Derivatization agents for laser desorption / ionization mass spectrometry

Abstract

In a first aspect, the present invention provides a compound of formula (I)C-L1-Z-(L2) p The present invention relates to a derivatization agent, preferably a derivatization agent for an analyte intended to be analyzed by LDI-MS, comprising the structural elements: -X, where C is a chromophore having an absorption maximum in the range of 280-400 nm; Z is a charge unit containing at least one permanently charged moiety; X is a reactive group; L1, L2 are each linker units; and p is either 0 or 1. A second aspect of the present invention relates to a kit comprising a derivatization agent according to the first aspect. In a third aspect, the present invention relates to the use of a derivatization agent according to the first aspect for mass spectrometric determination of an analyte molecule, wherein the mass spectrometric determination is LDI-MS. A fourth aspect of the present invention relates to a conjugate of a derivatization agent according to the first aspect and an analyte, comprising a conjugate of formula (II)C-L1-Z-(L2) p A fifth aspect of the invention relates to a conjugate having the structure -Xa-Ya-A, wherein C, L1, L2, p, Z, and N are as defined in the context of the first aspect; Xa is the remainder of reactive group X as defined in the context of the first aspect; A is an analyte, and Ya is the remainder of reactive group Y attached to analyte A, which has reacted with reactive group X of the derivatization agent to form a covalent bond between Xa and Ya. A fifth aspect of the invention relates to a method for the mass spectrometric determination of an analyte molecule, comprising the steps of: (a) providing an analyte of interest; (b) providing a derivatization agent comprising the structure of formula (I), as defined in the context of the first aspect; (c) reacting the analyte provided in accordance with (a) with the derivatization agent provided in accordance with (b), thereby forming a conjugate of the analyte and the derivatization agent; and (d) subjecting the conjugate formed in (c) to mass spectrometry, wherein the mass spectrometry is preferably LDI-MS.

Description

[Technical Field]

[0001] In a first aspect, the present invention provides a compound of formula (I)C-L1-Z-(L2) p -X, wherein C is a chromophore having an absorption maximum in the range of 280-400 nm; Z is a charge unit containing at least one permanently charged moiety; X is a reactive group; L1, L2 are each linker units; and p is either 0 or 1.

[0002] A second aspect of the present invention relates to a kit comprising a derivatization agent according to the first aspect. In a third aspect, the present invention relates to the use of a derivatization agent according to the first aspect for mass spectrometric determination of an analyte molecule, wherein the mass spectrometric determination is LDI-MS. A fourth aspect of the present invention relates to a conjugate of a derivatization agent and an analyte according to the first aspect, the conjugate having the formula (II)C-L1-Z-(L2) p A fifth aspect of the invention relates to a conjugate having the structure -Xa-Ya-A, wherein C, L1, L2, p, Z, and N are as defined in the context of the first aspect; Xa is the remainder of reactive group X as defined in the context of the first aspect; A is an analyte, and Ya is the remainder of reactive group Y attached to analyte A, which has reacted with reactive group X of the derivatization agent to form a covalent bond between Xa and Ya. A fifth aspect of the invention relates to a method for the mass spectrometric determination of an analyte molecule, comprising the steps of: (a) providing an analyte of interest; (b) providing a derivatization agent comprising the structure of formula (I), as defined in the context of the first aspect; (c) reacting the analyte provided in accordance with (a) with the derivatization agent provided in accordance with (b), thereby forming a conjugate of the analyte and the derivatization agent; and (d) subjecting the conjugate formed in (c) to mass spectrometry, wherein the mass spectrometry is preferably LDI-MS. [Background technology]

[0003] cutting edge Mass spectrometry (MS) is a widely used technique for the qualitative and quantitative analysis of chemicals ranging from small molecules to macromolecules. Generally, it is a highly sensitive and specific method, even capable of analyzing complex biological samples, such as environmental or clinical samples. However, measurement sensitivity remains an issue for some analytes, especially when analyzed from complex biological matrices such as serum.

[0004] MS is often combined with chromatographic techniques, particularly gas and liquid chromatography, such as HPLC, where analyte molecules of interest are separated by chromatography and individually subjected to mass spectrometry (Higashi et al. (2016) J. of Pharmaceutical and Biomedical Analysis 130 p.181-190).

[0005] However, there remains a need to increase the sensitivity of MS analytical methods, especially for the analysis of analytes in low abundance or when scarce material is available (such as biopsy tissue).

[0006] Several derivatization reagents aimed at improving the sensitivity of the measurement of these analytes are known in the art. These include, inter alia, reagents containing a charged unit and a neutral loss unit combined into a single functional unit (e.g., WO 2011 / 091436); other reagents for introducing a neutral loss unit and a charged unit are known, for example, from WO 2020 / 020850. Other reagents containing separate units are relatively bulky in structure, affecting 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, Cookson-type reagents, Amplifex Diene, Amplifex Keto, Girard T, and Girard P. All of these methods often suffer from drawbacks due to insufficient labeling efficiency, the generation of structural isomers due to coupling chemistry, suboptimal ionization efficiency, adverse effects on post-coupling chromatographic separation, suboptimal fragmentation behavior due to multiple fragmentation pathways, and the need for high collision energy. Therefore, there is an urgent need in the art for derivatization reagents that not only exhibit chemical structures that do not adversely affect the MS measurement workflow, but also enable highly sensitive detection of analytes from complex biological matrices. This is particularly important in random-access, high-throughput MS setups, where several different analytes exhibiting different chemical properties must be measured in a short period of time.

[0007] Chemical derivatization of analytes of interest can be used to enhance detection sensitivity in mass spectrometry applications. In most cases, to improve mass spectrometry response / sensitivity, charged (i) or chargeable (ii) compounds are used to generate (i) permanently charged or (ii) chargeable derivatized analytes. Most of these reagents are intended to improve ESI response but are not designed to generate specific product ions by CID for MS / MS applications. To address this issue, several derivatization reagents with structures suitable for MS / MS detection have been developed. In general, numerous derivatization reagents are available for liquid chromatography-based mass spectrometry (i.e., LC-MS, LC-MS / MS; see reviews in J. Sep. Sci. 2016, 39, 102-114; Biomed. Chromatogr. 2011; 25:1-10) and laser desorption-based applications in combination with an appropriate matrix (i.e., MALDI; see reviews in Trends in Analytical Chemistry 143(2021)116399; J. Mass Spectrom. 2021; 56:e4731). However, the vast number of available derivatization reagents lack suitable chromophores to enable efficient laser energy transfer in LDI applications and are therefore unsuitable for highly sensitive LDI measurements. As a result, these reagents must be used in combination with a matrix, which increases background and interferences and consequently reduces sensitivity. Few examples of matrix-free derivatization reagents for laser desorption applications have been reported, so-called "reactive matrix" (i.e., Anal. Chem. 2020, 92, 6224-6228; Chem Asian J. 2021, 16, 868-878 and Crit Rev Anal Chem. 2021 Dec 30; 1-17) or "LDI labeling" (i.e., Mass Spec Rev. 2019; 38: 3-21; Scientific Reports|5: 17853, ChemBioChem 2021, 22, 1430-1439;).Most of these molecules offer suitable chromophores but are not permanently charged, which is a disadvantage in terms of sensitivity and is not designed for MS / MS applications. While rare examples of permanently charged chromophore derivatization reagents have been reported (i.e., International Journal of Mass Spectrometry 353 (2013) 54-59), these reagents lack suitable neutral loss sites and are therefore unsuitable for MS / MS applications. In most cases, the derivatization reagents are small molecules, and if the analyte is also a small molecule, they still produce a low-molecular-weight derivatized analyte, or in either case, only a small mass shift after derivatization. This can be problematic for some mass spectrometry applications, as sensitive measurements in the low-molecular-weight range are often hindered by matrix-based MS interferences in this range.

[0008] Therefore, the problem underlying this invention was the need for a permanently charged chromophore derivatization reagent with a suitable neutral loss site. Summary of the Invention

[0009] First Aspect—Derivatizing Agent In a first aspect, the problem is solved by providing a derivatizing agent, preferably a derivatizing agent for analytes intended to be analyzed by laser desorption ionization mass spectrometry (LDI-MS), comprising a structural element of formula (I), C-L1-Z-(L2) p -X(I) During the ceremony, C is a chromophore having an absorption maximum in the range of 280-400 nm; Z is a charge unit containing at least one permanently charged moiety; X is a reactive group; L1 and L2 are each a linker unit; p is either 0 or 1; This was resolved by using a derivatizing agent.

