groups. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more thiol groups is selected from the group consisting of thiomandelic acid, DL-captopril, DL-thiorphan, N-acetylcysteine, D-penicillamine, glutathione, L-cysteine, zofenoprilat, tiopronin, dimercaprol, and succinimer.

[0105] In an embodiment of the first aspect of the present invention, the analyte molecule comprises one or more disulfide groups as functional groups. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more disulfide groups is selected from the group consisting of glutathione disulfide, dipyrithione, selenium sulfide, disulfiram, lipoic acid, L-cystine, fursultiamine, octreotide, desmopressin, vapreotide, terlipressin, linaclotide, and peginesatide. The selenium sulfide can be selenium disulfide, SeS2, or selenium hexasulfide, Se2S6.

[0106] 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.

[0107] In an embodiment of the first aspect of the invention, the analyte molecule comprises one or more phenolic groups as functional groups. In a particular embodiment of the first aspect of the invention, the analyte molecule comprising one or more phenolic groups is a steroid or steroid-like compound. In an embodiment of the first aspect of the invention, the analyte molecule comprising one or more phenolic groups is sp 2A steroid or steroid-like compound having a hybridized A ring and an OH group at position 3 of the A ring. In certain embodiments of the first aspect of the invention, the steroid or steroid-like analyte molecule is selected from the group consisting of estrogen, an estrogenic compound, estrone (E1), estradiol (E2), 17a-estradiol, 17b-estradiol, estriol (E3), 16-epiestriol, 17-epiestriol, and 16,17-epiestriol, and / or metabolites thereof. In embodiments, the metabolite is estriol, 16-epiestriol (16-epiE3), 17-epiestriol (17-epiE3), 16,17-epiestriol (16,17-epiE3), 16-ketoestradiol (16-ketoE2), 16a-hydroxyestrone (16a-OHEl), 2-methoxyestrone (2-MeOEl), 4-methoxyestrone (4-MeOEl), 2-hydroxyestrone-3-methyl ether (3-MeOEl), 2-methoxyestradiol (2-MeOE2), 4-methoxyestradiol (4-MeOE2), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2-hydroxyestradiol (2-OHE2), estrone (E1), estrone sulfate (E2s), 17a-estradiol (E2a), 17b-estradiol (E2B), estradiol sulfate (E2S), equilin (EQ), 17a-dihydroequilin (EQa), 17b-dihydroequilin (EQb), equilenin (EN), 17-dihydroequilenin (ENa), 17α-dihydroequilenin, 17β-dihydroequilenin (ENb), Δ8,9-dehydroestrone (dE1), Δ8,9-dehydroestrone sulfate (dE1), Δ9-tetrahydrocannabinol, and mycophenolic acid. β and b can be used interchangeably. α and a can be used interchangeably.

[0108] In an embodiment of the first aspect of the present invention, the analyte molecule comprises an amine group as a functional group. In an embodiment of the first aspect of the present invention, the amine group is an alkylamine group or an arylamine group. In an embodiment of the first aspect of the present invention, the analyte molecule comprising one or more amine groups is selected from the group consisting of proteins and peptides. In an embodiment of the first aspect of the present invention, the analyte molecule comprising an amine group is selected from the group consisting of 3,4-methylenedioxyamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, 3,4-methylenedioxymethamphetamine, amphetamine, methamphetamine, N-methyl-1,3-benzodioxolylbutanamine, 7-aminoclonazepam, 7-aminoflunitrazepam, 3,4-dimethylmethcathinone, 3-fluoromethamine, 3-methyl-4-methyl-2-propanol, 3-methyl-2-propanol, 3-methyl-4 ... Cathinone, 4-methoxymethcathinone, 4-methylethcathinone, 4-methylmethcathinone, amfepramone, butyrone, etcathinone, flephedrone, methcathinone, methylone, methylenedioxypyrovalerone, benzoylecgonine, dehydronorketamine, ketamine, norketamine, methadone, normethadone, 6-acetylmorphine, diacetylmorphine, morphine, norhydrocodone, oxycodone, oxymol and 2-methyl-1-(3,4-methylenedioxyphenyl)butane, N-methyl-1,3-benzodioxolylbutanamine, 2-amino-1-(3,4-methylenedioxyphenyl)butane, 1,3-benzodioxolylbutanamine, normeperidine, O-destramadol, desmetramadol, tramadol, lamotrigine, theophylline, amikacin, gentamicin, tobramycin, vancomycin, methotrexate, gabapentin, sisomicin, and 5-methylcytosine.

[0109] 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.

[0110] 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.

[0111] Such analyte molecules may be present in biological or clinical samples such as bodily fluids, e.g., blood, serum, plasma, urine, saliva, cerebrospinal fluid, etc., tissue or cell extracts. In embodiments of the first aspect of the invention, the analyte molecule(s) are present in a biological or clinical sample selected from the group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid, and dried blood spots. In some embodiments of the first aspect of the invention, the analyte molecule may be present in a purified or partially purified sample, e.g., a sample that is a purified or partially purified protein mixture or extract.

[0112] In an embodiment of the first aspect of the present invention, the reactive unit Q 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.

[0113] In an embodiment of the first aspect of the present invention, the reactive unit Q is a carbonyl-reactive unit, which 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.

[0114] In an embodiment of the first aspect of the present invention, the carbonyl-reactive unit is selected from the group consisting of: (i) a hydrazine unit, such as a HN—NH— or HN—NR— unit, where R is aryl or C1-4 alkyl, particularly C1 or C2 alkyl, and optionally substituted; (ii) a hydrazide unit, particularly a carbohydrazide or sulfohydrazide, in particular a HN—NH—C(O)— or HN—NR—C(O)— unit, where R is aryl or C1-4 alkyl, in particular C1 or C2 alkyl, optionally substituted; (iii) a hydroxylamino unit, such as a HN—O— unit; (iv) dithiol units, in particular 1,2-dithiol or 1,3-dithiol units; is selected from.

[0115] 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.

[0116] In an embodiment of the first aspect of the present invention, the analyte molecule comprises a ketone or aldehyde group and Q is a carbonyl-reactive unit, which is selected from the group: (i) a hydrazine unit, (ii) a hydrazide unit, (iii) a hydroxylamino unit, and (iv) Dithiol unit is selected from.

[0117] In an embodiment of the first aspect of the present invention, the reactive unit Q is a diene-reactive unit, which is capable of reacting with an analyte having a diene group. In an embodiment of the first aspect of the present invention, the diene-reactive unit is selected from the group consisting of Cookson-type reagents that can act as dienophiles, such as 1,2,4-triazoline-3,5-dione.

[0118] In an embodiment of the first aspect of the present invention, the reactive unit Q is a hydroxyl-reactive unit capable of reacting with an analyte having a hydroxyl group. In an embodiment of the first aspect of the present invention, the hydroxyl-reactive unit is selected from the group consisting of sulfonyl chloride, activated carboxylic acid ester (NHS or imidazolide), and fluoroaromatic / heteroaromatic capable of nucleophilic substitution of fluorine (T. Higashi, J. Steroid Biochem. Mol. Biol. (September 2016) Vol. 162, pp. 57-69). In an embodiment of the first aspect of the present invention, the reactive unit Q 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.

[0119] 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.

[0120] 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.

[0121] In an embodiment of the first aspect of the present invention, the phenol-reactive unit reacts with a phenol group on an analyte molecule. In an embodiment of the first aspect of the present invention, the phenol-reactive unit is selected from the group consisting of an N-hydroxysuccinimide (NHS) ester or sulfo-NHS ester, a pentafluorophenyl ester, a carbonylimidazole ester, a squaric acid ester, a hydroxybenzotriazole (HOBt) ester, a 1-hydroxy-7-azabenzotriazole (HOAt) ester, and an active ester unit such as a sulfonyl chloride unit. The phenol group present on the analyte molecule can be reacted with a highly reactive electrophile such as a triazolinedione (e.g., TAD) by reaction (H. Ban et al., J. Am. Chem. Soc. (2010) 132(5):1523-1525), or by diazotization, or alternatively by ortho-nitration, followed by reduction to an amine, which can then react with an amine-reactive reagent. In an embodiment of the first aspect of the present invention, the phenol-reactive unit is fluoro-1-pyridinium.

[0122] In an embodiment of the first aspect of the present invention, the reactive unit Q is an epoxide-reactive unit, which can react with an analyte containing an epoxide group. In an embodiment of the first aspect of the present invention, the epoxide-reactive unit is selected from the group consisting of amino, thiol, and a supernucleophilic N atom, enhanced by an α-effect via the adjacent O or N atom NH2-N / O molecule. In an embodiment of the first aspect of the present invention, the epoxide-reactive unit is selected from the group consisting of: (i) a hydrazine unit, such as HN-NH- or HN-NR 1 -unit (wherein R 1 is an aryl, an aryl containing one or more heteroatoms, or C 1~4 alkyl, especially C1 or C2 alkyl, optionally including, for example, halo, hydroxyl, and / or C 1~3 substituted with alkoxy) and (ii) a hydrazide unit, in particular a carbohydrazide or sulfohydrazide unit, in particular HN—NH—C(O)— or HN—NR 2 -C(O)- units, (In the formula, R 2 is an aryl, an aryl containing one or more heteroatoms, or C 1~4 alkyl, especially C1 or C2 alkyl, optionally including, for example, halo, hydroxyl, and / or C 1~3 substituted with alkoxy) and (iii) a hydroxylamino unit, such as a H2N-O- unit.

[0123] In an embodiment of the first aspect of the present invention, the reactive unit Q is a disulfide-reactive unit, which can react with an analyte containing a disulfide group. In an embodiment of the first aspect of the present invention, the disulfide-reactive unit is selected from the group consisting of thiols. In a further embodiment, the disulfide groups can be reduced to the respective thiol groups and then reacted with the thiol-reactive unit Q.

[0124] In an embodiment of the first aspect of the present invention, the reactive unit Q is a thiol-reactive group or an amino-reactive group, such as an activated ester group, such as an N-hydroxysuccinimide (NHS) ester or sulfo-NHS ester, a hydroxybenzotriazole (HOBt) ester, or a 1-hydroxy-7-aquabenzotriazole (HOAt) ester group.

[0125] In an embodiment of the first aspect of the present invention, the reactive unit Q is selected from 4-substituted 1,2,4-triazoline-3,5-dione (TAD), 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) or fluoro-substituted pyridinium.

[0126] In an embodiment of the first aspect of the present invention, the reactive unit Q 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.

[0127] 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 Q of the corresponding functional group of the analyte is selected from the options listed in the right column of Table 1. [Table 1]

[0128] In an embodiment of the first aspect of the invention, the compound comprises two charge units, designated Z1 and Z2. Z1 is a charge unit comprising at least one permanently charged moiety, in particular a permanently positively charged moiety or a permanently negatively charged moiety. Z2 is a charge unit comprising at least one permanently charged moiety, in particular a permanently positively charged moiety or a permanently negatively charged moiety.

[0129] In an embodiment of the first aspect of the invention, the charged units Z1 and / or Z2 are permanently charged, in particular under neutral conditions, in particular at pH values ​​between 6 and 8.

[0130] In an embodiment of the first aspect of the present invention, each of the charge units Z1 and Z2 is (i) comprises or consists of at least one or exactly one positively charged moiety. Alternatively, each of the charged units Z1 and Z2 is (ii) comprises or consists of at least one or exactly one negatively charged moiety. The compound comprises a net charge z1 of 2 or more than 2, for example 3, 4 or 5.

[0131] In an embodiment of the first aspect of the present invention, Z1 and Z2 are separated from each other by at least one atom, for example a C atom.

[0132] In an embodiment of the first aspect of the present invention, the charged unit Z1 or the charged unit Z2 is a positively charged unit. In an embodiment of the first aspect of the present invention, the positively charged unit Z1 and / or Z2 is selected such that the resulting compound has a pKa of 10 or greater, more particularly a pKa of 12 or greater. In an embodiment of the first aspect of the present invention, the positively charged unit Z1 and / or Z2 is selected from the group consisting of primary, secondary, tertiary, or quaternary ammonium, sulfonium, imidazolium, pyridinium, or phosphonium. In a particular embodiment of the first aspect, the positively charged moiety is tri-methyl-ammonium, N,N-dimethyl-piperidinium, or N-alkyl-quinuclidinium.

[0133] In an embodiment of the first aspect of the present invention, the charged unit Z1 or the charged unit Z2 is a negatively charged unit. In an embodiment of the first aspect of the present invention, the negatively charged unit Z1 and / or Z2 is selected such that the resulting compound has a pKb of 10 or more, more particularly a pKb of 12 or more. In an embodiment of the first aspect of the present invention, the negatively charged unit Z1 and / or Z2 is selected from the group consisting of phosphate, sulfate, sulfonate, or carboxylate.

[0134] In an embodiment of the first aspect of the present invention, L1 is a substituted or unsubstituted linker, in particular a group cleavable by fragmentation, such as a McLafferty fragmentation moiety, a Retro Diels Alder fragmentation moiety, or an aliphatic. In an embodiment of the first aspect of the present invention, L1 is not protonatable. In an embodiment of the first aspect of the present invention, L1 comprises 3 to 30 C atoms, in particular 5 to 20 C atoms, in particular 8 to 16 C atoms. In an embodiment, L1 comprises one or more heteroatoms, in particular N, O, or S. In an embodiment of the first aspect of the present invention, L1 comprises at least 4 heteroatoms, in particular 5, 6, or 7 heteroatoms, in particular N and / or O. In an embodiment of the first aspect of the present invention, L1 comprises 5 heteroatoms, in particular 3 O atoms and 2 N atoms.

[0135] In an embodiment of the first aspect of the present invention, the linker L1 comprises 1 to 10 C atoms and optionally one or more heteroatoms.

[0136] In an embodiment of the first aspect of the present invention, the linker L1 is selected from the group consisting of a McLafferty fragmentation unit, a Retro Diels Alder unit, a neutral loss cleavage unit, a bond dissociation unit, an alpha cleavage unit and a charge site rearrangement unit.

[0137] The McLafferty fragmentation unit is a carbonyl compound containing at least one gamma hydrogen.

[0138] Retro Diels alder units are Diels-Alder reaction products.

[0139] In an embodiment of the first aspect of the present invention, the neutral loss cleavage unit releases at least one neutral component upon ionization. The neutral component is a low molecular weight neutral component, particularly in the range of 10 to 100 Da, particularly 20 to 80 Da, particularly 25 to 65 Da. In particular, the molecular weight of the neutral component is 100 Da or less, particularly 80 Da or less, particularly 70 Da or less, particularly 50 Da or less, particularly 30 Da or less.

[0140] In an embodiment of the first aspect of the present invention, the neutral component is selected from the group consisting of N2, NO, NO2, S2, SO, SO2, CO, and CO2. In a particular embodiment, the neutral component is N2.

[0141] In an embodiment of the first aspect of the invention, loss of a neutral component reduces the mass / charge ratio (m / z) by -28 Da (if N2 or CO is lost), -30 Da (if NO is lost), -44 Da (in case CO2 is lost), -46 Da (if NO2 is lost), -48 Da (if SO2 is lost), or -64 Da (if S2 or SO2 is lost).

[0142] In an embodiment of the first aspect of the present invention, one neutral component is released. In an embodiment of the first aspect of the present invention, two neutral components are released. In particular, the second released neutral component is different from the first released neutral component. The release of the second neutral component occurs simultaneously with or after the release of the first neutral component. In particular, the release of the second neutral component occurs simultaneously with the release of the first neutral component, i.e., both neutral components are released simultaneously, i.e., in one single fragmentation event.

[0143] In an embodiment of the first aspect of the present invention, the neutral loss cleavage unit comprises or consists of a cyclic moiety capable of fragmentation. In an embodiment of the first aspect of the present invention, the neutral loss cleavage unit comprises or consists of a heterocyclic moiety, particularly a 4-, 5-, or 6-membered heterocyclic moiety, particularly capable of fragmentation by a retrocycloaddition reaction. In an embodiment of the first aspect of the present invention, the neutral loss cleavage unit comprises or consists of a 5-membered heterocyclic moiety, particularly a 4-, 5-, or 6-membered heterocyclic moiety having at least two adjacent heteroatoms, particularly two adjacent N atoms. In an embodiment of the first aspect of the present invention, the neutral loss cleavage unit comprises or consists of a triazole, tetrazole, oxadiazole, or thiadiazole moiety, or a hydrogenated derivative thereof. In an embodiment of the first aspect of the present invention, the neutral loss cleavage unit comprises or consists of a 1,2,3-triazole, a 1,4,5-triazole, a 3,4,5-triazole moiety, or a 2,3,4,5-tetrazole or a 2,3,5,6-tetrazole moiety.