[0010] The derivatization agent according to the present invention provides a solution to challenging high-sensitivity measurements, preferably, but not limited to, in the field of laser desorption / ionization (LDI)-based mass spectrometry (MS). The advantage of the derivatization agent is improved sensitivity in LDI-MS applications. The term "LDI-MS" includes (MA)LDI-MS, preferably (MA)LDI-MS / MS, and (SA)LDI-MS, preferably (SA)LDI-MS / MS, with "MALDI" (matrix-assisted laser desorption / ionization) and "SALDI" (surface-assisted laser desorption / ionization) being well-known and described in more detail below in the section relating to the third aspect of the present invention. The derivatization agent is distinguished from other reagents / solutions known in the art by its chemical structure concept and its principle of action. On the one hand, the derivatization agent possesses a suitable chromophore that enables efficient energy transfer during LDI. On the other hand, the derivatization agent adds a sufficiently large molecular weight to the analyte of interest, which results in a sufficient mass shift to overcome the high noise background of biological samples in the low molecular weight region. Generally, such high molecular weights are known to result in unfavorable ionization characteristics (low ionization efficiency, multiple fragmentation processes, etc.), but in this case, this is circumvented by the presence of a permanent positive charge (the Z unit in formula (I)). Thus, precursor ions (parent ions) can be detected with high sensitivity and optionally selected for fragmentation in MS / MS applications. Efficient fragmentation is ensured by the specific combination of the Z unit and the N unit, which, in some embodiments outlined in more detail below, is a quaternary amine group adjacent to the benzyl position, enabling a smooth and selective neutral loss fragmentation process. During MS / MS, the conjugate of the derivatization reagent and the analyte undergoes fragmentation by releasing large fragments and product ions (the analyte of interest modified with the benzyl cation).This large mass shift is also highly advantageous, as it ensures low background / interference for this novel reagent class, since such a specific and sensitive neutral loss pathway via a large molecular weight loss is highly unusual (see Anal. Chem. 2014, 86, 21, 10724-10731).

[0011] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It should also 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.

[0012] 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.

[0013] Each element of the present invention is described below. While these elements are listed with specific embodiments, it is understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application unless the context indicates otherwise.

[0014] The word "comprise," and variations such as "comprises" and "comprising," will be understood to 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.

[0015] 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.

[0016] Ratios, concentrations, amounts, and other numerical data may be expressed or presented herein in the form of a "range." It is understood that such range format is used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values ​​explicitly recited as boundaries of the range, but also all of the individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. By way of example, a numerical range of "4-20%" should be interpreted not only to include the explicitly recited value 4%-20%, but also to include each individual value and subrange within the stated range. Thus, this numerical range includes individual values ​​such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20%, etc., and subranges such as 4-10%, 5-15%, 10-20%, etc. This same principle applies to ranges reciting minimum or maximum values. Moreover, such interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0017] 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.

[0018] The term "mass spectrometry" or "MS" 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 producing ions of an analyte with a net charge equal to one or more electron units. Negative ions are those that have a net negative charge of one or more electronic units, while positive ions are those that have a net positive charge of one or more electronic 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.

[0019] "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).

[0020] Although ionization sources such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are known, a reliable tool for ionization is laser desorption ionization (LDI), for which a common laser is, for example, an ultraviolet (355 nm) Nd:YAG laser (neodymium-doped yttrium aluminum garnet; Nd:YAlO 12 )

[0021] Mass spectrometers separate and detect ions with slightly different masses, so they can easily distinguish between different isotopes of a given element.Therefore, mass spectrometry is 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 amino acid sequence.

[0022] 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. In the context of this disclosure, the terms "analyte," "analyte molecule," or "analyte of interest" are used interchangeably to refer to chemical species that are analyzed by mass spectrometry. Chemical species, or analytes, suitable for analysis via 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, cytosolic proteins, etc.), metabolites or hormones (e.g., testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (e.g., vitamin D), molecules characteristic of specific modifications of other molecules (e.g., sugar moieties or phosphoryl residues on proteins, methyl residues on genomic DNA), or substances internalized by an organism (e.g., therapeutic drugs, drugs of abuse, toxins, etc.), or metabolites of such substances. Such analytes can serve as biomarkers. In the context of the present invention, the term "biomarker" refers to a substance in a living system that is used as an indicator of the biological state of that system. Most sample workflows in MS further include sample preparation and / or enrichment steps, for example, by separating the analyte(s) of interest from the matrix using gas or liquid chromatography.

[0023] In some preferred embodiments of the derivatizing agent, the absorption maximum of chromophore C is an adsorption maximum determined by UV / VIS spectroscopy. Preferably, the absorption maximum of chromophore C is in the range of 290 to 380 nm, more preferably in the range of 300 to 360 nm, and more preferably in the range of 305 to 330 nm.

[0024] In some preferred embodiments of the derivatizing agent, the chromophore C has the structure of formula (C): [ka] In the formula, R 1 , R 2 , R 3 , R 4 , R 5 is a hydrogen atom; a hydroxyl group; NR x R y group [R x and R y are independently a hydrogen atom or a C1-C5 alkyl group; independently selected from the group consisting of C1-C5 alkyl, C5-C10 (hetero)aryl and -O-C1-C3 alkoxy groups; R 6 is either absent (i.e., there is a direct single covalent bond between the aromatic ring and the C(=O) group) or -CR 7 =CR 8 - group, wherein R 7 is a hydrogen atom or a C1-C3 alkyl group, and R 8 is selected from the group consisting of a hydrogen atom, a C1-C5 alkyl group, and an electron-withdrawing group; the dotted line represents a bond to the linker, and is preferably a single bond.

[0025] In some preferred embodiments of the derivatizing agent of formula (C), R 1 , R 2 , R 3 , R 4 , R 5 represents a hydrogen atom, a hydroxyl group, and NR x R y group [R x and R y are independently a hydrogen atom or a C1-C5 alkyl group, and an —O—C1-C3 alkoxy group. More preferably, R 1 , R 2 , R 3 , R 4 , R 5 are independently a hydrogen atom, a hydroxyl group, or NR x R y group [R x and R y are independently selected from the group consisting of a hydrogen atom or a C1-C5 alkyl group, and an —O—C1-C3 alkoxy group, provided that R 1, R 2 , R 3 , R 4 , R 5 provided that at least one of R is either a hydroxyl group or an —O—C1-C3 alkoxy group. 8 is preferably selected from a cyano group, a nitro group, a carboxyl group, a halogen atom (preferably a fluoro atom, a chloro atom, a bromine atom or an iodine atom), and an aryl group, where "aryl" is preferably selected from the group of C5-C10 (hetero)aryl groups, more preferably phenyl, and R 8 The electron-withdrawing group is more preferably cyano (C≡N group).

[0026] In some preferred embodiments of the derivatizing agent, the chromophore C has the structure of formula (C1), (C2), or (C3): [ka] In the formula, R 1 , R 2 , R 3 , R 4 is a hydrogen atom, Hydroxyl group NR x R y group [R x and R y are independently selected from the group consisting of a hydrogen atom or a C1-C5 alkyl group, a C1-C5 alkyl group, a C5-C10 (hetero)aryl group and an —O—C1-C3 alkoxy group.

[0027] For (C1), (C2) and (C3), in some preferred embodiments, R 1 , R 2 , R 3 , R 4 , R 5 represents a hydrogen atom, a hydroxyl group, and NR x R y group [R x and R yare independently a hydrogen atom or a C1-C5 alkyl group, and an —O—C1-C3 alkoxy group. 1 , R 2 , R 3 , R 4 , R 5 are independently a hydrogen atom, a hydroxyl group, or NR x R y group [R x and R y are independently selected from the group consisting of a hydrogen atom or a C1-C5 alkyl group, and an —O—C1-C3 alkoxy group, provided that R 1 , R 2 , R 3 , R 4 , R 5 provided that at least one of R is either a hydroxyl group or an —O—C1-C3 alkoxy group. 8 is preferably selected from a cyano group, a nitro group, a carboxyl group, a halogen atom (preferably a fluoro atom, a chloro atom, a bromine atom or an iodine atom), and an aryl group, where "aryl" is preferably selected from the group of C5-C10 (hetero)aryl groups, more preferably phenyl, and R 8 The electron-withdrawing group is more preferably cyano (C≡N group).

[0028] In some preferred embodiments of the derivatizing agent, the chromophore C has the structure of formula (C1), where R 1 and R 4 are both methoxy groups, and R 3 are hydroxyl groups (these residues R 2 , R 3 and R 4 (wherein C1 is the remainder of sinapinic acid). In some preferred embodiments of the derivatizing agent, the chromophore C has the structure of formula (C2), where R 2 and R 4 are both hydroxyl groups (these residues R 2 , R 4(wherein C2 has the residue of 2,5-dihydroxybenzoic acid). In some preferred embodiments of the derivatizing agent, the chromophore C has the structure of formula (C3), where R 3 is a hydroxyl group (this residue R 3 C3 is the remainder of α-cyano-4-hydroxy-cinnamic acid).

[0029] In some preferred embodiments of the derivatizing agent, the linker L1 is (C1-C5 alkylene-O-) m a group wherein m is an integer in the range of 1 to 10, a C1-C20 alkylene group, a C1-C20 alkylene group-heteroaryl group, and a (C1-C5 alkylene)-O-(C1-C5 alkylene) group, optionally linked to or intersected by a unit selected from the group consisting of a (hetero)aryl group, N2, NO, NO2, S2, SO, SO2, CO, and CO2, which unit, when present, is preferably a heteroaryl group, more preferably from triazole, phenyltriazole, tetrazole, and phenyltetrazole. The "heteroaryl" is preferably a C1-C10 heteroaryl having at least one heteroatom as part of the ring structure, the at least one heteroatom being preferably selected from N, O, and S. More preferably, the heteroaryl is selected from the group consisting of triazole, tetrazole, tetrazine, oxadiazole, thiadiazole, and any hydrogenated derivative thereof, more preferably 1,2,3-triazole, 1,2,4-triazole, 1,4,5-triazole, 3,4,5-triazole, 1,2,3,4-tetrazole, 2,3,4,5-tetrazole, 2,3,5,6-tetrazole, and 1,2,4,5 tetrazine. In some preferred embodiments of the derivatizing agent, the linker L1 has the structure (L1a): q -(L1b) r -(L1c) s wherein q, r, and s are each 0 or 1, provided that at least one of q, r, and s is 1; L1a is (C1-C5 alkylene-O-) mL1b is a unit selected from the group consisting of N2, NO, NO2, S2, SO, SO2, CO, CO2, triazole, phenyltriazole, tetrazole and phenyltetrazole, where the unit is preferably triazole or tetrazole, more preferably unit N is triazole, more preferably unit N is a 1,2,3 triazole ring which is bonded to linker L1a (if present) via a single bond at position 1 (N atom) of the triazole ring and to linker L1c (if present) via a single bond at position 4 (C atom) of the triazole ring; L1c (if present) is (C1-C5 alkylene-O-) m In some preferred embodiments of the derivatizing agent, the linker L1 has the following structure: [ka] where the dotted lines indicate the bonds to (C) and (Z), respectively.

[0030] In some preferred embodiments of the derivatization agent, the charged unit Z is positively or negatively charged, preferably positively charged. In some preferred embodiments of the derivatization agent, the charged unit Z is positively charged, preferably a tetraalkylammonium group, more preferably -CHN + In some preferred alternative embodiments of the derivatizing agent, the charged unit Z is negatively charged, and the negatively charged unit Z is preferably selected from the group consisting of phosphate, sulfate, sulfonate and carboxylate.

[0031] In some preferred embodiments of the derivatizing agent, the linker L2 comprises 1 to 10 C atoms and optionally one or more heteroatoms. Preferably, the linker L2 is a C1-C5-alkylene-C5-C10 aromatic ring, preferably having a C(=O) unit as a substituent of the aromatic ring, more preferably a C1-C3-alkylene-C6 aromatic ring, preferably having a C(=O) unit as a substituent of the aromatic ring, and the C6 aromatic ring is more preferably a substituted or unsubstituted benzene ring, more preferably a benzene ring having a C(=O) group para to the position to which the C1-C3-alkylene is bonded, more preferably a benzene ring having a C(=O) group at a position on the benzene ring that does not have any further substituents.

[0032] In some preferred embodiments of the derivatizing agent, the reactive group X is selected from the group consisting of a carbonyl-reactive unit, a diene-reactive unit, a hydroxyl-reactive unit, an amino-reactive unit, an imine-reactive unit, a thiol-reactive unit, a diol-reactive unit, a phenol-reactive unit, an epoxide-reactive unit, a disulfide-reactive unit, and an azide-reactive unit.

[0033] In some preferred embodiments of the derivatizing agent, the reactive unit X is a carbonyl-reactive unit, which can react with any type of molecule having a carbonyl group. The carbonyl-reactive unit is preferably 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 some preferred embodiments of the derivatizing agent, the carbonyl-reactive unit can have either an adjacent O or N atom NH2-N / O, or a hypernucleophilic N atom enhanced by the a-effect via a dithiol molecule.

[0034] In some preferred embodiments of the derivatizing agent, the carbonyl-reactive unit is selected from the following groups: (i) a hydrazine unit, such as HN-NH-, or HN-NR a-Unit [R a is aryl, aryl containing one or more heteroatoms, or C alkyl, particularly C or C alkyl optionally substituted, for example, with halo, hydroxyl, and / or C alkoxy; (ii) a hydrazide unit, in particular a carbohydrazide or sulfohydrazide unit, in particular HN—NH—C(O)—, or HN—NR b -C(O)- units, [R b is aryl, aryl containing one or more heteroatoms, or C alkyl, particularly C or C alkyl optionally substituted, for example, with halo, hydroxyl, and / or C alkoxy; (iii) a hydroxylamino unit, e.g., a HN—O— unit, and (iv) Dithiol units, in particular 1,2-dithiol or 1,3-dithiol units.

[0035] In some preferred embodiments of the derivatizing agent where the carbonyl-reactive unit is a carboxyl-reactive unit, the carboxyl-reactive unit reacts with a carboxyl group on the 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.

[0036] In some preferred embodiments of the derivatization agent, the reactive unit X is a diene-reactive unit that can react with an analyte having a diene group. In some preferred embodiments of the derivatization agent, 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.

[0037] In some preferred embodiments of the derivatization agent, the reactive unit X is a hydroxyl-reactive unit, which can react with an analyte having a hydroxyl group. In some preferred embodiments of the derivatization agent, 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. 2016 Sep;162:57-69). In some preferred embodiments of the derivatization agent, the reactive unit X is a diol-reactive unit that reacts with a diol group on an analyte molecule. In some preferred embodiments of the derivatization agent, the reactive unit is a 1,2-diol-reactive unit, the 1,2-diol-reactive unit comprises a boronic acid. In further embodiments, the diol can be oxidized to the respective ketone or aldehyde and then reacted with the ketone / aldehyde-reactive unit X. In some preferred embodiments of the derivatization agent, the amino-reactive unit reacts with an amino group on an analyte molecule. In some preferred embodiments of the derivatizing agent, the amino-reactive unit is selected from the group consisting of active ester groups such as N-hydroxysuccinimide (NHS) esters or sulfo-NHS esters, pentafluorophenyl esters, carbonylimidazole esters, squaric acid esters, hydroxybenzotriazole (HOBt) esters, 1-hydroxy-7-azabenzotriazole (HOAt) esters, and sulfonyl chloride units.

[0038] In some preferred embodiments of the derivatization agent, the thiol-reactive unit reacts with a thiol group on the analyte molecule. In some preferred embodiments of the derivatization agent, the thiol-reactive unit is selected from the group consisting of a haloacetyl group, in particular selected from the group consisting of an unsaturated imide unit such as a Br / I-CH2-C(=O)- unit, an acrylamide / ester unit, a maleimide, a methylsulfonylphenyloxadiazole, and a sulfonyl chloride unit.

[0039] In some preferred embodiments of the derivatization agent, the phenol-reactive unit reacts with a phenol group on an analyte molecule. In some preferred embodiments of the derivatization agent, the phenol-reactive unit is selected from the group consisting of an active ester unit, such as an N-hydroxysuccinimide (NHS) ester or sulfo-NHS ester, a pentafluorophenyl ester, a carbonylimidazole ester, a squaric acid ester, a hydroxybenzotriazole (HOBt) ester, a 1-hydroxy-7-azabenzotriazole (HOAt) ester, and a sulfonyl chloride unit. The phenol group present on the analyte molecule can be reacted with a triazoledione by reaction (H. Ban et al. J. Am. Chem. Soc., 2010, 132(5), pp. 1523-1525), or by diazotization, or alternatively by ortho-nitration, followed by reduction to an amine, which can then be reacted with an amine-reactive reagent.