[0144] The bond-dissociating unit is, for example, a chemical bond that can dissociate in a charged species under mass spectrometry conditions.

[0145] An alpha cleavage is, for example, a carbon-carbon bond adjacent to a particular functional group.

[0146] Charge site rearrangement is when an electron from a bond adjacent to a charged atom moves to that atom, neutralizing the original charge and transferring it to a different site.

[0147] In an embodiment of the first aspect of the present invention, Q is covalently bonded to Z1 or Z2. In particular, Q is directly bonded to Z1 or directly bonded to Z2.

[0148] In an embodiment of the first aspect of the present invention, L1 covalently links Z1 and Z2. In particular, L1 is directly linked to Z1 and directly linked to Z2 according to the following formula: Z1-L1-Z2.

[0149] In an embodiment of the first aspect of the present invention, the compound is fragmented between Z1 and Z2. In particular, the compound is fragmented by cleavage of the linker L1.

[0150] In an embodiment of the first aspect of the present invention, the compound has the following formulae 1-I to 1-III: (Z1-L1-Z2-Q) N( N≧1)(1-I), (Q-Z1-L1-Z2) N (N≧1)(1-II), (Z1-Z2-Q) N (N≧1)(1-III).

[0151] In this context, "N" refers to the net charge of the compound.

[0152] In the context of the disclosure below and / or above, "N≧1" means that N is greater than or equal to +1 (plus one). Additionally or alternatively, "N≧1" means that N is greater than or equal to −1 (minus one).

[0153] In an embodiment of the first aspect of the present invention, the compound comprises Formula 1-I and is selected from the group: [ka] is selected from.

[0154] Examples of these embodiments are doubly charged, especially doubly positively charged.

[0155] In an embodiment of the first aspect of the present invention, Z1 and Z2 each independently comprise a chelate complex, a phosphonium, an ammonium, a carbenium, a pyridinium, a sulfonium, a 3- to 10-membered charged heterocycle containing any of N, S, H, and C atoms, or a derivative thereof.

[0156] In an embodiment of the first aspect of the present invention, the compound has the formula 1-IV: (Z1-(L1-Z2) M -(L2-Zo)K -Q) N Including, During the ceremony, Zo is a charge unit, including in particular a positively or negatively or neutrally charged unit, L2 is a substituted or unsubstituted linker, in particular L2 is L1 or L2 is a fragmentation cleavable group; M>0, K>0 and N≧1. L1, Z1, Z2 and Q have the same meanings as above.

[0157] In this context, "N" refers to the net charge of the compound. In particular, if Zo=0 then N=M+1(-1), and if Zo is charged then N=M+1(-1)-K.

[0158] In embodiments of the first aspect of the present invention, Zo is a charged unit. Zo can be selected from the group consisting of primary, secondary, tertiary, or quaternary ammonium, carbenium, sulfonium, imidazolium, pyridinium, a 3- to 10-membered charged heterocycle containing N, S, H, and C atoms or derivatives thereof, a chelate complex, or a phosphonium. In certain embodiments of the first aspect, the positively charged moiety is tri-methyl-ammonium, N,N-dimethyl-piperidinium, or N-alkyl-quinuclidinium.

[0159] In an embodiment of the first aspect of the present invention, L2 is a substituted or unsubstituted linker. L2 is a fragmentation cleavable group, for example, a McLafferty fragmentation moiety, a Retro Diels Alder fragmentation moiety, benzyl, or aliphatic.

[0160] In an embodiment of the first aspect of the present invention, M is 0, 1, 2, 3, 4 or 5.

[0161] In an embodiment of the first aspect of the present invention, K is 0, 1, 2, 3, 4 or 5.

[0162] In an embodiment of the first aspect of the present invention, the compound comprises Formula 1-IV (M=1 and K=1). For example, the compound may be any of the following groups: [ka] is selected from.

[0163] In an embodiment of the first aspect of the present invention, the compound has the formula 1-V: (Z1-(L3-Z2) M -(L2-Zo) K -Q) N Including, During the ceremony, Zo is a charge unit, in particular comprising either a positive or negative or neutral charge unit, in particular comprising either a positive or negative charge unit; L2 is a substituted or unsubstituted linker, in particular L2 is L1 or L2 is a fragmentation cleavable group; L3 is a substituted or unsubstituted linker, in particular a non-cleavable group by fragmentation, such as benzyl or diazo; M>0, K>0 and N≧1. L1, Z1, Z2 and Q have the same meanings as above.

[0164] In an embodiment of the first aspect of the present invention, L3 is a substituted or unsubstituted linker. L3 is selected from a group that can be cleaved by fragmentation, such as a McLafferty fragmentation moiety, a Retro Diels Alder fragmentation moiety, benzyl, or aliphatic. Alternatively, L3 is a group that cannot be cleaved by fragmentation, such as a benzyl group, a diazo group, or an aliphatic group consisting of at least one or two carbon atoms (C1-C2) and aliphatic groups consisting of C1-C2, among others.

[0165] In an embodiment of the first aspect of the invention, the compound comprises Formula 1-V, where M=1 and K=1. For example, the compound is selected from Label 6 or Label 7.

[0166] In an embodiment of the first aspect of the present invention, the compound has the formula 1-VI: ((Z1) M -L1-Z2-(L2 / L3) K -Zo) N -Q Including, During the ceremony, Zo is a charge unit, including in particular a positively or negatively or neutrally charged unit, L2 is a substituted or unsubstituted linker, in particular L2 is L1 or L2 is a fragmentation cleavable group; L3 is a substituted or unsubstituted linker, in particular a non-cleavable group by fragmentation, such as benzyl or diazo; M>0, K≧0 and N≧1. L1, Z1, Z2 and Q have the same meanings as above.

[0167] In an embodiment of the first aspect of the present invention, the compound comprises Formula 1-VI: [ka] is.

[0168] In an embodiment of the first aspect of the present invention, the compound has the formula 1-VII: (Z1-L1-Z2-Q) N Including, wherein N≧1. L1, Z1, Z2 and Q have the same meanings as above.

[0169] In an embodiment of the first aspect of the present invention, the compound has the formula 1-VIII: [ka] Including, During the ceremony, Zo is a charge unit, including in particular a positively or negatively or neutrally charged unit, L2 is a substituted or unsubstituted linker, in particular L2 is L1 or L2 is a fragmentation cleavable group; L3 is a substituted or unsubstituted linker, in particular a non-cleavable group by fragmentation, such as benzyl or diazo; N>1, M>0 and K>0. Z1, Z2 and Q have the same meanings as above.

[0170] In an embodiment of the first aspect of the present invention, the compound has the formula 1-IX: [ka] Including, During the ceremony, Zo is a charge unit, including in particular a positively or negatively or neutrally charged unit, L2 is a substituted or unsubstituted linker, in particular L2 is L1 or L2 is a fragmentation cleavable group; L3 is a substituted or unsubstituted linker, in particular a non-cleavable group by fragmentation, such as benzyl or diazo; N≧1. Z1, Z2 and Q have the same meanings as above.

[0171] In an embodiment of the first aspect of the present invention, the compound comprises formula 1-IX: [ka] is selected from.

[0172] Labels 9 and 10 are doubly negatively charged.

[0173] In an embodiment of the first aspect of the present invention, the compound has the formula 1-X: [ka] Including, During the ceremony, Zo is a charge unit, including in particular a positively or negatively or neutrally charged unit, N≧1 and M>0. L1, Z1, Z2 and Q have the same meanings as above.

[0174] In an embodiment of the first aspect of the present invention, the compound has the formula 1-XI: [ka] Including, During the ceremony, Zo is a charge unit, including in particular a positively or negatively or neutrally charged unit, Z3 is a multiply charged unit, in particular a multiply charged metal unit; L3 is a substituted or unsubstituted linker, in particular a group that cannot be cleaved via fragmentation, such as a benzyl group, a diazo group, or an aliphatic group consisting of at least one or two carbon atoms (C1-C2) and an aliphatic group consisting of C1-C2.

[0175] Lig1 is a multiply charged metal complex binding ligand, particularly dependent on a high binding constant logKb>5, where N≧1. L1 and Q have the same meanings as above.

[0176] In an embodiment of the first aspect of the present invention, Lig1 is a multiply charged metal complex binding ligand and is selected from the following group: acetylacetonate (acac), 2-(2-aminoethylamino)ethanol, 2,2′-bis(diphenylphosphino)-6,6′-dimethoxy-1,1′-biphenyl, 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl, 1,2-bis[4,5-dihydro-3H-biphenyl] ... Naphtho[1,2-c:2',1'-e]phosphepino]benzene (BINAPHANE), 1,1'-bi-2-naphthol (BINOL), 5,5'-di-tert-butyl-2,2'-bipyridine, bis(oxazoline) ligand (BOX), 2,2'-bipyridine, bis(diphenylphosphino)butane, 1,5-cyclooctadiene, benzyl(methyl)phenylphosphine, 1,2-bis (2,5-diethylphosphorano)ethane, tert-butoxycarbonyl-4-diphenylphosphino-2-(diphenylphosphinomethyl)pyrrolidine, bis(4-isopropyl-4,5-dihydrooxazol-2-yl)phenylamine (bopa-ip), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N-heterocyclic carbenes, 1,10-phenanthroline, porphine, terpyridine (terpy), triphenylphosphine, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), bis(diphenylphosphino)methane (dppm), 1,2-bis(diphenylphosphino)ethane (dppe), 1,3-bis(diphenylphosphino)propane (dppp), crown ethers, [2.2.2] Cryptand, cyclopentadienyl anion, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethylenediaminotriacetate (TED), ethylenebis(oxyethylenetrilo)tetraacetate (egta4-), iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), tris(o-tolyl)phosphine, tris(2-aminoethyl)amine, cyclohexyl-o-anisylmethylphosphine (CAMP), phenyl-o-anisylmethylphosphine (PAMP), tropylium ion, citrate, cyclooctene, cyclooctatetraene (COT), cyclopentadienyl ion (Cp), 1,2,3,4,5-pentamethylcyclopentadienyl ion (Cp. * ), 1,4-diazabicyclo[2.2.2]octane (DABCO), dibenzylideneacetone (dba), 4-dimethylaminopyridine (DTPA), neocuproine, bis(2,5-dimethylphosphorano)benzene, (3,5-dioxa-4-phosphacyclohepta[2,1-a;3,4-a']dinaphthalen-4-yl)dimethylamine (MonoPhos), 1,3-diketimine acid ligands, bicyclo[2.2.1]hepta-2,5-diene, acetate, oxalate, 8-hydroxyquinoline, phthalocyanine, picolinate The ligand is selected from the group consisting of methyl amine, 2-phenylpyridine, pyrazine, salen ligand, 1,4,7-triazacyclononane (TACN), tartrate, trispyrazolylborate, tetraphenylporphyrin (TPP), 3,3',3''-phosphonetriyltris(benzenesulfonic acid) trisodium salt (tppts), 5-(3-pyridyl)-1H-tetrazole, 2-(1H-imidazol-2-yl)pyridine, 2-(1H-1,2,4-triazol-3-yl)pyridine, picoline, and 2,2'-bipyridine-4-butanoic acid.

[0177] In an embodiment of the first aspect of the present invention, the compound comprises formula 1-XI: [ka] is.

[0178] In an embodiment of the first aspect of the present invention, the compound has the formula 1-XII: [ka] Including, During the ceremony, Zo is a charge unit, including in particular a positively or negatively or neutrally charged unit, Z3 is a multiply charged unit, in particular a multiply charged metal unit; L3 is a substituted or unsubstituted linker, in particular a non-cleavable group by fragmentation, such as benzyl or diazo; Lig1 is a multiply charged metal complex binding ligand, specifically, with a binding constant logK B Depends on >5 Lig2 is a multiply charged metal complex binding ligand, and in particular, the binding constant depends on: 1 ≦ log K B ≦5 N≧1. Q has the same meaning as above.

[0179] In an embodiment of the first aspect of the invention, Lig1 is present twice in the complex.

[0180] In an embodiment of the first aspect of the present invention, Lig1 is equal to Lig2.

[0181] In an embodiment of the first aspect of the present invention, Lig1 may be selected from the group above.

[0182] In an embodiment of the first aspect of the present invention, Lig2 is a multiply charged metal complex binding ligand. Lig2 is selected from the group: 2-(2-aminoethylamino)ethanol, 1,1'-bi-2-naphthol (BINOL), bis(oxazoline)-ligand (BOX), tert-butoxycarbonyl-4-diphenylphosphino-2-(diphenylphosphinomethyl)pyrrolidine, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N-heterocyclic carbenes, aminopolycarboxylic acids, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), chlorine The amine may be selected from dimethylformamide, dimethylformamide, dimethylformamide (DMF), ...

[0183] In an embodiment of the first aspect of the present invention, the compound has the formula 1-XIII: (Z1-L3-Z2-L1-Z3-L2-Zo-Q) N Each of Z1, L3, Z2, L1, Z3, L2, Zo, Q and N has the same meaning as above.

[0184] In an embodiment of the first aspect of the invention, the compound is [ka] is.

[0185] In an embodiment of the first aspect of the present invention, M is 1, K is 1 and N is 2.

[0186] In an embodiment of the first aspect of the present invention, each of M, N, and K is an integer greater than 0. This means that the portion in brackets in Formulas 1-I to 1-XIII can be repeated M or K times. N is the net charge of the resulting molecule.

[0187] In an embodiment of the first aspect of the present invention, L1 and L2 may be fragmented in an ion source with in-source fragmentation or in a collision cell, ie at different potentials (voltages).

[0188] In an embodiment of the first aspect of the present invention, the compound comprises a counterion for forming a salt, the counterion preferably being selected from the following group: Cl - , Br - , F - , Formate, PF6 - , sulfonates, phosphates, acetates.

[0189] In an embodiment of the first aspect of the present invention, the compound does not contain trifluoroacetic acid (TFA). TFA, as a strong coordinating anion, can compensate for the net charge per TFA molecule and thus inhibit double charged moieties by forming singly charged species as TFA adducts.

[0190] In an embodiment of the first aspect of the invention, the compound is selected from the group: [ka] is selected from.

[0191] In an embodiment of the first aspect of the invention, the compound is doubly charged, in particular doubly permanently positively charged.

[0192] In an embodiment of the first aspect of the invention, the compound is selected from the group: [ka] Selected from

[0193] In a second aspect, the present invention relates to a composition comprising a compound 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.

[0194] In a third aspect, the present invention relates to a kit comprising a compound disclosed in detail above with respect to the first aspect of the present invention or a composition of the second aspect of the present invention disclosed in detail above herein. All embodiments described for the first aspect and / or the second aspect of the present invention apply to the third aspect of the present invention, and vice versa.

[0195] In a fourth aspect, the present invention relates to a complex for the quantitative detection of an analyte using mass spectrometry determination, the complex being formed by an analyte and a compound covalently bonded to each other, the complex comprising a permanent charge, particularly a permanent net positive charge, the complex having a mass m3 and a net charge z3, the complex being capable of forming at least one daughter ion having a mass m4 < m3 and a net charge z4 < z3 after fragmentation by mass spectrometry determination, and m3 / z3 < m4 / z4.

[0196] 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, seco-steroids, molecules characterized by specific modifications of other molecules, substances internalized by organisms, metabolites of such substances, and combinations thereof. All embodiments described for the first aspect and / or the second aspect and / or the third aspect of the present invention apply to the fourth aspect of the present invention, and vice versa.

[0197] In an embodiment of the fourth aspect of the present invention, a complex resulting from the formation of a covalent bond between the compound and a functional group present in the analyte molecule. Depending on the reactive unit Q of the compound and the functional group of the analyte molecule, one skilled in the art can fully determine the covalent bond formed between the two.

[0198] In an embodiment of the fourth aspect of the present invention, m3 ≧ 100, for example, m3 is 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150.

[0199] In an embodiment of the fourth aspect of the present invention, each or both of z3 and z4 is a permanent net charge.

[0200] In an embodiment of the fourth aspect of the present invention, each or both of z3 and z4 is a permanently positive net charge.

[0201] In an embodiment of the fourth aspect of the present invention, each or both of z3 and z4 is a permanently negative net charge.

[0202] In an embodiment of the fourth aspect of the present invention, the daughter ions comprise the analyte or a fragment thereof.

[0203] In an embodiment of the fourth aspect of the present invention, the daughter ions comprise an analyte and a fragment of a compound, the fragment of the compound being bound to the analyte via a covalent bond, and in particular the fragment of the compound having one permanent charge, in particular one permanently positive net charge or one permanently negative net charge.

[0204] In an embodiment of the fourth aspect of the invention, the complex is capable of forming further daughter ions, each comprising a fragment of the compound, each having an mx / zx value where x>4, and each of the mx / zx values ​​of the further daughter ions being less than the m3 / z3 value. The further daughter ions may further comprise the analyte or a fragment thereof.

[0205] In an embodiment of the fourth aspect of the present invention, z3=2, and after fragmentation, the complex can form a daughter ion with z4=1 and a further daughter ion, the further daughter ion having a net charge z5 (z5=1), and the daughter ion or further daughter ion comprises the analyte or a fragment thereof. The doubly charged complex is fragmented into at least two daughter ions between the two charges of the complex, i.e., a daughter ion and a further daughter ion. The daughter ion and the further daughter ion are each singly charged, for example, each singly positively charged or each singly negatively charged.

[0206] In embodiments of the fourth aspect of the invention, the analyte is selected from the group consisting of nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids, molecules characterized by a specific modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof. All embodiments of the analyte mentioned for the first aspect of the invention apply to the fourth aspect of the invention, and vice versa.

[0207] Furthermore, it is also considered within the scope of the present invention that a functional group present on the analyte molecule may first be converted into another readily available group by reaction with a reactive unit Q of the compound.

[0208] In an embodiment of the fourth aspect of the present invention, the analyte is selected from the group consisting of nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids, molecules characterized by a particular modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

[0209] In an embodiment of the fourth aspect of the present invention, the analyte molecule comprises a functional group selected from the group consisting of a carbonyl group, a diene group, a hydroxyl group, an amine group, an imine group, a thiol group, a diol group, a phenol group, an epoxy group, a disulfide group, and an azide group, each of which is capable of forming a covalent bond with a reactive unit Q of the compound.

[0210] 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.

[0211] In an embodiment of the fourth aspect of the invention, the complex comprises two permanent positive charges spaced apart by a linker L1, in particular each of the two permanent positive charges is a net positive permanent charge.

[0212] In an embodiment of the fourth aspect of the invention, the conjugate comprises at least three units Z1, Z2, Q' and an optional further unit L1, which units are covalently linked to each other, Q' is a reactive unit that forms a covalent bond with the analyte; Z1 is a charge unit comprising at least one permanently charged moiety, in particular a permanently positively charged moiety or a permanently negatively charged moiety, Z2 is a charge unit comprising at least one permanently charged moiety, in particular a permanently positively charged moiety or a permanently negatively charged moiety; and L1 is a substituted or unsubstituted linker, in particular a fragmentation cleavable group, such as a McLafferty fragmentation moiety, a Retro Diels Alder fragmentation moiety, benzyl, or aliphatic; and The net charge of the complex is greater than 1. Z1, Z2 and L1 may have the same meanings as stated for Z1, Z2 and L1 in relation to the embodiments of the first aspect of the invention.

[0213] In an embodiment of the fourth aspect of the invention, Q' results from the formation of a covalent bond between the reactive unit Q of the compound of the first aspect of the invention and a functional group present on the analyte molecule. Depending on the reactive unit Q of the compound of the first aspect of the invention and the functional group of the analyte molecule, one skilled in the art will be well able to determine the covalent bond formed between the two.

[0214] In an embodiment of the fourth aspect of the invention, the net charge of the complex is 2, 3, 4 or 5.

[0215] In an embodiment of the fourth aspect of the present invention, the conjugate has the following formula 2-I to 2-XI: (Z1-L1-Z2-Q') N (2-I) (Q'-Z1-L1-Z2) N (2-II) (Z1-Z2-Q') N (2-III) (Z1-(L1-Z2) M -(L2-Zo) K -Q') N (2-IV) (Z1-(L3-Z2) M -(L2-Zo) K -Q') N (2-V) ((Z1) M -L1-Z2-(L2 / L3) K -Zo) N -Q' (2-VI) [ka] is selected from the analyte is covalently bound to Q'; Z1 is a charge unit comprising at least one permanently charged moiety, in particular a permanently positively charged moiety or a permanently negatively charged moiety, L1 is a substituted or unsubstituted linker, in particular a fragmentation cleavable group, such as a McLafferty fragmentation moiety, a Retro Diels Alder fragmentation moiety, benzyl, or aliphatic; Z2 is a charge unit comprising at least one permanently charged moiety, in particular a permanently positively charged moiety or a permanently negatively charged moiety; Zo is a charge unit, including in particular a positively or negatively or neutrally charged unit, Z3 is a multiply charged unit, in particular a multiply charged metal unit; L3 is a substituted or unsubstituted linker, in particular a non-cleavable group by fragmentation, such as benzyl or diazo; Lig1 is a multiply charged metal complex binding ligand, specifically, with a binding constant logK B >5 depends on L2 is a substituted or unsubstituted linker, in particular L2 is L1 or L2 is a fragmentation cleavable group; Lig2 is a multiply charged metal complex binding ligand, and in particular, the binding constant depends on: 1 ≦ log K B ≦5, M>0, K>0 and N≧1.

[0216] In an embodiment of the fourth aspect of the invention, the binding compound is covalently bound via a carbonyl group, a hydroxyl group or a diene group of the analyte to form the complex, where binding compound refers to a compound of the compound-analyte complex.

[0217] In a fifth aspect, the present invention relates to the use of a compound for the mass spectrometric determination of an analyte. Preferably, the mass spectrometric determination comprises a tandem mass spectrometric determination, in particular a triple quadrupole mass spectrometric determination. All embodiments mentioned for the first aspect of the invention and / or the second aspect of the invention and / or the third aspect of the invention and / or the fourth aspect of the invention apply to the fifth aspect of the invention, and vice versa.

[0218] In an embodiment of the fifth aspect of the present invention, the signal to noise ratio of the mass spectrometry spectrum of the compound of the first aspect of the present invention or the conjugate of the fourth aspect of the present invention is lower compared to the mass spectrometry spectrum of an exemplary conjugate or exemplary compound having a maximum single permanent net charge of 1 or less.

[0219] In a sixth aspect, the present invention relates to a method for the mass spectrometric determination of an analyte, comprising: (a) reacting an analyte with a compound as disclosed herein with respect to the first aspect of the invention, whereby a complex as disclosed herein with respect to the fourth aspect of the invention is formed; (b) subjecting the complex from step (a) to mass spectrometric analysis.

[0220] In an embodiment of the sixth aspect of the present invention, the mass spectrometric analysis step (b) comprises: (i) subjecting ions of said complex to a first stage of mass spectrometry analysis, thereby characterizing said ions of said complex according to their mass / charge (m / z) ratio; (ii) causing fragmentation of ions of the complex, whereby the first entity, particularly the low molecular weight entity, is released and daughter ions of the complex are produced, the daughter ions of the complex having an m / z ratio different from that of the ion of the complex; (iii) subjecting the daughter ions of the complex to a second stage of mass spectrometry analysis, thereby characterizing the daughter ions of the complex according to their m / z ratio; and / or (ii) may further comprise alternative fragmentation of the ion of the complex, whereby a second entity different from the first entity is released to produce a second daughter ion of the complex; and (iii) may further comprise subjecting the first and second daughter ions of the complex to a second stage of mass spectrometry, whereby the first and second daughter ions of the complex are characterized according to their m / z ratios; The m / z ratio of the first daughter ion and / or the second daughter ion is greater than the m / z ratio of the ion of the complex.

[0221] In an embodiment of the sixth aspect of the present invention, a further step (a') before step (a) comprises (a') subjecting the ions of the complex or compound to an ion exchange of counterions, in which a particularly strongly coordinating anion, such as trifluoroacetate, is exchanged for chloride, bromide or a weakly coordinating counterion.

[0222] A weak counterion is an ion that does not bind to the analyte in the gas phase, and is dissociated by introduction into the mass spectrometer through the ion source.

[0223] By strongly coordinating anion, it is meant that the binding constant of a complex or compound containing a strongly coordinating anion as a counterion is greater than the binding constant of the corresponding complex or compound containing chloride as the counterion in the gas phase.

[0224] 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.

[0225] Step (a) can occur at different stages in the sample preparation workflow prior to determination by mass spectrometry. Samples containing analyte molecules can be pretreated and / or enriched by various methods. The pretreatment method depends on the type of sample, such as blood (fresh or dried), plasma, serum, urine, or saliva, while the enrichment method depends on the analyte of interest. Those skilled in the art will know which pretreated sample types are suitable for which sample types. Those skilled in the art will also know which enrichment methods are suitable for which analytes of interest.

[0226] In an embodiment of the sixth aspect of the present invention, step (a) of the method for the 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.

[0227] 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 water / ml sample, particularly 1:1 to 10:1 ml water / ml sample, particularly 2:1 to 5:1 ml water / ml sample.

[0228] In an embodiment of the sixth aspect of the present invention, the sample is a urine sample, and the sample is assigned to one of two other predetermined PT workflows, both of which include the addition of an internal standard and an enzymatic reagent followed by a predetermined incubation period, where the difference between the two workflows is the order in which the internal standard and the enzymatic reagent are added. The enzymatic reagent is typically a reagent used for glucuronide cleavage or protein cleavage, or any pretreatment of the analyte or matrix. In an additional step, a derivatization reagent, such as a compound of the present invention as disclosed herein above or below, is added, followed by an incubation period.

[0229] In an embodiment of the sixth aspect of the present invention, the enzyme reagent is selected from the group consisting of glucuronidase, (partial) exo- or endo-deglycosylating enzyme, or exo- or endo-preoteases. In an embodiment, the glucuronidase is added in an amount of 0.5 to 10 mg / mL, in particular in an amount of 1 to 8 mg / mL, in particular in an amount of 2 to 5 mg / mL.

[0230] In an embodiment of the sixth aspect of the invention, the sample is plasma or serum and is assigned to another predefined PT workflow which only involves the addition of an internal standard (ISTD) prior to a predefined incubation time.

[0231] As described hereinabove or below, the incubation time and temperature to be selected for the sample treatment, chemical reaction, or method step under consideration are well known to those skilled in the art. In particular, those skilled in the art know that incubation time and temperature are interdependent, e.g., higher temperatures typically result in shorter incubation periods, and vice versa. In embodiments of the sixth aspect of the present invention, the incubation temperature is in the range of 4 to 45°C, particularly in the range of 10 to 40°C, and particularly in the range of 20 to 37°C. In embodiments, the incubation time is in the range of 30 seconds to 1 minute, 30 seconds to 5 minutes, 30 seconds to 10 minutes, 1 minute to 10 minutes, or 1 minute to 20 minutes, 10 minutes to 30 minutes, 30 minutes to 60 minutes, or 60 minutes to 120 minutes. In certain embodiments, the incubation time is a multiple of 36 seconds.

[0232] Thus, step (a) of this method embodiment is carried out following any of the sample pretreatment processes disclosed above.

[0233] In an embodiment of the sixth aspect of the present invention, the reaction of the compound with the analyte molecule in step (a) is carried out before any enrichment process, and the compound is added to the pretreated sample of interest. Thus, a complex between the analyte molecule and the compound is formed after pretreatment and before the first enrichment process. Thus, the complex is subjected to the first and second enrichment processes before being subjected to mass spectrometric analysis in step (b).

[0234] 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).

[0235] In an embodiment of the sixth aspect of the present invention, the first enrichment workflow comprises adding a solid phase, in particular solid beads, carrying an analyte-selective group to the pretreated sample. In an embodiment of the sixth aspect of the present invention, the first enrichment workflow comprises adding magnetic or paramagnetic beads carrying an analyte-selective group to the pretreated sample. In an embodiment of the sixth aspect of the present invention, the addition of the magnetic beads comprises stirring or mixing. This is followed by a predetermined incubation period for capturing the analyte(s) of interest on the beads. In an embodiment of the sixth aspect of the present invention, the workflow comprises a washing step (W1) after incubation with the magnetic beads. Depending on the analyte(s), one or more additional washing steps (W2) are performed. One washing step (W1, W2) consists of a series of steps including magnetic bead separation by a magnetic bead manipulation unit including a magnet or electromagnet, liquid aspiration, addition of a washing buffer, resuspension of the magnetic beads, another magnetic bead separation step, and another liquid aspiration. Furthermore, the washing steps may differ in terms of the type of solvent (water / organic / salt / pH), apart from the volume and number or combination of washing cycles. The selection of each parameter is well known to those skilled in the art. The final washing step (W1, W2) is followed by the addition of an elution reagent, followed by resuspension of the magnetic beads and a predetermined incubation period to release the analyte(s) of interest from the magnetic beads. Subsequently, the unbound magnetic beads are separated, and the supernatant containing the derivatized analyte(s) of interest is captured.

[0236] In an embodiment of the sixth aspect of the present invention, a first enrichment workflow comprises adding magnetic beads carrying a matrix-selective group to a pretreated sample. In an embodiment of the sixth aspect of the present invention, the addition of the magnetic beads comprises stirring or mixing. This is followed by a predetermined incubation period to capture the matrix on the beads. Here, the analytes of interest do not bind to the magnetic beads and remain in the supernatant. The magnetic beads are then separated, and the supernatant containing the enriched analyte(s) of interest is collected.

[0237] 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.

[0238] 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.

[0239] In an embodiment of the sixth aspect of the present invention, analyte-specific magnetic beads are used and a compound as disclosed herein above or below is added to the sample of interest after the washing steps (W1, W2) have been completed either before, together with or following the elution reagent, followed by an incubation period (defined time and temperature).

[0240] In an embodiment of the sixth aspect of the present invention, the unbound magnetic beads are then separated and the supernatant containing the complexes of step (a) is collected. In an embodiment of the sixth aspect of the present invention, the supernatant containing the complexes of step (a) is transferred to a second enrichment workflow, in particular either directly to an LC station or after a dilution step by adding a diluent.

[0241] In an embodiment of the sixth aspect of the invention, matrix-specific magnetic beads are used and a compound as disclosed herein above or below is added to the sample of interest before or after the magnetic beads are separated. In an embodiment of the sixth aspect of the invention, the supernatant containing the complexes of step (a) is transferred to a second enrichment workflow, in particular either directly to an LC station or after a dilution step by adding a diluent.

[0242] Therefore, in an embodiment of the sixth aspect of the present invention, it is concluded that the reaction between the compound and the analyte molecule in step (a) is carried out following a first enrichment process, and the compound is added to the sample of interest after the first enrichment process, particularly the first enrichment process using magnetic beads. Thus, the sample is first pretreated as described herein above, and then subjected to a first enrichment process, particularly the first enrichment process using magnetic beads carrying an analyte-selective group as described herein above, and the compound is added before, simultaneously with, or after elution from the beads. Thus, a complex between the analyte molecule and the compound is formed after the first enrichment process and before the second enrichment process. Thus, the complex is subjected to a second enrichment process before being subjected to mass spectrometric analysis in step (b).

[0243] In another embodiment of the sixth aspect of the invention, step (a) of the method is performed after a second analyte enrichment workflow. The second enrichment workflow uses chromatographic separation to further enrich the analyte of interest in the sample. In an embodiment of the sixth aspect of the invention, the chromatographic separation is gas or liquid chromatography, both methods being familiar to those skilled in the art. In an embodiment of the sixth aspect of the invention, the liquid chromatography is selected from the group consisting of HPLC, rapid LC, micro LC, flow injection, and trapping and elution.

[0244] In an embodiment of the sixth aspect of the invention, step (a) of the method is carried out simultaneously with or after chromatographic separation. In an embodiment of the sixth aspect of the invention, the compound is added to the column together with the elution buffer. In an alternative embodiment, the compound is added post-column.

[0245] 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.