[0040] In some preferred embodiments of the derivatization agent, the reactive unit X is an epoxide-reactive unit, which can react with an analyte having an epoxide group. In some preferred embodiments of the derivatization agent, the epoxide-reactive unit is selected from the group consisting of amino, thiol, and a supernucleophilic N atom, enhanced by the a-effect via the adjacent O or N atom NH-N / O molecule.

[0041] In some preferred embodiments of the derivatizing agent, the epoxide-reactive unit is selected from the following groups: (i) a hydrazine unit, such as HN-NH-, or HN-NR a -Unit [R a is aryl, aryl containing one or more heteroatoms, or C alkyl, particularly C or C alkyl optionally substituted, for example, with halo, hydroxyl, and / or C alkoxy; (ii) a hydrazide unit, in particular a carbohydrazide or sulfohydrazide unit, in particular HN—NH—C(O)—, or HN—NR b -C(O)- units, [R b is aryl, aryl containing one or more heteroatoms, or Ci-4 alkyl, particularly Ci or C2 alkyl optionally substituted with, for example, halo, hydroxyl, and / or C1-3 alkoxy; and (iii) a hydroxylamino unit, such as a H2N-O- unit.

[0042] In some preferred embodiments of the derivatization agent, the reactive unit X is a disulfide-reactive unit, which can react with an analyte containing a disulfide group. In some preferred embodiments of the derivatization agent, the disulfide-reactive unit is selected from the group consisting of thiols. In further embodiments, the disulfide groups can be reduced to the respective thiol groups and then reacted with the thiol-reactive unit X.

[0043] In some preferred embodiments of the derivatization agent, the reactive unit X is an azide-reactive unit that reacts with an azide group on an analyte molecule. In some preferred embodiments of the derivatization agent, the azide-reactive unit reacts with the azide group via azide-alkyne cycloaddition. In some preferred embodiments of the derivatization agent, 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 further embodiments 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 X.

[0044] Second Aspect—Kit A second aspect of the present invention relates to a kit comprising a derivatizing agent according to the first aspect. All details, embodiments and preferred embodiments disclosed above in the section relating to the first aspect also apply to the kit of the second aspect.

[0045] A "kit" is any article of manufacture (e.g., package or container) containing at least one reagent, e.g., an agent for the treatment of a disorder, or a probe for specifically detecting a biomarker gene or protein of the invention. The kit is preferably promoted, distributed, or sold as a unit for carrying out the uses and / or methods of the invention, as described below in the sections relating to the third and fourth aspects of the invention.

[0046] Typically, the kit may further comprise a carrier means compartmentalized to receive one or more container means, such as vials and tubes, under close supervision. In particular, each of the container means contains one of the separate elements used in the method of the first embodiment. The kit may further comprise one or more other containers containing additional materials, including, but not limited to, buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. Labels may be provided on the container to indicate that the composition is to be used for a particular application, and may also indicate instructions for either in vivo or in vitro use. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disc), or directly on a computer or data processing device. Additionally, the kit may contain standard amounts of biomarkers, as described elsewhere herein, for calibration purposes.

[0047] "Package insert" is used to refer to instructions customarily included in commercial packaging of a therapeutic or drug product, which contain information about the indications, uses, dosage, administration, contraindications of such therapeutic or drug product, other therapeutic products with which the packaged product is combined, and / or warnings regarding their use.

[0048] Third Aspect—Use of Derivatizing Agents for Mass Spectrometric Determination of Analyte Molecules In a third aspect, the present invention relates to the use of a derivatization agent according to the first aspect for mass spectrometric determination of analyte molecules, wherein the mass spectrometric determination is laser desorption / ionization mass spectrometry (LDI-MS), preferably (MA)LDI-MS, more preferably (MA)LDI-MS / MS, or (SA)LDI-MS, more preferably (SA)LDI-MS / MS. All details, embodiments and preferred embodiments disclosed above in the sections relating to the first and second aspects also apply to the use of the third aspect. "MS" and "MS / MS" have the meanings explained above in the sections relating to the first aspect.

[0049] The term "LDI" is a common abbreviation for laser desorption / ionization (LDI), and when combined with an appropriate matrix, it is called matrix-assisted laser desorption / ionization (MALDI). Matrix materials and supports, such as metals, especially steel, plates, are known to those skilled in the art, as are the conditions used. The matrix typically consists of crystallizing molecules, the three most commonly used of which are sinapinic acid, alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA, alpha-cyano or alpha-matrix), and 2,5-dihydroxybenzoic acid (DHB). MALDI techniques typically employ the use of UV lasers, such as nitrogen lasers (337 nm) and frequency-tripled and frequency-quadrupled Nd:YAG lasers (355 nm and 266 nm, respectively). Infrared laser wavelengths used for infrared MALDI include 2.94 μm Er:YAG lasers, mid-infrared optical parametric oscillators, and 10.6 μm carbon dioxide lasers.

[0050] Surface-assisted laser desorption / ionization (SALDI) is a soft laser desorption technique that uses a medium to absorb energy from a laser and then transfer it to the target sample without the use of matrix molecules, in which the active surface of a specific substrate plays a crucial role. A key substrate is the solid surface of porous silicon. Porous silicon represented the first matrix-free SALDI surface analysis, allowing for the easy detection of intact molecular ions. Many different surfaces are known to function as SALDI substrates. Based on elemental composition, the majority of SALDI substrates reported in the literature can generally be classified into three main types: carbon-based, semiconductor-based, and metal-based. SALDI processes using inorganic matrices for preparation have been described in several studies, such as Law et al. (Anal. Bioanal. Chem. 2011, 399, 2597, DOI 10.1007 / s00216-010-4063-3). Preferably, in the context of the present invention, a SALDI-MS target plate based on a steel sheet having a functional amorphous aC:H:Si:X (X=heteroatom modified) plasma activated chemical vapor deposition (PACVD) surface coating as the top layer is used.

[0051] Laser desorption / ionization mass spectrometry (LDI-MS) measurements are preferably performed in positive ion mode using a 355 nm Nd:YAG laser. The laser repetition rate of the LDI-MS system is set appropriately for MALDI and SALDI experiments, respectively. Additional measurement parameters, such as migration pattern, migration speed, frequency, acquisition time, mass spectrum scan time, laser intensity, or voltage settings, are selected as needed and are known to those skilled in the art who are also familiar with the necessary software-based data analysis tools.

[0052] Fourth Aspect—Conjugates A fourth aspect of the present invention provides a conjugate of a derivatization agent and an analyte according to the first aspect of the present invention, having the structure of formula (II): C-L1-Z-(L2) p -Xa-Ya-A(II) wherein C, L1, L2, p, Z and N are as defined in the section relating to the first aspect; Xa is the remainder of the reactive group X as defined in the section relating to the first aspect; A is an analyte and Ya is the remainder of the reactive group Y attached to the analyte A which has reacted with the reactive group X of the derivatising agent to form a covalent bond between Xa and Ya. All details, embodiments and preferred embodiments described above in the sections relating to the first, second and third aspects of the invention also apply to the conjugate of the fourth aspect, and in particular all details, embodiments and preferred embodiments described above in the section relating to the first aspect also apply here.

[0053] In some preferred embodiments of the conjugate, the analyte is selected from the group consisting of nucleic acids (preferably selected from DNA, mRNA, miRNA, and rRNA), amino acids, peptides, proteins (preferably cell surface receptors or cytosolic proteins), metabolites, hormones (preferably selected from testosterone, estrogen, and estradiol), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (vitamin D), molecules characteristic of a particular modification of another molecule (preferably selected from a sugar moiety, a phosphoryl residue on a protein, or a methyl residue on genomic DNA), substances internalized by an organism (preferably selected from therapeutic agents, drugs of abuse, and toxins), and metabolites of such substances.