[0246] Therefore, in an embodiment of the sixth aspect of the present invention, it is concluded that the reaction between compound and analyte molecule in step (a) is followed by a second enrichment process, and compound is added to target sample after the second enrichment process using chromatography, particularly liquid chromatography.Therefore, in this case, the sample is first pretreated as described hereinabove, then is subjected to a first enrichment process, particularly using magnetic beads as described hereinabove, and then is subjected to chromatographic separation, particularly using liquid chromatography, followed by compound addition.Therefore, the complex between bound analyte molecule and bound compound is formed after the second enrichment process.Therefore, the complex is not subjected to an enrichment process before being subjected to mass spectrometric analysis in step (b).

[0247] In an embodiment of the invention, a clinical diagnostic system comprises a compound of the first aspect of the invention and / or a composition of the second aspect of the invention and / or a kit of the third aspect of the invention and / or a conjugate of the fourth aspect of the invention. Additionally or optionally, the compound of the first aspect of the invention is used for the mass spectrometric determination of an analyte, and the clinical diagnostic system comprises the mass spectrometric determination. Additionally or optionally, the method for the mass spectrometric determination of an analyte of the sixth aspect of the invention is carried out by the clinical diagnostic system.

[0248] A "clinical diagnostic system" is a laboratory automation device dedicated to analyzing samples for in vitro diagnostics. Clinical diagnostic systems can have different configurations depending on the need and / or desired laboratory workflow. Further configurations can be achieved by combining multiple devices and / or modules together. A "module" is a work cell, typically smaller in size than the entire clinical diagnostic system, with a dedicated function. This function can be analytical, but can also be pre-analytical or post-analytical, or the function can be a support function for any of the pre-analytical, analytical, or post-analytical functions. Specifically, a module can be configured to cooperate with one or more other modules to perform a dedicated task in a sample processing workflow, for example, by performing one or more pre-analytical and / or post-analytical steps. In particular, a clinical diagnostic system can include one or more analytical devices designed to perform respective workflows optimized for specific types of analysis, such as clinical chemistry, immunochemistry, coagulation, hematology, liquid chromatography separations, mass spectrometry, etc. Thus, a clinical diagnostic system can include one analytical device or any combination of such analytical devices with their respective workflows, and pre-analysis and / or post-analysis modules can be coupled to individual analytical devices or shared by multiple analytical devices. Alternatively, pre-analysis and / or post-analysis functions can be performed by units integrated into the analytical devices. A clinical diagnostic system can include functional units such as a liquid handling unit for pipetting, pumping, and / or mixing samples and / or reagents and / or system fluids, as well as functional units for sorting, storing, transporting, identifying, separating, and detecting. A clinical diagnostic system can include a sample preparation station for automated preparation of samples containing analytes of interest, a liquid chromatography (LC) separation station with multiple LC channels, and / or a sample preparation / LC interface for inputting the prepared sample into any one of the LC channels.The clinical diagnostic system may further include a controller programmed to assign samples to predefined sample preparation workflows, each of which includes a predefined series of sample preparation steps and requires a predefined time for completion depending on the analyte of interest. The clinical diagnostic system may further include a mass spectrometer (MS) and an LC / MS interface for connecting the LC separation station to the mass spectrometer. The terms "automatically" and "automated" as used herein are broad terms and should be given their ordinary and customary meaning to those skilled in the art and should not be limited to a specific or customized meaning. The terms may specifically, but are not limited to, refer to a process performed entirely by at least one computer and / or computer network and / or machine, particularly without manual action and / or user interaction.

[0249] A "sample preparation station" may be a pre-analytical module coupled to one or more analytical devices or units within an analytical device, designed to perform a series of sample processing steps aimed at removing or at least reducing interfering matrix components in the sample and / or concentrating the analyte of interest in the sample. Such processing steps may include one or more of the following processing operations performed sequentially, in parallel, or in a staggered manner on a sample or multiple samples: pipetting (aspiration and / or delivery) of fluids, pumping of fluids, mixing with reagents, incubation at a specific temperature, heating or cooling, centrifugation, separation, filtering, sieving, drying, washing, resuspension, aliquoting, transport, storage, etc.

[0250] A liquid chromatography (LC) separation station is an analytical device or module or unit within an analytical device designed to subject a prepared sample to chromatographic separation, e.g., to separate analytes of interest from matrix components, e.g., residual matrix components after sample preparation that may still interfere with subsequent detection, e.g., mass spectrometric detection, and / or to separate analytes of interest from each other to enable individual detection. According to one embodiment, the LC separation station is an intermediate analytical device or module or unit within an analytical device designed to prepare samples for mass spectrometry and / or transfer prepared samples to a mass spectrometer. In particular, the LC separation station is a multi-channel LC station comprising multiple LC channels.

[0251] The clinical diagnostic system, e.g., a sample preparation station, may also include a buffer unit for receiving multiple samples before a new sample preparation initiation sequence is initiated, and the samples may be individually randomly accessible, and their individual preparation may be initiated according to the sample preparation initiation sequence.

[0252] Clinical diagnostic systems utilize LC coupled to mass spectrometry, which is more convenient and reliable and therefore suitable for clinical diagnostics. In particular, high throughput, e.g., up to 100 samples / hour or more, can be achieved using random-access sample preparation and LC separation while enabling online connection to mass spectrometry. Furthermore, the process can be fully automated, increasing walk-away time and reducing the level of skill required.

[0253] In further embodiments, the present invention relates to the following aspects:

[0254] 1. A compound for the quantitative detection of an analyte using mass spectrometric determination, comprising: the compound comprises a permanent charge, in particular a permanent net charge, and is capable of covalently binding to the analyte; the compound has mass m1 and net charge z1; The compound is capable of forming at least one daughter ion having a mass m2 < m1 and a net charge z2 < z1 after fragmentation by mass spectrometry determination, a compound for which m1 / z1 < m2 / z2.

[0255] 2. m1 / z1 is at least 60 or more than that, for example, 66 for C7H2ON2 2+ and / or m2 / z2 is at least 70 or more than that, for example, 74 for C4H 12 N + The compound according to embodiment 1.

[0256] 3. The compound according to embodiment 1 or 2, which does not contain a sulfoxide unit (SO).

[0257] 4. The compound according to any one of embodiments 1 to 3, wherein z1 is an integer and is 2 or more than 2. [[ID=]]

[0258] 5. The compound according to any one of embodiments 1 to 4, wherein z1 is 2, 3, 4 or 5, preferably z1 is 2.

[0259] 6. The compound according to any one of embodiments 1 to 5, wherein z2 = z1 - 1, preferably z2 is 1.

[0260] 7. The compound according to any one of embodiments 1 to 6, wherein each or both of z1 and z2 is a permanent charge, particularly a permanent net positive charge.

[0261] 8. The compound according to any one of embodiments 1 to 7, wherein each or both of z, and z2 is a permanent positive charge, particularly a permanently positive net charge.

[0262] 9. The compound according to any one of embodiments 1 to 8, wherein each or both of z1 and z2 is a permanent negative charge, particularly a permanently negative net charge.

[0263] 10. The compound according to any one of aspects 1-9, wherein the net charge z1 is the sum of x times the positive permanent charge and y times the negative permanent charge of the compound.

[0264] 11. The compound of any one of aspects 1-10, wherein the net charge z2 is the sum of x times the positive permanent charge and y times the negative permanent charge of at least one daughter ion.

[0265] 12. The compound of any one of aspects 1-10, wherein the compound is capable of forming additional daughter ions, each of the additional daughter ions comprising a fragment or fragments of the compound, each having an mx / zx value where x>4, and each of the mx / zx values ​​of the additional daughter ions being less than the m1 / z1 value.

[0266] 13. A compound comprising at least three units Z1, Z2, Q and an optional further unit L1, wherein the units are covalently linked to each other; Q is a reactive unit capable of forming a covalent bond with the analyte; Z1 is a charge unit comprising at least one permanently charged moiety, in particular a permanently positively charged moiety or a permanently negatively charged moiety, Z2 is a charge unit comprising at least one permanently charged moiety, in particular a permanently positively charged moiety or a permanently negatively charged moiety; and L1 is a substituted or unsubstituted linker, in particular a fragmentation cleavable group, such as a McLafferty fragmentation moiety, a Retro Diels Alder fragmentation moiety, or an aliphatic; Aspect 13. The compound of any one of aspects 1 to 12, wherein the net charge of the compound is greater than one.

[0267] 14. The compound according to any one of aspects 1-13, wherein Z1 and Z2 are separated from each other by at least one atom.

[0268] 15. The compound according to any one of aspects 1-14, wherein Q is covalently bonded to Z1 or Q is covalently bonded to Z2.

[0269] 16. The compound according to any one of aspects 1-15, wherein L1 covalently links Z1 and Z2.

[0270] 17. A compound according to any one of aspects 1-16, which is fragmented between Z1 and Z2.

[0271] 18.Formula 1-I:(Z1-L1-Z2-Q) N( N ≥ 1) The compound of any one of Aspects 1 to 17, comprising: N≧1 can mean N≧+1 and / or N≧−1. N≧+1 can mean, for example, +1, +2, +3, +4, +5, +6, etc. N≧−1 can mean, for example, −1, −2, −3, −4, −5, −6, etc.

[0272] 19.Formula 1-II: (Q-Z1-L1-Z2) N (N≧1) 19. The compound of any one of aspects 1-18, comprising:

[0273] 20.Formula 1-III: (Z1-Z2-Q) N (N≧1) 20. The compound of any one of aspects 1-19, comprising:

[0274] 21. The compound of any one of aspects 1-20, wherein the reactive unit Q 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.

[0275] 22. The reactive unit Q is a carbonyl reactive group, in particular Q is selected from the group consisting of: (i) a hydrazine unit, such as a HN—NH— or HN—NR— unit, where R is aryl or C1-4 alkyl, particularly C1 or C2 alkyl, and optionally substituted; (ii) a hydrazide unit, particularly a carbohydrazide or sulfohydrazide, in particular a HN—NH—C(O)— or HN—NR—C(O)— unit, where R is aryl or C1-4 alkyl, in particular C1 or C2 alkyl, optionally substituted; (iii) a hydroxylamino unit, such as a HN—O— unit; (iv) dithiol units, in particular 1,2-dithiol or 1,3-dithiol units; 22. The compound according to any one of aspects 1 to 21, selected from:

[0276] 23. The compound according to any one of aspects 1-22, wherein the reactive unit Q is a thiol-reactive group or an amino-reactive group, such as an activated ester group, such as an N-hydroxysuccinimide (NHS) ester or sulfo-NHS ester, a hydroxybenzotriazole (HOBt) ester, or a 1-hydroxy-7-aquabenzotriazole (HOAt) ester group.

[0277] 24. The compound according to any one of aspects 1 to 23, wherein the reactive unit Q is selected from 4-substituted 1,2,4-triazoline-3,5-dione (TAD), 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), or fluoro-substituted pyridinium.

[0278] 25. The compound according to any one of aspects 1-24, wherein the linker L1 comprises 1 to 10 C atoms and optionally one or more heteroatoms.

[0279] 26. The compound according to any one of aspects 1 to 25, wherein linker L1 is selected from the group consisting of a McLafferty fragmentation unit, a Retro Diels Alder unit, a neutral loss cleavage unit, a bond dissociation unit, an alpha cleavage unit, and a charge site rearrangement unit.

[0280] 27. The compound according to any one of aspects 1 to 26, wherein Z1 and Z2 each independently comprise a chelate complex, a phosphonium, an ammonium, a carbenium, a pyridinium, a sulfonium, a 3- to 10-membered charged heterocycle containing any of N, S, H, and C atoms, or a derivative thereof.

[0281] 28.Formula 1-IV:(Z1-(L1-Z2) M -(L2-Zo) K -Q) N Including, During the ceremony, Zo is a charge unit, in particular including either a positively charged unit, a negatively charged unit or a neutrally charged unit; L2 is a substituted or unsubstituted linker, particularly L2 is L1, or particularly L2 is a fragmentation cleavable group; M>0, K>0 and N≧1; A compound according to any one of aspects 1 to 27.

[0282] 29.Formula 1-V:(Z1-(L3-Z2) M -(L2-Zo) K -Q) N Including, During the ceremony, Zo is a charge unit, in particular including either a positively charged unit, a negatively charged unit or a neutrally charged unit; L2 is a substituted or unsubstituted linker, particularly L2 is L1, or particularly L2 is a fragmentation cleavable group. L3 is a substituted or unsubstituted linker, in particular a non-cleavable group by fragmentation, such as benzyl or diazo; M>0, K>0 and N≧1; A compound according to any one of aspects 1 to 28.

[0283] 30.Formula 1-VI:((Z1) M -L1-Z2-(L2 / L3) K -Zo) N -Q Including, During the ceremony, Zo is a charge unit, in particular including either a positively charged unit, a negatively charged unit or a neutrally charged unit; L2 is a substituted or unsubstituted linker, particularly L2 is L1, or particularly L2 is a fragmentation cleavable group; L3 is a substituted or unsubstituted linker, in particular a non-cleavable group by fragmentation, such as benzyl or diazo; M>0, K≧0 and N≧1; A compound according to any one of aspects 1 to 29.

[0284] 31.Formula 1-VII:(Z1-L1-Z2-Q) N Including, A compound according to any one of aspects 1 to 30, wherein N≧1.

[0285] 32.Formula 1-VIII: [ka] Including, During the ceremony, Zo is a charge unit, in particular including either a positively charged unit, a negatively charged unit or a neutrally charged unit, L2 is a substituted or unsubstituted linker, particularly L2 is L1, or particularly L2 is a fragmentation cleavable group; L3 is a substituted or unsubstituted linker, in particular a non-cleavable group by fragmentation, such as benzyl or diazo; N≧1, M>0, and K>0; A compound according to any one of aspects 1 to 31.

[0286] 33.Formula 1-IX: [ka] Including, During the ceremony, Zo is a charge unit, in particular including either a positively charged unit, a negatively charged unit or a neutrally charged unit; L2 is a substituted or unsubstituted linker, particularly L2 is L1, or particularly L2 is a fragmentation cleavable group; L3 is a substituted or unsubstituted linker, in particular a non-cleavable group by fragmentation, such as benzyl or diazo; N≧1, A compound according to any one of aspects 1 to 32.

[0287] 34.Formula 1-X: [ka] Including, During the ceremony, Zo is a charge unit, in particular including either a positively charged unit, a negatively charged unit or a neutrally charged unit, N≧1 and M>0; A compound according to any one of aspects 1 to 33.

[0288] 35.Formula 1-XI: [ka] Including, During the ceremony, Zo is a charge unit, in particular including either a positively charged unit, a negatively charged unit or a neutrally charged unit, Z3 is a multiply charged unit, in particular a multiply charged metal unit; L3 is a substituted or unsubstituted linker, in particular a group that cannot be cleaved via fragmentation, such as a benzyl group, a diazo group, or an aliphatic group consisting of at least one or two carbon atoms (C1-C2) and aliphatic groups consisting of C1-C2, Lig1 is a multiply charged metal complex binding ligand, specifically dependent on a binding constant logKb>5. N≧1, A compound according to any one of aspects 1 to 34.

[0289] 36. Equation 1-XII: [ka] Including, During the ceremony, Zo is a charge unit, in particular including either a positively charged unit, a negatively charged unit or a neutrally charged unit, Z3 is a multiply charged unit, in particular a multiply charged metal unit; L3 is a substituted or unsubstituted linker, in particular a non-cleavable group by fragmentation, such as benzyl or diazo; Lig1 is a multiply charged metal complex binding ligand, specifically, with a binding constant logK B >5 depends on Lig2 is a multiply charged metal complex binding ligand, specifically, with a binding constant of 1 ≤ log K B Depends on ≦5, N≧1, A compound according to any one of aspects 1 to 35.

[0290] 37.Formula 1-XIII:(Z1-L3-Z2-L1-Z3-L2-Zo-Q) N wherein each of Z1, L3, Z2, L1, Z3, L2, Zo, Q, and N can have the same meaning as recited in other embodiments.

[0291] 38. A compound according to any one of aspects 1 to 37, wherein each of M, N, and K is an integer and greater than 0. The portion within the parentheses of Formulas 1-I to 1-XIII can be repeated M times and K times, and N is the net charge of the compound.

[0292] 39. A compound according to any one of aspects 1 to 38, comprising a counterion for forming a salt, and the counterion is preferably selected from the following group: Cl - , Br - , F - , formate, PF6 - , sulfonate, phosphate, acetate.

[0293] 40. A compound according to any one of aspects 1 to 39, which does not contain trifluoroacetic acid (TFA).

[0294] 41. A composition comprising the compound according to any one of claims 1 to 40.

[0295] 42. A kit comprising the compound according to any one of claims 1 to 40, or the composition according to claim 41.

[0296] 43. A complex for quantitative detection of an analyte using mass spectrometry determination, wherein the complex is formed by the analyte and the compound covalently bonded to each other, and the complex contains a permanent charge, particularly a permanent net charge, the complex has a mass m3 and a net charge z3, the complex can form at least one daughter ion having a mass m4 < m3 and a net charge z4 < z3 after fragmentation by mass spectrometry determination, and m3 / z3 < m4 / z4.

[0297] 44. The complex according to aspect 43, wherein m3 ≧ 100.

[0298] 45. The complex according to aspects 43 to 44, wherein each or both of z3 and z4 are permanent net charges.

[0299] 46. ​​The complex according to any one of aspects 43 to 45, wherein each or both of z3 and z4 is a permanently positive net charge.

[0300] 47. The complex of any one of aspects 43 to 46, wherein each or both of z3 and z4 is a permanently negative net charge.

[0301] 48. The complex of any one of aspects 43 to 47, wherein the daughter ion comprises the analyte or a fragment thereof.

[0302] 49. The complex according to any one of aspects 43 to 48, which is a parent ion.

[0303] 50. The complex according to any one of aspects 43 to 49, wherein the daughter ion comprises the analyte and a fragment of the compound, the fragment of the compound being bound to the analyte via a covalent bond, and in particular the fragment of the compound bearing one permanent charge, in particular one permanent positive charge or one permanent negative charge.

[0304] 51. The complex of any one of aspects 43 to 50, wherein the complex is capable of forming additional daughter ions, each comprising a fragment of the compound, each having an mx / zx value where x>4, and wherein each of the mx / zx values ​​of the additional daughter ions is less than the m3 / z3 value.

[0305] 52. The complex of any one of aspects 43 to 51, wherein z3=2 and, after fragmentation, the complex is capable of forming a daughter ion with z4=1 and a further daughter ion, the further daughter ion having a net charge z5 (z5=1), and the daughter ion or the further daughter ion comprises the analyte or a fragment thereof.

[0306] 53. The conjugate according to aspects 43 to 52, wherein the analyte is selected from the group consisting of nucleic acids, amino acids, peptides, proteins, metabolites, hormones, fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids, molecules characterized by a specific modification of another molecule, substances internalized by an organism, metabolites of such substances, and combinations thereof.

[0307] 54. The conjugate according to any one of embodiments 43 to 53, wherein the conjugate comprises two permanent positive charges spaced apart by a linker L1.

[0308] 55. A compound comprising at least three units Z1, Z2, Q' and an optional further unit L1, wherein the units are covalently linked to each other; Q' is a reactive unit that forms a covalent bond with the analyte; Z1 is a charge unit comprising at least one permanently charged moiety, in particular a permanently positively charged moiety or a permanently negatively charged moiety, Z2 is a charge unit comprising at least one permanently charged moiety, in particular a permanently positively charged moiety or a permanently negatively charged moiety; and L1 is a substituted or unsubstituted linker, in particular a fragmentation cleavable group, such as a McLafferty fragmentation moiety, a Retro Diels Alder fragmentation moiety, or an aliphatic; 55. The conjugate of any one of embodiments 43 to 54, wherein the net charge of the conjugate is greater than one.

[0309] 56. The complex has the following formulas 2-I to 2-XI: (Z1-L1-Z2-Q') N (2-I) (Q'-Z1-L1-Z2) N (2-II) (Z1-Z2-Q') N (2-III) (Z1-(L1-Z2) M -(L2-Zo) K -Q') N (2-IV) (Z1-(L3-Z2) M -(L2-Zo) K -Q') N (2-V) ((Z1) M -L1-Z2-(L2 / L3) K -Zo) N -Q' (2-VI) [ka] is selected from the analyte is covalently bound to Q'; Z1 is a charge unit comprising at least one permanently charged moiety, in particular a permanently positively charged moiety or a permanently negatively charged moiety, L1 is a substituted or unsubstituted linker, in particular a fragmentation cleavable group, such as a McLafferty fragmentation moiety, a Retro Diels Alder fragmentation moiety, or an aliphatic; Z2 is a charge unit comprising at least one permanently charged moiety, in particular a permanently positively charged moiety or a permanently negatively charged moiety; Zo is a charge unit, including in particular a positively or negatively or neutrally charged unit, Z3 is a multiply charged unit, in particular a multiply charged metal unit; L3 is a substituted or unsubstituted linker, in particular a non-cleavable group by fragmentation, such as benzyl or diazo; Lig1 is a multiply charged metal complex binding ligand, specifically, with a binding constant logK B >5 depends on L2 is a substituted or unsubstituted linker, in particular L2 is L1 or L2 is a fragmentation cleavable group; Lig2 is a multiply charged metal complex binding ligand, and in particular, the binding constant depends on: 1 ≦ log K B ≦5, The conjugate according to any one of aspects 43 to 55, wherein M>0, K>0, and N≧1, preferably N>1. N>1 can mean N>+1, such as +2, +3, +4, +5, +6, and / or N>−1, such as −2, −3, −4, −5, −6, and the like.

[0310] 57. Use of a compound according to any one of claims 1 to 40 for the mass spectrometric determination of an analyte, preferably wherein the mass spectrometric determination comprises a tandem mass spectrometric determination, in particular a triple quadrupole mass spectrometric determination.

[0311] 58. The use of embodiment 57, wherein the signal-to-noise ratio of the mass spectrometry spectrum of the compound of any one of embodiments 1 to 40 or the complex of any one of embodiments 43 to 56 is lower compared to the mass spectrometry spectrum of an exemplary complex or exemplary compound having a maximum single permanent net charge of 1 or less.

[0312] 59. A method for the mass spectrometric determination of an analyte, comprising the steps of: (a) reacting an analyte with a compound according to any one of embodiments 1 to 40, whereby a complex according to any one of embodiments 43 to 56 is formed; (b) subjecting the complex from step (a) to mass spectrometric analysis; A method comprising:

[0313] 60. Step (b) is (i) subjecting ions of said complex to a first stage of mass spectrometry analysis, thereby characterizing said ions of said complex according to their mass / charge (m / z) ratio; (ii) causing fragmentation of the complex ion, whereby a first entity, particularly a low molecular weight entity, is released and daughter ions of the complex are produced, the daughter ions of the complex having a different m / z ratio than the complex ion; (iii) subjecting the daughter ions of the complex to a second stage of mass spectrometry analysis, thereby characterizing the daughter ions of the complex according to their m / z ratio; and / or (ii) may further comprise alternative fragmentation of the ion of the complex, whereby a second entity different from the first entity is released to produce a second daughter ion of the complex; and (iii) may further comprise subjecting the first and second daughter ions of the complex to a second stage of mass spectrometry, whereby the first and second daughter ions of the complex are characterized according to their m / z ratios; 60. The method of embodiment 59, wherein the m / z ratio of the first daughter ion and / or the second daughter ion is greater than the m / z ratio of the ion of the complex.

[0314] 61. A further step (a') before step (a) is 61. The method of any one of claims 59 to 60, comprising (a') subjecting the ion of the complex or the ion of the compound to ion exchange of a counterion, wherein a particularly strongly coordinating anion, such as trifluoroacetate, is exchanged for chloride, bromide, or a weakly coordinating counterion as the counterion.

[0315] 62. The compound or method of any one of aspects 1-61, wherein the fragmentation is a one-step process. [Example]

[0316] The following examples are offered to illustrate, but not to limit, the invention claimed herein.

[0317] FIG. 1 shows a schematic mass spectrum of a doubly charged labeled analyte relative to the precursor and its fragmentation into a singly charged analyte. In this case, the doubly charged complex (parent ion) fragments into two daughter ions. The first daughter ion is a singly charged molecule containing a labeled fragment and an analyte molecule and has an m / z value greater than the m3 / z3 value of the doubly charged complex. The second daughter ion is a singly charged labeled fragment and has an m / z value less than the m3 / z3 value of the doubly charged labeled analyte. The doubly charged labeled analyte results in an enhanced MS signal. In contrast, the known singly charged complex fragments only into a singly charged daughter ion, and the daughter ion has an m / z value less than the m / z value of the known singly charged complex. The known singly charged complex does not fragment into a daughter ion with an m / z value greater than the m3 / z3 value of the known singly charged complex. Known singly charged complexes do not exhibit such fragmentation patterns, MS signal enhancement, or better signal to noise ratios compared to doubly charged complexes.

[0318] By placing one permanent charge with one label on one reactive part of the analyte molecule, the fragmentation characteristics change from a high mass precursor ion to a low mass quantitation ion after the MSMS process. This fragmentation pattern is consistent with the formation of a single charge (permanent or pseudo-molecular ion) after the ionization process or chemical derivatization. + H;M +This occurs when ions such as Na are generated. Small molecules (up to approximately 2000 Da) in particular exhibit this fragmentation pattern of small fragment shifts from high m / z values ​​to lower m / z values. This results in false-positive signals from undesired ions, such as matrix or coeluting / isobasic compounds with similar fragmentation patterns as the molecule of interest (see also Figure 5). This results in similar signals during the MSMS process and therefore interferes with the quantitative signal of the desired analyte. Small amounts of clinically relevant analytes are generated as pseudomolecular ions in their doubly charged form after the ESI (electrospray ionization) process. These types of molecules consist primarily of peptides, such as Tyr-Met-Arg-Phe-NH2, or related substances, such as vancomycin. The charge on these types of molecules is derived from a double protonation process (in positive ion mode), which depends on the respective ion source parameters that regulate ionization.

[0319] Figure 2 shows a schematic diagram of an MS spectrum (intensity (%) vs. m / z). It depicts the fragmentation behavior of a singly charged comparative compound within a + / - 0.5 Dalton window (the window is represented between two dashed lines). A typical mass spectrometer with a resolving power of R = 2000 can separate isobaric species of 500 m / z and +- 0.5 Da, which is typical of a mass spectrometer in MSMS mode. When using this type of resolution to enhance the selectivity of precursor molecule separation, naturally occurring isotopes do not enter the MSMS collision process because the first isotope added to the ionized molecule is +0.5 Da. For higher mass molecules around m / z 2000, the first isotope peak enters the collision cell after precursor molecule separation. Most clinically relevant small molecule analytes are less than 2000 Da. Therefore, the intensity of naturally occurring isotopes is lost during the fragmentation process. For molecules containing CHNOS, this is up to about 30% intensity at a mass of about 500 Da.

[0320] Figure 3 shows a schematic representation of the MS spectrum (intensity (%) vs. m / z) describing the fragmentation behavior of a doubly charged compound or complex of the invention in a + / - 0.5 Dalton window (the window is represented between the two dashed lines).

[0321] Doubly charged (and respectively multiply charged) molecules are advantageous compared to singly charged molecules (see Figure 2). A fixed isolation in the mass spectrometer is established due to the effect that initially the naturally occurring isotopes are separated into monoisotopic peaks by one charge of +1 Da and two charges of +0.5 Da. Therefore, approximately 30% more ions are available for further MSMS processing.

[0322] 4A-4E show the LLOQ (lower limit of quantitation) of the doubly charged complex (analyte: estradiol or its derivative; compound: doubly permanently positively charged compound, estradiol conjugated with label 6, also called RHA256). With a permanently positive label, a better signal-to-noise ratio of analyte versus analyte-compound can be achieved, which is directly related to the enhanced detection limit and therefore the respective enhancement factor.

[0323] Figures 5a-5c show the background noise of blank samples using the transitions of singly charged molecules (RHA139F2 (Figure 5a) and RHA171F2 (Figure 5b)) compared with a doubly charged molecule (estradiol conjugated with Label 6 (Figure 5c)). The unusual fragmentation pathways from lower m / z precursors or lower m / z compounds to higher m / z quantitation ions (daughter ions) result in significantly lower background noise. The resulting detection limit of 0.4 pg / ml compared to the equivalent underivatized workflow (estradiol's native detection limit of approximately 5 ng / ml in positive mode and approximately 30 pg / ml in negative ion mode) demonstrates the enhanced detection limit of the label. The background noise of the mass spectra of Label 6 and conjugated estradiol is better than that of RHA139F2 and RHA171F2. The structures of RHA139F2, RHA171F2, and estradiol conjugated with Label 6 are as follows: [ka]

[0324] Upon fragmentation of a doubly permanently charged molecule or complex, the two permanently installed charges are distributed after fragmentation between the label information-bearing ion and the analyte information-bearing ion, and these two ions can function very well as modifier and quantifier ions because they appear at least as intense without loss of ions.

[0325] Using this concept, which is nearly free of all interfering substances, improves the selectivity of the MSMS process and depletes background ions (see Figures 4 and 5).

[0326] Figure 6 shows the mass spectrum of a doubly positively charged complex (analyte-label 6 complex). The complex contains estradiol or a derivative thereof as the analyte of interest and estradiol conjugated with label 6 as the doubly permanently positively charged compound. The unfragmented complex has an m / z value of 256.16 (parent ion). The singly permanently positively charged daughter ion, containing estradiol and a fragment of the compound, has an m / z value of 349.20, which is greater than the m / z value of the parent ion. Two additional daughter ions, each containing a fragment of the compound, have m / z values ​​of 198.09 and 104.05, respectively.

[0327] If the concept of multiple positive charges and one negative charge (or vice versa) is used within one label and the opposite charges are separated after the MSMS process (depending on the resulting charge of the precursor), quantification of the analyte can be performed in an ionization mode different from that for precursor isolation. This can be done by fast switching between positive and negative ionization modes.

[0328] The labels described herein, which can bear multiple permanent charges (either x positive charges, y negative charges, or (xy) net positive and negative charges), exhibit fragmentation behavior into one or more fragments, carrying information about the portions of the label and the analyte molecular ion after fragmentation. Thus, MS signal enhancement of analytes can be important, for example, for low-abundance analytes.

[0329] Figure 7 shows a schematic diagram of peak "splitting," illustrating the ability of a chromatographic system to separate different isomers resulting from a derivatization reaction of an analyte molecule from one another.

[0330] FIG. 8 shows a schematic of the workflow for determining the enhancement factor of a labeled analyte compared to an unlabeled analyte.

[0331] 9A and 9B show the detection of the signal quenching effect of TFA on doubly charged derivatives / compounds.

[0332] FIG. 9A shows the respective MSMS transitions of label 15 (doubly charged molecular ion to singly charged fragment) as a derivatizing agent / compound for 500 pg / ml testosterone without TFA. [ka]

[0333] FIG. 9B shows the respective MSMS transitions of label 15 (doubly charged molecular ion to singly charged fragment) as a derivatization agent / compound for 500 pg / ml testosterone with TFA.

[0334] The addition or presence of TFA quenches the doubly charged ions, resulting in a significantly lower signal compared to a system without TFA.

[0335] A conjugate containing labeled 15 as the compound and testosterone as the analyte with or without TFA is prepared as follows:

[0336] Analytical derivatization of testosterone using label 15. A 500 ng / mL solution of testosterone (S1) was prepared in methanol. A solution (S2) containing an excess of either the derivatization reagent or label 15 compared to solution (S1), diluted in methanol (molar ratio >1000), was added, and the solution was acidified with glacial acetic acid (20% v / v). Solutions S1 and S2 were mixed to obtain solution S3, which was kept at 65 °C for 2 h and then at room temperature for 12 h. A dilution step with methanol was performed to reach a concentration of 500 pg / mL in a total volume of 1 mL.

[0337] The molecules so prepared were split 1:1 (v / v) in their metabolism solutions (S3-A) and (S3-B): Solution S3-B was spiked with 40 μl of water / TFA, while S3-A was spiked with 40 μl of water only.

[0338] Both solutions were measured in ESI-positive-full scan mode with a mass to display of m / z=302.71 Da, corresponding to the doubly charged derivative of testosterone.