[0054] The analyte may be present in a sample of interest, such as a biological sample or a clinical sample. The terms "sample" and "sample of interest" are used interchangeably herein and refer to a portion or piece of a tissue, organ, or individual, usually smaller than the tissue, organ, or individual intended to represent the entire tissue, organ, or individual. Upon analysis, the sample provides information about the state of the tissue, or the health or disease state of the organ or individual. Examples of samples include, but are not limited to, liquid samples such as blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva, and lymphatic fluid, or solid samples such as dried blood spots and tissue extracts. Another example of a sample is a cell culture or tissue culture.

[0055] 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).

[0056] 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 means necessary to enable subsequent analysis of the desired analyte by mass spectrometry. Pretreatment means typically include, but are not limited to, elution of solid samples (e.g., elution of dried blood spots), addition of a hemolyzing reagent (HR) to whole blood samples, and addition of an enzyme reagent to urine samples. Similarly, addition of an internal standard (ISTD) is considered sample pretreatment.

[0057] The term "hemolytic reagent (HR)" refers to a reagent that lyses cells present in a sample, and in the context of the present invention, refers to a hemolytic reagent, particularly 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, liquids with high osmolality (e.g., 8M urea), ionic liquids, and various detergents.

[0058] 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 spectrometry detection workflow (i.e., including any pretreatment, enrichment, and actual detection steps). The ISTD exhibits similar properties to the analyte of interest, but is clearly distinguishable from it. For example, during a chromatographic separation, such as gas or liquid chromatography, the ISTD has approximately the same retention time as the analyte of interest from the sample. Thus, both the analyte and the ISTD enter the mass spectrometer simultaneously. The ISTD, however, exhibits a different molecular weight than the analyte of interest from the sample. This allows ions from the ISTD and the analyte to be distinguished in mass spectrometry using their different mass-to-charge (m / z) ratios. Both are subjected to fragmentation to produce daughter ions. These daughter ions can be distinguished by their m / z ratios and their respective parent ions. As a result, the signals from the ISTD and the analyte can be determined and quantified separately. Because the ISTD is added in a known amount, the signal intensity of an analyte from 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., containing a label such as 2H, 13C, or 15N).

[0059] 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 process that occurs following sample pretreatment and provides a sample containing enriched analytes compared to the initial sample. The first enrichment workflow may involve 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 may be made of a variety of different materials. The beads may have various sizes and may have porous or non-porous surfaces.

[0060] In the context of the present disclosure, the term "second enrichment process" or "second enrichment workflow" refers to an enrichment process that occurs following sample pretreatment and a first enrichment process and provides a sample containing enriched analytes relative to the initial sample and the sample after the first enrichment process.

[0061] In some preferred embodiments of the conjugate, the reactive group Y is selected from the group consisting of a carbonyl group, a diene group, a hydroxyl group, an amine group, an imine group, a thiol group, a diol group, a phenol group, an epoxide group, a disulfide group, and an azide group.

[0062] In some preferred embodiments of the conjugate, the analyte molecule, prior to reaction with the derivatizing agent, comprises a functional group selected from the group above, each of the functional groups set forth in this group being capable of forming a covalent bond with the reactive unit X of the derivatizing agent. Furthermore, it is also considered within the scope of the present invention that a functional group present on the analyte molecule is first converted into another group that is readily available by reaction with the reactive unit X of the derivatizing agent.

[0063] In some embodiments, the analyte molecule contains 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 prior to reaction with a derivatizing agent. In embodiments in which the carbonyl group is an amide group, those skilled in the art will recognize that, although the amide group itself is a stable group, it can be hydrolyzed to a carboxylic acid group and an amino group. Hydrolysis of the amide group can be achieved by acid / base catalysis or by enzymatic processes, both of which are well known to those skilled in the art. In embodiments in which the carbonyl group is a masked aldehyde group or a masked keto group, the respective group is either a hemiacetal group or an acetal group, particularly a cyclic hemiacetal group or an acetal group. In some embodiments, the acetal group is converted to an aldehyde group or a keto group prior to reaction with a derivatizing agent.

[0064] In some embodiments, the carbonyl group is a keto group. The keto group can be transferred to an imine group of the intermediate before reacting with the reactive unit of the derivatizing agent. In some embodiments, the analyte molecule containing one or more keto groups is preferably a ketosteroid. In certain embodiments, 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, DHEA (dehydroepiandrosterone), 17-OH pregnenolone, 17-OH progesterone, 17-OH progesterone, androsterone, epiandrosterone, and Δ4 androstenedione), 11-desoxycortisol corticosterone, 21-deoxycortisol, 11-deoxycorticosterone, allopregnenolone, and aldosterone.

[0065] In some embodiments, the carbonyl group is a carboxyl group. The carboxyl group is reacted directly with a derivatizing agent or converted to an activated ester group before reacting with a derivatizing agent. In some embodiments, the analyte molecule containing one or more carboxyl groups is selected from the group consisting of D8-tetrahydrocannabinolic acid, benzoylecgonine, salicylic acid, 2-hydroxybenzoic acid, gabapentin, pregabalin, valproic acid, vancomycin, methotrexate, mycophenolic acid, montelukast, repaglinide, furosemide, telmisartan, gemfibrozil, dichlorofenac, ibuprofen, indomethacin, zomepirac, isoxepak, and penicillin. In some embodiments, the analyte molecule comprising one or more carboxyl groups is preferably an amino acid preferably 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.

[0066] In some embodiments, the carbonyl group is an aldehyde group. The aldehyde group can be transferred to an imine group of an intermediate before reacting with the reactive unit of the derivatizing agent. In some embodiments, the analyte molecule containing one or more aldehyde groups is preferably selected from the group consisting of pyridoxal, N-acetyl-D-glucosamine, alcaftadine, streptomycin, and josamycin.

[0067] In some embodiments, the carbonyl group is a carbonyl ester group. The analyte molecule containing one or more ester groups is preferably selected from the group consisting of cocaine, heroin, Ritalin, aceclofenac, acetycholine, amcinonide, amiloxate, amylocaine, anileridine, aranidipine, and artesunate, pethidine.

[0068] In some embodiments, the carbonyl group is an anhydride group. The analyte molecule containing one or more anhydride groups is preferably selected from the group consisting of cantharidin, succinic anhydride, trimellitic anhydride, and maleic anhydride.

[0069] In some embodiments, the analyte molecule comprises one or more diene groups as functional groups, particularly conjugated diene groups. The analyte molecule comprising one or more diene groups is preferably a secosteroid. In some embodiments, the secosteroid is selected from the group consisting of cholecalciferol (vitamin D3), ergocalciferol (vitamin D2), calcidiol, 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, 3-epi-25-hydroxyvitamin D2, 3-epi-25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D2, and 24,25-dihydroxyvitamin D3, vitamin A, tretinoin, isotretinoin, alitretinoin, natamycin, sirolimus, amphotericin B, nystatin, everolimus, temsirolimus, and fidaxomicin.

[0070] In some embodiments, the analyte molecule comprises one or more hydroxyl groups as functional groups. The analyte molecule then preferably comprises a single hydroxyl group or two hydroxyl groups. In embodiments where multiple hydroxyl groups are present, the two hydroxyl groups (1,2 diol) may be located adjacent to each other or may be separated by one, two, or three C atoms (1,3-diol, 1,4-diol, 1,5-diol, respectively). In certain embodiments, the analyte molecule comprises a 1,2 diol group. In embodiments where only one hydroxyl group is present, the analyte is preferably selected from the group consisting of primary alcohols, secondary alcohols, and tertiary alcohols. In some embodiments in which the analyte molecule contains one or more hydroxyl groups, the analyte is preferably selected from the group consisting of benzyl alcohol, menthol, L-carnitine, pyridoxine, metronidazole, isosorbide mononitrate, guaifenesin, clavulanic acid, migitol, zalcitabine, isoprenaline, acyclovir, methocarbamol, tramadol, venlafaxine, atropine, clofedanol, α-hydroxyalprazolam, α-hydroxytriazolam, lorazepam (forazepam), oxazepam, tamazepam, ethyl glucuronide, ethylmorphine, morphine, morphine-3-glucuronide, buprenorphine, codeine, dihydrocodeine, p-hydroxypropoxyphene, O-desmethyltramadol, dihydroquinidine, and quinidine.In some embodiments where the analyte molecule comprises multiple hydroxyl groups, the analyte is preferably selected from the group consisting of vitamin C, glucosamine, mannitol, tetrahydrobiopterin, cytarabine, azacytidine, ribavirin, floxuridine, gemcitabine, streptozocin, adenosine, bivalavirin, cladribine, estriol, trifluridine, clofarabine, nadolol, zanamivir, lactulose, adenosine monophosphate, idoxuridine, regadenoson, lincomycin, clindamycin, canaglifodine, tobramycin, Selected from the group consisting of netilmicin, kanamycin, ticagrelor, epirubicin, doxorubicin, arbekacin, steptomycin, quavacin, amikacin, neomycin, framycetin, paromomycin, erythromycin, clarithromycin, azithromycin, vindesine, digitoxin, digoxin, metrizamide, acetyldigitoxin, deslanoside, fludalazine, clofarabine, gemcitabine, cytarabine, capecitabine, vidarabine, trifluridine, idoxuridine, and plicamycin.