[0339] As can be seen in Figure 9A (ESI-MS of m / z = 302.7 Da using solution S3-A; upper chromatogram; and ESI-MS of m / z = 302.7 Da using solution S3-B; lower chromatogram), the peak height in the chromatogram using S3-B is significantly lower than that in S3-A. Quenching of the doubly charged (changed) peak can be observed with the addition of a strongly coordinating anion such as TFA, which inhibits the doubly charged (changed) molecular ion, thus resulting in the generation of the pseudo-molecular ion [M + TFA], which is unfavorable for quantitative analysis.

[0340] Chromatography and MS parameters Polarity ES+ Calibration Static 2 Soft transmit mode disabled Capillary (kV) 3.00 3.14 Corn (V) 50.00 144.92 Source Offset (V) 30.0 Source temperature (℃) 140 140 Desolvation temperature (℃)350 350 Cone gas flow rate (L / Hr) 150 149 Desolvation gas flow rate (L / Hr) 1000 990 Collision gas flow (mL / min) 0.15 0.14 Nebulizer gas flow (Bar) 7.00 6.52 LM1 resolution 3.0 HM1 resolution 15.0 Ion Energy 1-0.2 MS mode collision energy 4.00 MSMS mode collision energy 2.00 MS mode entry 1.00 MS mode exit 1.00 Gas On MS Mode Entrance 1.00 Gas On MS Mode Exit 1.00 Gas On MSMS Mode Entrance 1.00 Gas On MSMS Mode Exit 1.00 Gas off MS mode entrance 30.00 Gas off MS mode exit 30.00 Gas Off MSMS Mode Entrance 30.00 Gas Off MSMS Mode Exit 30.00 ScanWave MS mode entrance 1.00 ScanWave MS mode exit 1.00 ScanWave MSMS Mode Entrance 1.00 ScanWave MSMS Mode Exit 1.00 LM2 resolution 3.0 HM2 resolution 15.0 Ion Energy2 0.2 Gain 1.00 Multiplier 513.80 Active Reservoir C Cone Energy Lamp: Disabled Probe temperature lamp: Disabled Collision Energy Lamp: Disabled Equipment parameters-function 2: Parameter file - E:\Regulated projects\ESI-Derivatization_PDA.PRO\ACQUDB\cz20Mrz2019-testo-label-general_tuning.IPR Polarity ES+ Calibration Static 2 Soft transmit mode disabled Capillary (kV) 3.00 3.14 Corn (V) 50.00 144.92 Source Offset (V) 30.0 Source temperature (℃) 140 140 Desolvation temperature (℃)350 350 Cone gas flow rate (L / Hr) 150 149 Desolvation gas flow rate (L / Hr) 1000 990 Collision gas flow (mL / min) 0.15 0.14 Nebulizer gas flow (Bar) 7.00 6.52 LM1 resolution 3.0 HM1 resolution 15.0 Ion Energy 1-0.2 MS mode collision energy 4.00 MSMS mode collision energy 2.00 MS mode entry 1.00 MS mode exit 1.00 Gas On MS Mode Entrance 1.00 Gas On MS Mode Exit 1.00 Gas On MSMS Mode Entrance 1.00 Gas On MSMS Mode Exit 1.00 Gas off MS mode entrance 30.00 Gas off MS mode exit 30.00 Gas Off MSMS Mode Entrance 30.00 Gas Off MSMS Mode Exit 30.00 ScanWave MS mode entrance 1.00 ScanWave MS mode exit 1.00 ScanWave MSMS Mode Entrance 1.00 ScanWave MSMS Mode Exit 1.00 LM2 resolution 3.0 HM2 resolution 15.0 Ion Energy2 0.2 Gain 1.00 Multiplier 513.80 Active Reservoir C Cone Energy Lamp: Disabled Probe temperature lamp: Disabled Collision Energy Lamp: Disabled Engineer Settings: MS1 low mass position 673 MS1 high mass position 335 MS1 low mass resolution 215 MS1 high mass resolution 2152 MS1 resolution linearity 531 MS1 High Mass DC Balance 0.07 MS1 DC polarity negative MS2 low mass position 672 MS2 high mass position 291 MS2 low mass resolution 219 MS2 high mass resolution 2162 MS2 resolution linearity 528 MS2 High Mass DC Balance 0.50 MS2 DC polarity negative Inter-scan delay: Automatic Mode MS scan delay (sec) 0.003 Polarity / Mode Switch Inter-Scan Delay (sec) 0.020 Enhanced inter-scan delay (sec) 0.020 Inter-Channel Delay - See Table MS1 Delay Table: R Delay <=1.250 0.001 <=4.000 0.002 <=10.000 0.003 <=20.000 0.004 > 20.000 0.005 --------------Method Parameter Execution-------------- Waters Acquity SDS Duration: 6.50 minutes comment: Solvent Selection A:A2 Solvent Selection B: B1 Low pressure limit: 0.000 bar High pressure limit: 1034.200 bar Solvent A: Water + NH4Ac + 0.1% formic acid Solvent name B: MeOH + NH4Ac + 0.1% formic acid Switch 1: No change Switch 2: No change Switch 3: No change Seal cleaning: 5.0 minutes Chart Out 1: System Pressure Chart Out 2: %B System pressure data channel: Yes Flow Data Channel: None %Data Channel: None %B Data Channel: Yes Primary A pressure data channel: None Accumulator A pressure data channel: None Primary B pressure data channel: None Accumulator B pressure data channel: None Degasser pressure data channel: None Slope Table Time (minutes) Flow rate %A %B curve 1.Initial 0.400 60.0 40.0Initial 2.0.50 0.400 60.0 40.0 6 3.3.00 0.400 10.0 90.0 6 4.5.00 0.400 10.0 90.0 6 5.5.10 0.400 60.0 40.0 6 6.6.50 0.400 60.0 40.0 6 Run event: Yes Gradient start (for injection): 0 uL 2D Iterations: None Waters Acquity PDA Duration: 6.50 minutes PDA detector type: UPLC LG 500nm Lamp: On Sampling rate: 10 points / second Filter time constant: 0.2000 seconds Exposure time: automatic msec Quadratic filter area interpolation: None Use of UV blocking filters: No 3D Channel... Range: 200-400 Resolution: 2.4nm Initial Switch 1: No change Initial Switch 2: No change Waters ACQUITY FTN AutoSampler Duration: 6.50 minutes comment: Preload: Disabled Offline Loop: Automatic min Cleaning solvent: ACN: Water Pre-injection cleaning time: 15.0 seconds Post-injection cleaning time: 15.0 seconds Purging solvent name: ACN:water Dilution: Ineffective Dilution volume: 0uL Delay time: 0 minutes Dilution needle placement: Automatic mm Target column temperature: 40.0℃ Column temperature alarm band: Disabled Target sample temperature: 6.0 C Sample temperature alarm band: Disabled Syringe withdrawal speed: Automatic Needle placement: 0.5mm Pre-suction air gap: Automatic Post-suction air gap: Automatic Column temperature data channel: None Room temperature data channel: Yes Sample temperature data channel: Yes Specimen Organizer Temperature Data Channel: None Sample pressure data channel: None Preheater temperature data channel: None Seal Force Data Channel: None No injection mode: No Automatic Additive Mixing Stroke Cycle: Automatic Automatic addition mixing stroke volume: Automatic uL Active Preheater: Use the console configuration Run Events: None Sample run injection parameters Injection volume (ul)-5.00 Feature 1 Active scans: 738 Cycle time (seconds): Automatic Delay between scans (seconds): Automatic Inter-channel delay (seconds): Automatic Span (Da): 0.500 Start time and end time (minutes): 0.000 to 5.000 Ionization mode: ES+ Data type: SIR or MRM enhancement Function type: 1 channel MRM Chan reaction dwell (sec) Kohnbolt. Col. energy delay (sec) Compound 1:289.25 > 108.78 0.200 Tune 25.0 Automatic Testosterone Feature 2 Active scans: 737 Cycle time (seconds): Automatic Delay between scans (seconds): Automatic Inter-channel delay (seconds): Automatic Span (Da): 0.500 Start time and end time (minutes): 0.000 to 5.000 Ionization mode: ES+ Data type: SIR or MRM enhancement Function type: 1 channel MRM Chan reaction dwell (sec) Kohnbolt. Col. Energy delay (sec) Compound Formula | Mass 1:624.40 > 203.00 0.200 Tune 40.0 Automatic DMA041 CE40 420.2 Feature 3 Active scans: 3901 Function Type: Diode Array Wavelength range (nm): 200-400

[0341] From the linear calibration curves, the respective detection limits were obtained using the procedure described in DIN EN ISO 32645. Enhancement factors can be calculated based on the limit of detection (LOD) of the labeled analyte, e.g., testosterone, compared to the LOD of the underivatized analyte, e.g., testosterone. The principle workflow is shown in Figure 8.

[0342] 10A-10D show the fragmentation patterns of the analyte-label 17 complex at different fragmentation energies (5 V-25 V).

[0343] Figure 10D shows the fragmentation pattern of the complex at 5 V. The parent ion with m3 / z3 value 261 fragments into the first daughter ion with m4 / z4 value 462. Additional ions are shown, such as the TFA adduct with m / z value 635 and the second daughter ion with m / z value 453. By increasing the fragmentation energy from 5 V to 25 V (Figures 10A-10D), the intensity of the first daughter ion increases, while the intensities of the TFA adduct and parent ion decrease. The best performance of the fragmentation energy can be determined. Methods for selecting the best performance of the fragmentation energy, for example, by using a computer (software or manual), are well known to those skilled in the art. The optimized fragmentation energy can be adjusted by routine measurements.

[0344] FIG. 11 shows the fragmentation pattern of the analyte-label 15 complex.

[0345] A complex is formed by the analyte testosterone and compound label 15 covalently bound to each other. The complex (parent ion) contains two permanent charges and has an m / z ratio of 291. The complex fragments into at least four daughter ions, each with an m / z ratio greater than that of the parent ion (m / z: 302, 328, 510, and 524). Possible structures of these daughter ions are shown in Figure 11. Additional daughter ions with m / z ratios less than m / z are detected.

[0346] The labels described herein, which are capable of establishing two permanent charges, exhibit fragmentation behavior into one or more fragments and carry information about the portion of the label and the portion of the analyte molecular ion after fragmentation. Thus, MS signal enhancement of an analyte can be important, for example, for low-abundance analytes.

[0347] General synthesis protocol 1: [ka] T = precursor of reactive functional group Q R1 and R2 can be independently selected from the following groups: R1 = methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexoxy, phenoxy, methylthioxy, ethylthioxy, propylthioxy, fenthioxy, acyl, formyl, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, benzyloxycarbonyl, benzoyloxy, acetoxy, fluoro, chloro, bromo, iodo, hydroxy, phenyl, benzyl, methyl, ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aryl, heteroaryl. R2 = methylene, ethylene, propylene, butylene, pentylene, hexylene, aryl, heteroaryl. Reaction conditions: a = alkylation steps, different solvents and temperatures: b = methylation step, different solvent, MeI; Conversion of T in c=Q: e.g., N2H4, MeOH or SOCl2, DCM or iodobenzene diacetate, MeOH

[0348] The choice of solvent and / or temperature will depend on the nature of the product being produced, and methods for selecting an appropriate solvent and / or temperature are well known to those skilled in the art.

[0349] Alkylation step a of general synthesis protocol: The methyl ester reagent (4.35 mmol) was dissolved in 15 mL of solvent and ethylenediamine reagent (10.95 mmol) was added. The reaction mixture was stirred (at room temperature or 70 °C) for 2-10 h and then concentrated in vacuo. The crude product was coevaporated with DMF (3 × 15 mL) and redissolved in a mixture of 20 mL of acetonitrile and 10 mL of ethyl acetate. The solution was stored at -20 °C for 16 h to precipitate the quaternary ammonium salt. The supernatant was removed, and the resulting solid was dried in vacuo.

[0350] General synthesis protocol methylation step B: The crude material (Step a) was dissolved in a mixture of solvents and methyl iodide (7.5 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours and then dried in vacuo. The crude bis-quaternary ammonium salt was then co-evaporated with acetonitrile / methanol (2 / 1, 2 x 30 mL).

[0351] General synthesis protocol step c: The crude material (step b) was dissolved in acetonitrile / methanol, and hydrazine hydrate (15 mmol) was added. The reaction mixture was stirred at room temperature for 4 hours and then dried in vacuo. The resulting crude product was purified by preparative RP-HPLC to give the desired product as a crystalline solid. Final conversion of the TFA salt to the corresponding formate salt was achieved by using a formic acid-activated anion exchange resin (Lewatit-MP-62 free base polymer).

[0352] General synthesis protocol 2: [ka] T = precursor of reactive functional group Q R1 and R2 can be independently selected from the following groups: R1 = methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexoxy, phenoxy, methylthioxy, ethylthioxy, propylthioxy, fenthioxy, acyl, formyl, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, benzyloxycarbonyl, benzoyloxy, acetoxy, fluoro, chloro, bromo, iodo, hydroxy, phenyl, benzyl, methyl, ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aryl, heteroaryl. R2 = methylene, ethylene, propylene, butylene, pentylene, hexylene, aryl, heteroaryl. Reaction conditions: a = alkylation step, different solvents and temperatures Conversion of T at b = Q: e.g., N2H4, MeOH or SOCl2, DCM or iodobenzene diacetate, MeOH

[0353] Alkylation step a of general synthesis protocol: Pyridine reagent (0.50 mmol) and (bromomethyl)trimethylammonium reagent (0.60 mmol) were dissolved in 1 mL of dry DMF. The reaction mixture was stirred at room temperature (rt) or 70 °C for 24-36 h. The solvent was removed in vacuo, and the residue was purified by preparative HPLC. Pure fractions were collected and concentrated in vacuo to give the product as a solid.

[0354] General synthesis protocol step b: PTAD-Py reagent (0.029 mmol) was dissolved in MeOH (500 μL). A solution of iodobenzene diacetate (0.033 mmol) in MeOH (500 μL) was added to the first solution. The reaction mixture was stirred at room temperature for 15 minutes. The solvent was removed in vacuo, and the residue was used directly without further purification.

[0355] General synthesis protocol 3: [ka] R1 and R2 can be independently selected from the following groups: R1 = methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexoxy, phenoxy, methylthioxy, ethylthioxy, propylthioxy, fenthioxy, acyl, formyl, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, benzyloxycarbonyl, benzoyloxy, acetoxy, fluoro, chloro, bromo, iodo, hydroxy, phenyl, benzyl, methyl, ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aryl, heteroaryl. R2 = methylene, ethylene, propylene, butylene, pentylene, hexylene, aryl, heteroaryl. Reaction conditions: a = alkylation step, different solvents and temperatures

[0356] Alkylation step a of general synthesis protocol: The 2-fluoropyridine reagent (0.36 mmol) and (bromomethyl)trimethylammonium reagent (0.46 mmol) were dissolved in 1 mL of dry DMF. The reaction mixture was stirred at room temperature or 90°C for 24-48 hours. The solvent was removed in vacuo, and the residue was purified by preparative HPLC. The pure fractions were collected and concentrated in vacuo to give the product as a colorless oil.