[0071] In some embodiments, the analyte molecule comprises one or more thiol groups (including, but not limited to, alkylthiol groups and thiol ary groups) as functional groups. The analyte molecule comprising one or more thiol groups is preferably selected from the group consisting of thiomandelic acid, DL-captopril, DL-thiorphan, N-acetylcysteine, D-penicillamine, glutathione, L-cysteine, zefenoprilat, tiopronin, dimercaprol, and succinimide.

[0072] In some embodiments, the analyte molecule comprises one or more disulfide groups as functional groups, and the analyte molecule comprising one or more disulfide groups is preferably selected from the group consisting of glutathione disulfide, dipyrithione, selenium sulfide, disulfiram, lipoic acid, L-cystine, fursultiamine, octreotide, desmopressin, vapreotide, terlipressin, linaclotide, and peginesatide.

[0073] In some embodiments, the analyte molecule comprises one or more epoxide groups as functional groups, and the analyte molecule comprising one or more epoxide groups is preferably selected from the group consisting of carbamazepine 10,11-epoxide, carfilzomib, furosemide epoxide, and fosfomycin, sevelamer, cerulenin, scopolamine, tiotropium, methylscopolamine bromide, eplerenone, mupirocin, natamycin, carfilzomib, and troleandomycin.

[0074] In some embodiments, the analyte molecule comprises one or more phenolic groups as functional groups. The analyte molecule comprising one or more phenolic groups is preferably a steroid or steroid-like compound. In some embodiments, the analyte molecule comprising one or more phenolic groups is preferably a sp 2The steroid or steroid-like compound has a hybridizing A ring and an OH group at position 3 of the A ring. The steroid or steroid-like analyte molecule is preferably selected from the group consisting of estrogen, estrogenic compounds, estrone (E1), estradiol (E2), 17a-estradiol, 17p-estradiol, estriol (E3), 16-epiestriol, 17-epiestriol, and 16,17-epiestriol, and / or their metabolites. In embodiments, the metabolite is estriol, 16-epiestriol (16-epiE3), 17-epiestriol (17-epiE3), 16,17-epiestriol (16,17-epiE3), 16-ketoestradiol (16-ketoE2), 16a-hydroxyestrone (16a-OHEl), 2-methoxyestrone (2-MeOEl), 4-methoxyestrone (4-MeOEl), 2-hydroxyestrone-3-methyl ether (3-MeOEl), 2-methoxyestradiol (2-MeOE2), 4-methoxyestradiol (4-MeOE2), 2-hydroxyestrone (20HE1), 4-hydroxyestrone estrone (4-OHE1), 2-hydroxyestradiol (2-OHE2), estrone (E1), estrone sulfate (E1s), 17a-estradiol (E2a), 17p-estradiol (E2b), estradiol sulfate (E2s), equilin (EQ), 17a-dihydroequilin (EQa), 17p-dihydroequilin (EQb), equilenin (EN), 17-dihydroequilenin (ENa), 17b-dihydroequilenin (ENb), A8,9-dehydroestrone (dE1), A8,9-dehydroestrone sulfate (dE1), D9-tetrahydrocannabinol, and mycophenolic acid.

[0075] In some embodiments, the analyte molecule comprises an amine group as a functional group. The amine group is preferably an alkylamine group or an arylamine group. In some embodiments, the analyte comprising one or more amine groups is selected from the group consisting of proteins and peptides. The analyte molecule comprising an amine group is preferably 3,4-methylenedioxyamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, 3,4-methylenedioxymethamphetamine, amphetamine, methamphetamine, N-methyl-1,3-benzodioxolylbutanamine, 7-aminoclonazepam, 7-aminoflunitrazepam, 3,4-dimethylmethcathinone, 3-fluoromethcathinone. , 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, oxy Selected from the group consisting of simorphone, phencyclidine, norpropoxyphene, amitriptyline, clomipramine, dothiepin, doxepin, imipramine, nortriptyline, trimipramine, fentanyl, glycylxylidide, lidocaine, monoethylglycylxylidide, N-acetylprocainamide, procainamide, pregabalin, 2-methylamino-1-(3,4-methylenedioxyphenyl)butane, 2-amino-1-(3,4-methylenedioxyphenyl)butane, normeperidine, O-destramadol, tramadol, lidocaine, N-acetylprocainamide, procainamide, gabapentin, lamotrigine, theophylline, amikacin, gentamicin, tobramycin, vancomycin, methotrexate, gabapentin, sisomicin, and 5-methylcytosine.

[0076] In some embodiments, the analyte molecule is a carbohydrate or a substance having a carbohydrate moiety, such as a glycoprotein or a nucleoside. The analyte molecule is preferably a monosaccharide, particularly selected from the group consisting of ribose, desoxyribose, arabinose, ribulose, glucose, mannose, galactose, fucose, fructose, N-acetylglucosamine, N-acetylgalactosamine, neuraminic acid, N-acetylneurominic acid, etc. In some embodiments, the analyte molecule is an oligosaccharide, particularly selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and polysaccharides. In some embodiments, disaccharides are preferably selected from the group consisting of sucrose, maltose, and lactose. In some embodiments, the analyte molecule is a substance containing the above-mentioned monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide moiety.

[0077] In some embodiments, the analyte molecule comprises an azide group as a functional group selected from the group consisting of alkyl or aryl azides, and the analyte molecule comprising one or more azide groups is preferably selected from the group consisting of zidovudine and azidocillin.

[0078] The functional group Y of the analyte reacts or can react with the reactive group X of the derivatizing agent; the reactive group X of the derivatizing agent is disclosed in detail in the section relating to the first aspect of the present invention, and suitable bonds with the functional group Y of the analyte are also described. It will be understood by those skilled in the art that the remaining Xa and Ya of X and Y, respectively, form a covalent bond in each case. For example, if the reactive group X of the derivatizing agent is a carbonyl-reactive unit such as a hydrazine unit, particularly an -NH-NH group, and the functional group of the analyte is a carbonyl group such as a keto group C(=O), an -NH-N= structure is formed, with the remaining Xa of X being -NH-N=, and the remaining Ya of Y being a carbon atom with a double bond to the nitrogen atom.

[0079] As already mentioned above, the chromophore C possessed by the derivatization agent is a suitable chromophore that enables efficient energy transfer during LDI. Furthermore, the derivatization agent adds a sufficiently large molecular weight to the analyte of interest, resulting in a sufficient mass shift above the high noise background of biological samples in the low molecular weight range. While such high molecular weights are generally known to result in unfavorable ionization characteristics (low ionization efficiency, multiple fragmentation processes, etc.), this is avoided in this case by the presence of a permanent positive charge (the Z unit in formula (I)). Therefore, precursor ions (parent ions) can be detected with high sensitivity and, optionally, selected for fragmentation in MS / MS applications.

[0080] In some preferred embodiments of the conjugate, the conjugate has a molecular weight in the range of ≧500 g / mol, preferably ≧700 g / mol and / or an M in the mass spectrum at m / z ≧500, preferably ≧700. + The molecular weight of the conjugate is preferably determined by the M peak in the obtained mass spectrum. + The molecular weight of the derivatizing agent coupled to the analyte of interest is so high that the peak is outside the range of the low molecular weight background, i.e., the M + Very high so as to bring the peak outside the low molecular weight background range.

[0081] In some preferred embodiments of the conjugate, the conjugate comprises a neutral loss unit C-L1-Z, where C, L1, and Z are as defined above, having a molecular weight of ≥300 g / mol and / or a peak in the mass spectrum at m / z ≥ 300, preferably a molecular weight of ≥ 320 g / mol and / or a peak in the mass spectrum at m / z ≥ 320, more preferably a molecular weight of ≥ 350 g / mol and / or a peak in the mass spectrum at m / z ≥ 350, more preferably a molecular weight of ≥ 370 g / mol and / or a peak in the mass spectrum at m / z ≥ 370, and more preferably a molecular weight of ≥ 380 g / mol and / or a peak in the mass spectrum at m / z ≥ 380.