[0357] General synthesis protocol 4: [ka] T = precursor of reactive functional group Q R1 and R2 can be independently selected from the following groups: R1 = methylene, ethylene, propylene, butylene, pentylene, hexylene, aryl, heteroaryl. R2 = methylene, ethylene, propylene, butylene, pentylene, hexylene, aryl, heteroaryl. Reaction conditions: a = alkylation step, different solvents and temperatures b = methylation step, different solvent, MeI; Conversion of T in c=Q: e.g., N2H4, MeOH or SOCl2, DCM or iodobenzene diacetate, MeOH

[0358] Example 1: Label 13 [ka]

[0359] Synthesis of Label 13 Synthesis of [4-[2-(azaniumamino)-2-oxo-ethyl]phenyl]methyl-dimethyl-[2-(trimethylammonio)ethyl]ammonium; 2,2,2-trifluoroacetate [ka]

[0360] Bromomethylphenylacetic acid (4.35 mmol) was dissolved in 15 mL of methanol. Trimethylsilyl chloride (0.87 mmol) was added, and the reaction was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo, and the residue was coevaporated with methanol (2 × 15 mL). The crude methyl ester was dissolved in 15 mL of acetonitrile, and tetramethylethylenediamine (10.95 mmol) was added. The reaction mixture was stirred at 70 °C for 5 hours and then concentrated in vacuo. The crude product was coevaporated with DMF (3 × 15 mL) and redissolved in a mixture of 20 mL of acetonitrile and 10 mL of ethyl acetate. The solution was stored at -20 °C for 16 hours to precipitate the quaternary ammonium salt. The supernatant was removed, and the resulting solid was dried in vacuo. The crude material was dissolved in a mixture of acetonitrile / methanol (2 / 1, 30 mL), and methyl iodide (7.5 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours and then dried in vacuo. The crude bisquaternary ammonium salt was then co-evaporated with acetonitrile / methanol (2 / 1, 2x 30 mL) and dissolved in acetonitrile / methanol (1 / 4, 40 mL), and hydrazine hydrate (15 mmol) was added. The reaction mixture was stirred at room temperature for 4 h and then dried in vacuo. The resulting crude product was purified by preparative RP-HPLC to give the desired product (16% over four steps) as a crystalline solid. Final conversion of the TFA salt to the corresponding formate salt was achieved by using a formic acid-activated anion exchange resin (Lewatit-MP-62 free base polymer). HPLC-MS(m / z)[M 2+ +TFA] + Calculated value 407.5, detected value 407.3

[0361] Preparation of labeled 13-analyte derivatives (conjugates) and their analysis by MS [ka]

[0362] Labeled 13 (120 mg, 190 μmol) and testosterone (100 mg, 350 μmol, 1.8 equiv.) were dissolved in 5 mL of ACN / MeOH / AcOH (10 / 90 + 5% by volume), and the mixture was stirred at room temperature. After 16 h, the reaction mixture was concentrated in vacuo, and the crude product was subjected to HPLC purification (Triart C18 20 × 250 mm, linear gradient: 20% to 100% B in 30 min; A = water; B = ACN). Lyophilization afforded the desired conjugate. HPLC-MS(m / z)[M2+FA] + Calculated value 609.9, detected value 609.8

[0363] Example 2: Label 14 [ka]

[0364] Synthesis of Label 14 Synthesis of [4-[[4-(3,5-dioxo-1,2,4-triazolidin-4-yl)pyridin-1-ium-1-yl]methyl]phenyl]methyl-trimethyl-ammonium trifluoroacetate: [ka]

[0365] 4-(Pyridin-4-yl)-1,2,4-triazolidine-3,5-dione (0.50 mmol) and 4-(bromomethyl)benzyltrimethylammonium bromide (0.60 mmol) [synthesized as previously reported in Polym. Chem. 2014, 5, 1180-1190] were dissolved in 1 mL of dry DMF. The reaction mixture was stirred at 70 °C for 36 h. The solvent was removed in vacuo, and the residue was purified by preparative HPLC. Pure fractions were collected and concentrated in vacuo to give the product (78 mg, 24% yield) as a solid.

[0366] 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; 20~60 min: 70% H2O 0.1% TFA,30% CH3CN 0.1% TFA; 60~64 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 64~74 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 74~79 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 79~90 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1 H NMR (400 MHz, methanol-d4) δ ppm 3.10 (s, 9 H), 4.55 (s, 2 H), 5.85 (s, 2 H), 7.61-7.70 (m, 4 H), 8.85-8.89 (m, 2 H), 9.01-9.06 (m, 2 H). HPLC-MS(m / z)[M] 2+ Calculated value 170.59, Found value 170.79.

[0367] Preparation of labeled 14-analyte derivatives (conjugates) and their analysis by MS [ka]

[0368] 25-Hydroxyvitamin D monohydrate (0.024 mmol) and [4-[[4-(3,5-dioxo-1,2,4-triazolidin-4-yl)pyridin-1-ium-1-yl]methyl]phenyl]methyl-trimethyl-ammonium trifluoroacetate (0.029 mmol) were dissolved in MeOH (500 μL). A solution of iodobenzene diacetate (0.033 mmol) in MeOH (500 μL) 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 product was obtained as the chloro salt after ion exchange.

[0369] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~20 min: 5% H2O 0.1% TFA,95% CH3CN 0.1% TFA; 20~40 min: 5% H2O 0.1% TFA; 95% CH3CN 0.1% TFA; 40~45 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 45~50 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 50~55 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 55~65 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1 H NMR(400 MHz, methanol-d4)δ ppm 0.46(s,2 H)0.59(s,1 H)0.90-0.98(m,4 H)1.14(s,3 H)1.15(s,3 H)1.25-1.47(m,10 H)1.58-2.30(m,12 H)2.37-2.46(m,1 H)2.97-3.86(m,1 H)3.12(s,9 H)3.82-4.02(m,2 H)4.15-4.26(m,1 H)4.57(s,2 H)4.76-4.82(m,1 H)5.10-5.17(n,1 H)5.87(s,2 H)7.59-7.73(m,4 H)8.78-8.90(m,2 H)9.02-9.16(m,2 H). HPLC-MS(m / z)[M] 2+ Calculated value 376.75, detected value 370.06.

[0370] Example 3: Label 15

[0371] Synthesis of Label 15 Step 1: Synthesis of [4-[[3-(2-methoxy-2-oxoethyl)pyridin-1-ium-1-yl]methyl]phenyl]methyltrimethyl-ammonium bistrifluoroacetate: [ka]

[0372] A mixture of methyl 2-(3-pyridyl)acetate (0.161 mmol) and 3-bromopropyl(trimethyl)ammonium bromide (0.146 mmol) was dissolved in dry DMF (1 mL). The solution was stirred at 70 °C for 20 h. The solvent was removed and purified by preparative HPLC to give 30.8 mg of the desired product as a colorless oil.

[0373] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~40 min: 90% H2O 0.1% TFA,10% CH3CN 0.1% TFA; 40~44 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 44~52 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 52~54 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 54~59 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 59~60 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1 H NMR(400 MHz, methanol-d4)δ ppm 3.10(s,9 H)3.73(s,3 H)4.02(s,2 H)4.55(s,2 H)5.92(s,2 H)7.58-7.72(m,4 H)8.10(dd,J=7.97,6.21 Hz,1 H)8.57(d,J=8.16 Hz,1 H)9.02(d,J=6.15 Hz,1 H)9.11(s,1 H).

[0374] 13 C NMR(150 MHz,methanol-d4)δ ppm 36.13(1 C),51.67(1 C)51.76(3 C),63.60(1 C),68.28(1 C),127.80(1 C),129.33(1 C),129.36(2 C),133.73(2 C),135.95(1 C),136.75(1 C),143.14(1 C),145.41(1 C),147.30(1 C),169.97(1 C). HPLC-MS (m / z) [M+TFA] + Calculated value 427.18, detected value 427.61.

[0375] Step 2: Synthesis of [4-[[3-[2-(azaniumamino)-2-oxoethyl]pyridin-1-ium-1-yl]methyl]phenyl]methyl-trimethylammonium tristrifluoroacetate: [ka]

[0376] [4-[[3-(2-Methoxy-2-oxoethyl)pyridin-1-ium-1-yl]methyl]phenyl]methyltrimethyl-ammonium bistrifluoroacetate (0.057 mmol) was dissolved in dry MeOH (1 mL) and hydrazine hydrate (0.617 mmol) was added. The solution was stirred at 50 °C for 3 h. The solution was then cooled to room temperature, the solvent was removed in vacuo, and the crude mixture was purified by preparative HPLC to give 21.6 mg of the desired product as a colorless oil.

[0377] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~40 min: 90% H2O 0.1% TFA,10% CH3CN 0.1% TFA; 40~44 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 44~52 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 52~54 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 54~59 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 59~60 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1 H NMR(400 MHz,methanol-d4)δ ppm 3.10(s,9 H)3.98(s,2 H)4.55(s,2 H)5.92(s,2 H)7.60-7.69(m,4 H)8.10(dd,J=7.97,6.21 Hz,1 H)8.57(d,J=8.03 Hz,1 H)9.02(d,J=6.15 Hz,1 H)9.14(s,1 H). 13 C NMR(150 MHz,METHANOL-d4)δ ppm 35.50(1 C),51.77(3 C),63.61(1 C),68.26(1 C),127.84(1 C),129.32(1 C),129.39(2 C),133.72(2 C),135.89(1 C),136.35(1 C),143.25(1 C),145.37(1 C),147.16(1 C),167.93(1 C).

[0378] The compounds may also be obtained as different types of salts.

[0379] Example 4: Label 16 [ka]

[0380] Synthesis of Label 16 Synthesis of [4-[[4-(dimethylamino)-2-fluoro-pyridin-1-ium-1-yl]methyl]phenyl]methyl-trimethyl-ammonium trifluoroacetate: [ka]

[0381] 2-Fluoro-N,N-dimethylpyridin-4-amine (0.36 mmol) and 4-(bromomethyl)benzyltrimethylammonium bromide (0.46 mmol) [synthesized as previously reported in Polym. Chem. 2014, 5, 1180-1190] were dissolved in 1 mL of dry DMF. The reaction mixture was stirred at 90 °C for 48 h. The solvent was removed in vacuo, and the residue was purified by preparative HPLC. Pure fractions were collected and concentrated in vacuo to give 66 mg (35% yield) of the product as a colorless oil.

[0382] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~60 min: 70% H2O 0.1% TFA,30% CH3CN 0.1% TFA; 60~64 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 64~74 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 74~79 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 79~90 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA.

[0383] 1 H NMR(400 MHz,ACETONITRILE-d3)δ ppm 3.04(s,9 H)3.18(s,3 H)3.23(s,3 H)4.50(s,2 H)5.41(d,J=2.01 Hz,2 H)6.69(dd,J=9.29,2.89 Hz,1 H)6.87(dd,J=7.72,2.82 Hz,1 H)7.48(d,J=8.03 Hz,2 H)7.54-7.65(m,2 H)8.07(dd,J=7.78,6.15 Hz,1 H). 19F NMR (376 MHz, acetonitrile-d3) δ ppm -90.63 (dd, J = 8.94, 6.56 Hz, 1 F) -75.66 (s, 1 F). HPLC-MS(m / z)[M] 2+ Calculated value 151.60, found value 151.77.

[0384] Preparation of labeled 16-analyte derivatives (conjugates) and their analysis by MS Synthesis of estradiol conjugates: [ka]

[0385] Estradiol (0.09 mmol) and [4-[[4-(dimethylamino)-2-fluoro-pyridin-1-ium-1-yl]methyl]phenyl]methyl-trimethyl-ammonium trifluoroacetate (0.11 mmol) were dissolved in CH3CN (1 ml). DIPEA (0.14 mmol) was then added, and the reaction mixture was stirred at room temperature for 30 minutes. The solvent was removed under vacuum, and the residue was purified by preparative HPLC. Pure fractions were collected and lyophilized to give the product as a white solid.

[0386] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~60 min: 50% H2O 0.1% TFA,50% CH3CN 0.1% TFA; 60~64 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 64~74 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 74~79 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 79~90 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1H NMR(400 MHz, methanol-d4)δ ppm 0.77(s,3 H)1.15-1.59(m,7 H)1.62-1.77(m,1 H)1.86-2.10(m,4 H)2.19-2.30(m,1 H)2.30-2.40(m,1 H)2.81-2.87(m,2 H)2.96(s,3 H)3.09(s,9 H)3.23(s,3 H)3.66(t,J=8.60 Hz,1 H)4.53(s,2 H)5.54(s,2 H)5.85(d,J=2.76 Hz,1 H)6.79-6.85(m,2 H)6.87(dd,J=7.78,2.76 Hz,1 H)7.40(d,J=8.41 Hz,1 H)7.52(d,J=8.16 Hz,2 H)7.61(d,J=8.28 Hz,2 H)8.19(d,J=7.78 Hz,1 H). HPLC-MS(m / z)[M] 2+ Calculated value 277.69, detected value 278.05.

[0387] The compounds may also be obtained as different types of salts.

[0388] Label 16b: Additional synthetic conjugates: [ka]

[0389] Yield: 21.0 mg as a colorless solid.

[0390] 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; 64~80 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 80~83 minutes: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 83~89 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 89~90 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA.

[0391] 1 H NMR(400 MHz, methanol-d4)δ ppm 0.78(s,3 H)1.16-1.61(m,7 H)1.64-1.88(m,1 H)1.80-2.12(m,4 H)2.21-2.31(m,1 H)2.38(br s,1 H)2.87(br s,2 H)3.12(s,9 H)3.67(t,J=8.53 Hz,1 H)4.60(s,2 H)5.94(s,2 H)6.90-7.00(m,2 H)7.24(d,J=8.66 Hz,1 H)7.43-7.53(m,1 H)7.62-7.79(m,5 H)8.44(t,J=8.09 Hz,1 H) 8.93 (d, J = 6.27 Hz, 1 H). HPLC-MS(m / z)[M] 2+ Calculated value 256.17, detected value 256.69.

[0392] Example 5: Label 17 [ka]

[0393] Synthesis of Label 17 Step 1: Synthesis of 3-[3-(2-methoxy-2-oxoethyl)pyridin-1-ium-1-yl]propyltrimethylammonium bistrifluoroacetate: [ka]

[0394] A mixture of methyl 2-(3-pyridyl)acetate (1.610 mmol) and 3-bromopropyl(trimethyl)ammonium bromide (1.628 mmol) was dissolved in dry DMF (1 mL). The solution was stirred at 100 °C for 18 h. The solvent was removed and purified by preparative HPLC to give 292.6 mg of the desired product as a black oil.

[0395] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~60 min: 70% H2O 0.1% TFA,30% CH3CN 0.1% TFA; 60~64 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 64~80 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 80~83 minutes: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 83~89 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 89~90 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1 H NMR(400 MHz,methanol-d4)δ ppm 1.95-2.02(m,2 H)3.22(s,3 H)3.60-3.64(m,2 H)3.75(s,3 H)4.05-4.08(m,2 H)4.81(s,2 H)8.13(dd,J=6.15,7.93 Hz,1 H)8.60(d,J=8.10 Hz,1 H)9.07(d,J=6.29 Hz,1 H)9.18(s,1H). 13C NMR(101 MHz,methanol-d4)δ ppm 24.83(1 C)36.33(1 C)51.73(1 C)52.62(1 ​​C)52.66(1 C)52.70(1 C)57.82(1 C)62.35(1 C)127.69(1 C)136.55(1 C)143.24(1 C)145.34(1 C)147.31(1 C)170.02(1 C).

[0396] Step 2: Synthesis of 3-[3-[2-(azaniumamino)-2-oxoethyl]pyridin-1-ium-1-yl]propyltrimethyl-ammonium tristrifluoroacetate: [ka]

[0397] 3-[3-(2-Methoxy-2-oxoethyl)pyridin-1-ium-1-yl]propyltrimethylammonium bistrifluoroacetate (0.205 mmol) was dissolved in dry MeOH (2 mL) and hydrazine hydrate (2.058 mmol) was added. The solution was stirred at 0 °C for 4 h. The solvent was removed in vacuo, and the crude mixture was subjected to purification by preparative HPLC to give 66.9 mg of the desired product as a brownish solid.

[0398] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~30 min: 90% H2O 0.1% TFA,10% CH3CN 0.1% TFA; 30~34 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 34~50 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 50~53 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 53~59 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 59~60 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1 H NMR(400 MHz,methanol-d4)δ ppm 2.60-2.69(m,2 H)3.21(s,3 H)3.58-3.64(m,2 H)3.94-3.97(m,2 H)4.82(s,2 H)8.12(dd,J=6.20,7.99 Hz,1 H)8.58(d,J=8.28 Hz,1 H)9.05(d,J=5.82 Hz,1H)9.21(s,1 H).

[0399] Preparation of labeled 17-analyte derivatives (conjugates) and their analysis by MS Synthesis of testosterone conjugates: [ka]

[0400] 3-[3-[2-(azaniumamino)-2-oxoethyl]pyridin-1-ium-1-yl]propyltrimethyl-ammonium tristrifluoroacetate (0.113 mmol) and testosterone (0.104 mmol) were dissolved in MeOH (1 mL) and stirred at room temperature for 2.5 days. After removing the solvent in vacuo, the crude mixture was purified by preparative HPLC to give 15.9 mg of a colorless solid.

[0401] HPLC method C-18 column: 0 min: 100% H2O, 0% CH3CN; 0–45 min: 5% H2O, 95% CH3CN; 45–49 min: 5% H2O; 95% CH3CN; 49–50 min: 40% H2O; 60% CH3CN; 50–55 min: 40% H2O; 60% CH3CN; 55-60 min: 40% H2O; 60% CH3CN. HPLC-MS(m / z)[M 2+ +TFA] + Calculated value 635.38, found value 635.56.