[0082] The term "neutral loss unit" refers to a unit that can release an uncharged component, i.e., release a neutral component. Typically, a neutral component comprises a single atom or multiple atoms. A neutral loss unit may be neutral, positively, or negatively charged. A neutral loss unit is capable of fragmentation under MS conditions, resulting in the release of at least one neutral component. After release of the neutral component, the remaining portion of the neutral loss unit retains its original charge. Thus, if the neutral loss unit is uncharged, it remains neutral after the loss of the neutral component. If the neutral loss unit is positively charged, it remains positively charged after the loss of the neutral component. If the neutral loss unit is negatively charged, it remains negatively charged after the loss of the neutral component. Typically, the release of the neutral component occurs in a single fragmentation event. The term "fragmentation" refers to the dissociation of a single molecule into two or more separate molecules. As used herein, the term fragmentation refers to a specific fragmentation event, where the destruction point of the parent molecule where the fragmentation event occurs is clearly defined, and the two or more daughter molecules resulting from the fragmentation event are well characterized.Methods for determining the destruction point of the parent molecule and the two or more daughter molecules obtained are well known to those skilled in the art.The daughter molecules obtained can be stable, or can dissociate during subsequent fragmentation events.Fragmentation can occur via collision-induced dissociation (CID), electron-capture dissociation (ECD), electron-transfer dissociation (ETD), negative electron-transfer dissociation (NETD), electron-detachment dissociation (EDD), photodissociation, particularly infrared multiphoton dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD), surface-induced dissociation (SID), high-energy C-trap dissociation (HCD), and charge-remote fragmentation.

[0083] As already shown above, efficient fragmentation is ensured by the specific combination of the Z and N units, which, in some embodiments, is a quaternary amine group adjacent to the benzyl position, enabling a smooth and selective neutral loss fragmentation process. During MS / MS, the conjugate of the derivatization reagent and analyte undergoes fragmentation by releasing a large fragment and product ion (the analyte of interest modified with a benzyl cation). This large mass shift is also highly advantageous, as such a specific and sensitive neutral loss pathway via a large molecular weight loss is highly unusual, ensuring low background / interference for this novel reagent class.

[0084] Fifth Aspect—Method for Mass Spectrometric Determination of Analyte Molecules In a fifth aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) providing an analyte of interest; (b) providing a derivatizing agent comprising the structure of formula (I) as defined in the section relating to the first aspect; (c) reacting the analyte provided according to (a) with the derivatization agent provided according to (b), thereby forming a preferably covalent conjugate of the analyte and the derivatization agent; and (d) subjecting the conjugate formed in (c) to mass spectrometry. The present invention relates to a method for the mass spectrometric determination of an analyte molecule, comprising:

[0085] Preferably, the mass spectrometry is laser desorption ionization mass spectrometry (LDI-MS), more preferably (MA)LDI-MS, more preferably (MA)LDI-MS / MS, or (SA)LDI-MS, more preferably (SA)LDI-MS / MS.

[0086] All details, embodiments and preferred embodiments described in the sections above for the first, second, third and fourth aspects of the invention, in particular the details, embodiments and preferred embodiments described in the section relating to the first aspect of the invention, also apply to the fifth aspect of the invention.

[0087] The present invention is further described by the following embodiments and combinations of embodiments, as indicated by their respective dependencies and back references. In particular, in each example where various embodiments are listed, in connection with a term such as "described in any one of embodiments 1 to 4," it is meant that all embodiments within this scope are clearly disclosed to one skilled in the art, i.e., this expression is understood by a person skilled in the art to be synonymous with "described in any one of embodiments 1, 2, 3, and 4."

[0088] 1. A derivatization agent, preferably for analytes intended to be analyzed by laser desorption / ionization mass spectrometry (LDI-MS), comprising a structural element of formula (I): C-L1-Z-(L2) p -X(I) During the ceremony, C is a chromophore having an absorption maximum in the range of 280-400 nm; Z is a charge unit containing at least one permanently charged moiety; X is a reactive group; L1 and L2 are each a linker unit; p is either 0 or 1, a derivatizing agent.

[0089] 2. The derivatization agent of embodiment 1, wherein the absorption maximum of chromophore C is an adsorption maximum as determined by UV / VIS spectroscopy.

[0090] 3. The derivatizing agent according to embodiment 1 or 2, wherein the absorption maximum of chromophore C is in the range of 290 to 380 nm, preferably in the range of 300 to 360 nm, more preferably in the range of 305 to 330 nm.

[0091] 4. Chromophore C has the structure of formula (C): [ka] In the formula, R 1 , R 2 , R 3 , R 4 , R 5 is a hydrogen atom; a hydroxyl group; NR x R y group [R x and R y are independently a hydrogen atom or a C1-C5 alkyl group; independently selected from the group consisting of C1-C5 alkyl, C5-C10 (hetero)aryl and -O-C1-C3 alkoxy groups; R 6 is either absent (i.e., there is a direct single covalent bond between the aromatic ring and the C(=O) group) or -CR 7 =CR 8 - group, wherein R 7 is a hydrogen atom or a C1-C3 alkyl group, and R 8 is selected from the group consisting of a hydrogen atom, a C1-C5 alkyl group, and an electron-withdrawing group; and the dotted line represents a bond to the linker, which is preferably a single bond.

[0092] 5. Chromophore C has the structure of formula (C1), (C2) or (C3): [ka] In the formula, R 1 , R 2 , R 3 , R 4 is a hydrogen atom, Hydroxyl group NR x R y group [R x and R y are independently a hydrogen atom or a C1-C5 alkyl group], a C1-C5 alkyl group, a C5-C10 (hetero)aryl group, and an —O—C1-C3 alkoxy group.

[0093] 6. Chromophore C has the structure of formula (C1), where R 1 and R 4 are both methoxy groups, and R 3 are hydroxyl groups (these residues R 2 , R 3 and R 4 wherein C1 is the remainder of sinapinic acid), a derivatizing agent according to embodiment 5.

[0094] 7. Chromophore C has the structure of formula (C2), wherein R 2 and R 4 are both hydroxyl groups (these residues R 2 , R 4 wherein C2 is the remainder of 2,5-dihydroxybenzoic acid), the derivatizing agent of embodiment 5.

[0095] 8. Chromophore C has the structure of formula (C3), wherein R 3 is a hydroxyl group (this residue R 3 wherein C3 is the remainder of α-cyano-4-hydroxy-cinnamic acid), a derivatizing agent according to embodiment 5.

[0096] 9. The linker L1 is (C1-C5 alkylene-O-)m The derivatizing agent according to any one of embodiments 1 to 8, wherein m is an integer in the range of 1 to 10, a C1-C20 alkylene group, a C1-C20 alkylene group-heteroaryl group, and a (C1-C5 alkylene)-O-(C1-C5 alkylene) group, optionally linked to or intersected by a unit selected from the group consisting of a (hetero)aryl group, N2, NO, NO2, S2, SO, SO2, CO, and CO2, which unit, when present, is preferably a heteroaryl group, more preferably from triazole, phenyltriazole, tetrazole, and phenyltetrazole.

[0097] 10. The linker L1 has the structure (L1a) q -(L1b) r -(L1c) s wherein q, r, and s are each 0 or 1, provided that at least one of q, r, and s is 1; L1a is (C1-C5 alkylene-O-) m L1b is a unit selected from the group consisting of N2, NO, NO2, S2, SO, SO2, CO, CO2, triazole, phenyltriazole, tetrazole and phenyltetrazole, where the unit is preferably triazole or tetrazole, more preferably the neutral loss unit N is a triazole, more preferably the neutral loss unit N is a 1,2,3 triazole ring which is bonded to the linker L1a (if present) via a single bond at the 1-position (N atom) of the triazole ring and to the linker L1c (if present) via a single bond at the 4-position (C atom) of the triazole ring; L1c (if present) is (C1-C5 alkylene-O-) m 10. The derivatizing agent of any one of embodiments 1 to 9, wherein m is an integer ranging from 1 to 10, a C1-C10 alkylene group, and a (C1-C5 alkylene)-O-(C1-C5 alkylene) group.