[0402] The compounds may also be obtained as different types of salts.

[0403] Synthesis of 18: [ka]

[0404] 4-(Pyridin-4-yl)-1,2,4-triazolidine-3,5-dione (0.18 mmol) and 1,4-bis(bromomethyl)-2-methoxybenzene [previously reported in J. Am. Chem. Soc. 1996, 118, 4271] (0.18 mmol) were dissolved in 3 mL of dry DMF. The reaction mixture was stirred at room temperature (rt) over the weekend. Then, TMA solution (0.35 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. The solvent was removed in vacuo, and the crude product was purified by preparative HPLC. Pure fractions were collected and concentrated in vacuo. The residue was dissolved in dilute HCl (aq) and lyophilized (repeated twice). The product was obtained as a mixture of two isomers (ratio of approximately 7:3 by NMR) (17% yield) as a white solid.

[0405] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~10 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 10~60 min: 70% H2O 0.1% TFA,30% CH3CN 0.1% TFA; 60~64 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 64~74 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 74~79 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 79~90 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. Major isomer: 1 H NMR (400 MHz, methanol-d₄) δ ppm 3.13 (s, 9 H), 3.91 (s, 3 H), 4.54 (s, 2 H), 5.77 (s, 2 H), 7.23-7.31 (m, 2 H), 7.70 (d, J = 8.28 Hz, 1 H), 8.76-8.84 (m, 2 H), 8.95-9.02 (m, 2 H). Minor isomer: 1 H NMR(400 MHz, methanol-d4)δ ppm 3.10(s,9 H)3.96(s,3 H)4.54(s,2 H)5.82(s,2 H)7.18(dd,J=7.84,1.69 Hz,1 H)7.36(d,J=1.51 Hz,1 H)7.56(d,J=7.78 Hz,1 H)8.84-8.93(m,2 H)9.02-9.09(m,2 H). HPLC-MS(m / z)[M] 2+ Calculated value 185.60, found value 185.78.

[0406] Synthesis of Vitamin D derivatized with Label 18: [ka]

[0407] 25-Hydroxyvitamin D monohydrate (0.019 mmol) and 18 (0.020 mmol) were dissolved in MeOH (500 μL). A solution of iodobenzene diacetate (0.023 mmol) in MeOH was added to the first solution. The reaction mixture was stirred at room temperature for 15 minutes. Complete conversion of vitamin D to the corresponding product was observed. The solvent was removed in vacuo, and the crude product was purified by preparative HPLC. Pure fractions were collected and concentrated in vacuo. The residue was dissolved in dilute HCl (aq) and lyophilized (repeated twice). The product was obtained as a white solid.

[0408] 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. HPLC-MS(m / z)[M] 2+ Calculated value 384.8, detected value 385.05.

[0409] Synthesis of Labeled 19: [ka]

[0410] 2-Fluoro-N,N-dimethylpyridin-4-amine (0.18 mmol) and 1,4-bis(bromomethyl)-2-methoxybenzene [previously reported in J. Am. Chem. Soc. 1996, 118, 4271] (0.18 mmol) were dissolved in 3 mL of dry DMF. The reaction mixture was stirred at room temperature over the weekend. Then, TMA solution (0.36 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo, and the residue was purified by preparative HPLC. Pure fractions were collected and concentrated in vacuo to give the product as a mixture of two isomers (ratio of approximately 7:3 by NMR) (14% yield) as a colorless oil.

[0411] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~60 min: 70% H2O 0.1% TFA,30% CH3CN 0.1% TFA; 60 - 64 minutes: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 64 - 74 minutes: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 74 - 79 minutes: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA; 79 - 90 minutes: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA.

[0412] Main isomer: 1 H NMR (400 MHz, acetonitrile - d3) δ ppm 3.05 (s, 9H) 3.16 (s, 3H) 3.21 (s, 3H) 3.87 (s, 3H) 4.46 (s, 2H) 5.29 (d, J = 1.63 Hz, 2H) 6.63 (dd, J = 9.41, 2.89 Hz, 1H) 6.77 (dd, J = 7.78, 2.89 Hz, 1H) 7.16 (dd, J = 7.72, 1.44 Hz, 1H) 7.19 (d, J = 1.13 Hz, 1H) 7.47 (dd, J = 7.78, 1.00 Hz, 1H) 7.92 (dd, J = 7.78, 6.15 Hz, 1H).

[0413] Minor isomer: 1 H NMR (400 MHz, acetonitrile - d3) δ ppm 3.04 (s, 9H) 3.16 (s, 3H) 3.21 (s, 3H) 3.87 (s, 3H) 4.43 (s, 2H) 5.28 (d, J = 1.63 Hz, 2H) 6.62 (br dd, J = 9.29, 2.89 Hz, 1H) 6.76 (br dd, J = 7.72, 2.82 Hz, 1H) 7.08 - 7.22 (m, 2H) 7.42 - 7.50 (m, 1H) 7.91 (br dd, J = 7.78, 6.02 Hz, 1H). 19 F NMR (376 MHz, ACETONITRILE - d3) δ ppm - 75.39, - 90.11. HPLC - MS (m / z) [M + TFA] + Calculated value 446.21, detected value 446.39.

[0414] Synthesis of estradiol conjugated with label 19: [ka]

[0415] Estradiol (0.022 mmol) and 19 (0.026 mmol) were dissolved in CHCN (1 mL). KCO (0.066 mmol) was then added, and the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo, and the residue was purified by preparative HPLC. The pure fractions were collected and concentrated in vacuo. The residue was dissolved in dilute HCl (aq) and lyophilized (repeated twice). The product was obtained as a white solid as a mixture of two isomers (ratio of approximately 7:3 by NMR).

[0416] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~60 min: 50% H2O 0.1% TFA,50% CH3CN 0.1% TFA; 60~64 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 64~74 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 74~79 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA.

[0417] Major isomer: 1H NMR(400 MHz, methanol-d4)δ ppm 0.78(s,3 H)1.16-1.59(m,8 H)1.63-1.79(m,1 H)1.87-2.10(m,3 H)2.19-2.29(m,1 H)2.32-2.40(m,1 H)2.83-2.85(m,2 H)2.94(br s,3 H)3.12(s,9 H)3.21(br s,3 H)3.67(t,J=8.66 Hz,1 H)3.94(s,3 H)4.52(s,2 H)5.46(s,2 H)5.82(d,J=2.76 Hz,1 H)6.77-6.83(m,3 H)7.16(dd,J=7.65,1.51 Hz,1 H)7.24(d,J=1.25 Hz,1 H)7.41(br d,J=8.03 Hz,1 H)7.44(d,J=7.78 Hz,1 H)8.14(d,J=7.91 Hz,1 H).

[0418] Minor isomer: 1 H NMR(400 MHz, methanol-d4)δ ppm 0.78(s,3 H)1.16-1.59(m,8 H)1.63-1.79(m,1 H)1.87-2.10(m,3 H)2.19-2.29(m,1 H)2.32-2.40(m,1 H)2.83-2.85(m,2 H)2.97(br s,3 H)3.08(s,3 H)3.23(br s,1 H)3.67(t,J=8.66 Hz,1 H)3.88(s,3 H)4.52(s,2 H)5.52(s,2 H)5.88(br d,J=2.76 Hz,1 H)6.83-6.89(m,1 H)7.20-7.22(m,1 H)7.41(br d,J=8.03 Hz,1 H)7.51(br d,J=7.78 Hz,1 H)8.21(br d,J=7.78 Hz,1 H). HPLC-MS(m / z)[M] 2+ Calculated value 292.7, detected value 293.04.

[0419] Synthesis of Label 20: [ka]

[0420] 2-Fluoro-N,N-dimethylpyridin-4-amine (0.78 mmol) and (3-bromopropyl)trimethylammonium bromide (0.94 mmol) were dissolved in 1 mL of dry DMF. The reaction mixture was stirred at 70 °C over the weekend. The solvent was removed in vacuo, and the residue was purified by preparative HPLC. Pure fractions were collected and concentrated in vacuo to give the product as a colorless oil (46% yield).

[0421] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~60 min: 70% H2O 0.1% TFA,30% 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. 1 H NMR(400 MHz,D2O)δ ppm 2.18-2.35(m,2 H)3.03(s,9 H)3.08(s,3 H)3.13(s,3 H)3.31-3.39(m,2 H)4.10-4.21(m,2 H)6.59(dd,J=9.35,2.82 Hz,1 H)6.74(dd,J=7.78,2.76 Hz,1 H)7.77(dd,J=7.72,6.09 Hz,1 H). 19 F NMR(376 MHz,D2O)δ ppm-91.38--91.27,-75.63. 13 C NMR (101 MHz, deuterium oxide) δ ppm 23.01(1 C)25.37(1 C)39.94(1 C)47.81(1 C)53.03(3 C)62.73(1 C)91.04(1 C)91.31(1 C)106.32(1 C)139.45(1 C)159.87(1 C). HPLC-MS(m / z)[M] 2+ Calculated value 120.60, detected value 120.59.

[0422] Synthesis of estradiol conjugated with label 20: [ka]

[0423] Estradiol (0.073 mmol) and label 20 (0.11 mmol) were dissolved in CHCN (1 mL). KCO (0.22 mmol) was then added, and the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo, and the residue was purified by preparative HPLC. The pure fractions were collected and concentrated in vacuo. The residue was dissolved in diluted HCl (aq) and lyophilized (repeated twice). The product was obtained as a white solid.

[0424] HPLC method C-18 column: 0 min: 100% H2O 0.1% TFA,0% CH3CN 0.1% TFA; 0~60 min: 50% H2O 0.1% TFA,50% CH3CN 0.1% TFA; 60~64 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 64~74 min: 2% H2O 0.1% TFA; 98% CH3CN 0.1% TFA; 74~79 min: 60% H2O 0.1% TFA; 40% CH3CN 0.1% TFA. 1H NMR(400 MHz, methanol-d4)δ ppm 0.79(s,3 H)1.22-1.55(m,8 H)1.64-1.80(m,1 H)1.85-2.12(m,3 H)2.20-2.34(m,1 H)2.34-2.49(m,3 H)2.91(br dd,J=8.60,4.08 Hz,2 H)2.96(s,3 H)3.18(s,9 H)3.22(s,3 H)3.49-3.55(m,2 H)3.68(t,J=8.60 Hz,1 H)4.37(t,J=7.28 Hz,2 H)5.84(d,J=2.76 Hz,1 H)6.84(dd,J=7.65,2.76 Hz,1 H)7.06-7.10(m,2 H)7.49(d,J=8.41 Hz,1 H)8.03(d,J=7.65 Hz,1 H). HPLC-MS(m / z)[M] 2+ Calculated value 246.68, detected value 247.00.

[0425] This patent application claims priority to European patent application 20175802.6, the contents of which are incorporated herein by reference.

Claims

1. 1. A compound for the quantitative detection of an analyte using mass spectrometric determination, comprising: the compound comprises a permanent charge and is capable of covalently binding to the analyte; the compound has a mass m1 and a net charge z1; the compound is capable of forming, after fragmentation as determined by mass spectrometry, at least one daughter ion having a mass m<m and a net charge z<z; m1 / z1<m2 / z2, The compound is represented by the following formulas (1-I) to (1-VI) and (1-VIII) to (1-XIII): (Z1-L1-Z2-Q) Ｎ (1-I)、 (Q-Z1-L1-Z2) Ｎ (1-II)、 (1000) Ｎ (1-999)、 (Z1-(L1-Z2) Ｍ -(L2-Zo) Ｋ -V) Ｎ (1-1V) ((Z11-(L3-Z2) Ｍ -(L2-Zo) Ｋ -V) Ｎ (1-V) (((Z1) Ｍ -L1-Z2-(L2 / L3) Ｋ -Zo) Ｎ --Q(1-VI)、 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 ((Z1-L3-Z2-L1-Z3-L2-Zo-Q) Ｎ (1-XII) [Zo is a positively charged unit, Z3 is a multiply charged unit, L2 is a substituted or unsubstituted linker; L3 is a substituted or unsubstituted linker; Lig1 is a multiply charged metal complex binding ligand; Lig2 is a multiply charged metal complex binding ligand; M>0, K>0, and N>1; Z1 is a permanently positively charged moiety or a permanently negatively charged moiety; Z2 is a permanently positively charged moiety or a permanently negatively charged moiety; L1 is a fragmentation cleavable group; The net charge of the compound is greater than 1, where: Q is a reactive unit capable of forming a covalent bond with an analyte molecule, such as: (i) a hydrazine unit, (ii) a hydrazide unit, (iii) a hydroxylamino unit, and (iv) a dithiol unit, or the reactive unit Q is selected from 4-substituted 1,2,4-triazoline-3,5-dione (TAD), 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), or fluoro-substituted pyridinium; A compound comprising one or more of:

2. 2. The compound of claim 1, wherein the fragmentation is a one-step process.

3. 3. A compound according to claim 1 or 2, wherein z1 is 2, 3, 4 or 5, and / or m1 / z1 is at least 60, and / or m2 / z2 is at least 70.

4. The compound according to any one of claims 1 to 3, wherein z2 = z1-1.

5. The compound of any one of claims 1 to 4, wherein each or both of z1 and z2 is a permanent charge.

6. The compound of any one of claims 1 to 5, wherein each or both of z1 and z2 is a permanent positive charge.

7. 7. The compound of claim 1, wherein the compound is capable of forming further daughter ions, each of the further daughter ions comprising a fragment of the compound or multiple fragments of the compound, each having an mx / zx value where x>4, and each of the mx / zx values ​​of the further daughter ions being less than their m1 / z1 values.

8. The compound of any one of claims 1 to 7, which is free of trifluoroacetic acid (TFA).

9. A composition comprising a compound according to any one of claims 1 to 8.

10. A kit comprising the compound according to any one of claims 1 to 8 or the composition according to claim 9.

11. 1. A conjugate for quantitative detection of an analyte using mass spectrometric determination, comprising: the complex is formed by the analyte and the compound covalently bound to each other, the complex comprising a permanent charge; the complex has a mass m3 and a net charge z3; the complex is capable of forming, after fragmentation as determined by mass spectrometry, at least one daughter ion having a mass m<m and a net charge z<z; m3 / z3<m4 / z4, The complex is represented by the following formulas (2-I) to (2-XI): (Z1-L1-Z2-Q’) Ｎ (2-I) (Q’-Z1-L1-Z2) Ｎ (2-II) (1000) Ｎ (2-999) (Z1-(L1-Z2) Ｍ -(L2-Zo) Ｋ -Q') Ｎ (2-1V) ((Z11-(L3-Z2) Ｍ -(L2-Zo) Ｋ -Q') Ｎ (2-V) (((Z1) Ｍ -L1-Z2-(L2 / L3) Ｋ -Zo) Ｎ -Q' (2-VI) 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 wherein the analyte is covalently attached to Q′; Z1 is a permanently positively charged moiety or a permanently negatively charged moiety; Z2 is a permanently positively charged moiety or a permanently negatively charged moiety; L1 is a fragmentation cleavable group; Zo is a charge unit, Z3 is a multiply charged unit, L2 is a substituted or unsubstituted linker; L3 is a substituted or unsubstituted linker; Lig1 is a multiply charged metal complex binding ligand; Lig2 is a multiply charged metal complex binding ligand; M>0, K>0 and N>1. A complex selected from:

12. 12. The complex of claim 11, wherein z3=2 and, after fragmentation, the complex is capable of forming the daughter ion with z4=1 and a further daughter ion, the further daughter ion having a net charge z5 (z5=1), and the daughter ion or the further daughter ion comprises the analyte or a fragment thereof.

13. Use of a compound according to any one of claims 1 to 8 for the mass spectrometric determination of said analyte.

14. The use of claim 13, wherein the determination by mass spectrometry includes determination by tandem mass spectrometry or determination by triple quadrupole mass spectrometry.

15. 1. A method for the mass spectrometric determination of an analyte, comprising: (a) reacting the analyte with a compound according to any one of claims 1 to 8, whereby a complex according to any one of claims 11 to 12 is formed; (b) subjecting the complex from step (a) to mass spectrometric analysis; A method comprising:

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