[0098] 11. The linker L1 has the following structure: [ka] 11. The derivatizing agent of any one of embodiments 1 to 10, wherein the dotted lines indicate the bonds to (C) and (Z), respectively.

[0099] 12. The derivatizing agent according to any one of embodiments 1 to 11, wherein the charged unit Z is positively or negatively charged, preferably positively charged.

[0100] 13. The charged unit Z is positively charged and is preferably a tetraalkylammonium group, more preferably -CHN + The derivatizing agent of any one of embodiments 1 to 12, which is a (CH3)2CH2- group.

[0101] 14. The derivatizing agent according to any one of embodiments 1 to 12, wherein the charged unit Z is negatively charged, and the negatively charged unit Z is preferably selected from the group consisting of phosphate, sulfate, sulfonate and carboxylate.

[0102] 15. The derivatization agent according to any one of embodiments 1 to 12, wherein the linker L2 comprises 1 to 10 C atoms and optionally one or more heteroatoms.

[0103] 16. The derivatizing agent according to embodiment 15, wherein the linker L2 is preferably a C1-C5-alkylene-C5-C10 aromatic ring having a C(=O) unit as a substituent of the aromatic ring, and the linker L2 is preferably a C1-C3-alkylene-C6 aromatic ring having a C(=O) unit as a substituent of the aromatic ring, and the C6 aromatic ring is more preferably a substituted or unsubstituted benzene ring, more preferably a benzene ring having a C(=O) group para to the position to which the C1-C3-alkylene is attached, and more preferably a benzene ring having a C(=O) group at a position on the benzene ring that has no further substituents.

[0104] 17. The derivatizing agent of any one of embodiments 1-16, wherein the reactive group X is selected from the group consisting of a carbonyl-reactive unit, a diene-reactive unit, a hydroxyl-reactive unit, an amino-reactive unit, an imine-reactive unit, a thiol-reactive unit, a diol-reactive unit, a phenol-reactive unit, an epoxide-reactive unit, a disulfide-reactive unit, and an azide-reactive unit.

[0105] 18. A kit comprising the derivatizing agent of any one of embodiments 1 to 17.

[0106] 19. Use of a derivatization agent according to any one of embodiments 1 to 17 for mass spectrometric determination of analyte molecules, wherein the mass spectrometric determination is laser desorption / ionization mass spectrometry (LDI-MS), preferably (MA)LDI-MS, more preferably (MA)LDI-MS / MS, or (SA)LDI-MS, more preferably (SA)LDI-MS / MS.

[0107] 20. A conjugate of a derivatization agent and an analyte according to any one of embodiments 1 to 17, having the structure of formula (II): C-L1-Z-(L2) p -Xa-Ya-A(II) wherein C, L1, L2, p, Z, and N are as defined in any one of embodiments 1 to 17; Xa is the remainder of the reactive group X as defined in any one of embodiments 1 to 17; A is an analyte, and Ya is the remainder of the reactive group Y attached to the analyte A, which has reacted with the reactive group X of the derivatizing agent to form a covalent bond between Xa and Ya.

[0108] 21. The conjugate according to embodiment 20, wherein the analyte is selected from the group consisting of nucleic acids (preferably selected from DNA, mRNA, miRNA and rRNA), amino acids, peptides, proteins (preferably cell surface receptors or cytosolic proteins), metabolites, hormones (preferably selected from testosterone, estrogen and estradiol), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids (vitamin D), molecules characteristic of a particular modification of another molecule (preferably selected from sugar moieties, phosphoryl residues on proteins, methyl residues on genomic DNA), substances internalized by an organism (preferably selected from therapeutic agents, drugs of abuse, toxins) and metabolites of such substances.

[0109] 22. The conjugate of embodiment 20 or 21, wherein the reactive group Y is selected from the group consisting of a carbonyl group, a diene group, a hydroxyl group, an amine group, an imine group, a thiol group, a diol group, a phenol group, an epoxide group, a disulfide group, and an azide group.

[0110] 23. Molecular weight in the range of ≥ 500 g / mol, preferably ≥ 700 g / mol and / or M in the mass spectrum of m / z ≥ 500, preferably ≥ 700 + 23. The conjugate of any one of embodiments 20 to 22, having a peak.

[0111] 24. A conjugate according to any one of embodiments 20 to 22, comprising a neutral loss unit C-L1-Z having a molecular weight of ≥ 300 g / mol and / or a peak in the mass spectrum at m / z ≥ 300, preferably having a molecular weight of ≥ 320 g / mol and / or a peak in the mass spectrum at m / z ≥ 320, more preferably having a molecular weight of ≥ 350 g / mol and / or a peak in the mass spectrum at m / z ≥ 350, more preferably having a molecular weight of ≥ 370 g / mol and / or a peak in the mass spectrum at m / z ≥ 370, more preferably having a molecular weight of ≥ 380 g / mol and / or a peak in the mass spectrum at m / z ≥ 380.

[0112] 25. A method for mass spectrometric determination of an analyte molecule, comprising: (e) providing an analyte of interest; (f) providing a derivatizing agent comprising the structure of formula (I) as defined in any one of embodiments 1 to 17; (g) reacting the analyte provided according to (a) with the derivatization agent provided according to (b), thereby forming a preferably covalent conjugate of the analyte and the derivatization agent; and (h) subjecting the conjugate formed in (c) to mass spectrometry.

[0113] 26. A method for mass spectrometric determination, wherein the mass spectrometry is laser desorption ionization mass spectrometry (LDI-MS), preferably (MA)LDI-MS, more preferably (MA)LDI-MS / MS, or (SA)LDI-MS, more preferably (SA)LDI-MS / MS. [Brief explanation of the drawings]

[0114] [Figure 1] Shown is the full scan MALDI mass spectrum (m / z 200 to m / z 1000) of an equimolar (3 µM) analyte mixture consisting of derivatized compounds 6a, 6b, 6c and native testosterone in a sinapinic acid MALDI matrix. [Figure 2] A dilution series of equimolar (3 μM, 300 nM, 30 nM, and 3 nM) analyte mixtures consisting of derivatized compounds [6a], [6b], and [6c] compared to underivatized testosterone [TH] in the presence of a sinapinic acid MALDI matrix is ​​shown. Detected counts are displayed on a logarithmic scale relative to the corresponding molar concentrations. [Figure 3] Shown is a full-scan SALDI mass spectrum (m / z 50–m / z 1000) of an equimolar (3 μM) analyte mixture consisting of derivatized compounds [6a]+, [6b]+, and [6c]+ compared to underivatized testosterone [TH]+ without any additional MALDI matrix. *: in-source fragments of [6a]+, [6b]+, and [6c]+. [Figure 4] A dilution series of equimolar (3 μM, 300 nM, 30 nM, and 3 nM) analyte mixtures consisting of derivatized compounds [6a], [6b], and [6c] compared to underivatized testosterone [TH] in the presence of a sinapinic acid MALDI matrix is ​​shown. Detected counts are displayed on a logarithmic scale relative to the corresponding molar concentrations. DETAILED DESCRIPTION OF THE INVENTION

[0115] The present invention will be further illustrated by the following Reference Examples, Comparative Examples and Examples. [Example]

[0116] (Comparative) Example 1: Synthesis of [4-(hydrazinecarbonyl)phenyl]-N,N,N-trimethylmethanaminium bromide (alkyne 3) [ka] Bromide 1 (2.00 g, 8.73 mmol) and N,N-dimethylpropargylamine (4.8 mL, 64.1 mmol) were dissolved in 30 mL of EtOH, and the reaction mixture was stirred at room temperature for 16 h. The precipitate was collected by filtration, washed with EtOH and EtO, and dried in vacuo to give the desired product 2 as a yellow solid (2.73 g, quantitative yield). 1 H-NMR (400MHz, methanol-d4): δ[ppm]=3.19(s,3H),3.68(t,1H),3.93(s,3H),4.31(d,2H),4.74(s,2H),7.75(d,2H),8.16(d,2H). 13 C-NMR (101 MHz, methanol-d4): δ [ppm] = 49.47, 51.56, 53.43, 65.89, 71.10, 82.48, 129.87, 131.72, 132.37, 132.90, 166.08. ESI-MS: 232.2 ([M + ] + , calculated value: 232.3)

[0117] Hydrazine monohydrate (6.8 ml, 64.1 mmol) was added to a solution of tertiary amide 2 (2.00 g, 6.41 mmol) in 30 ml MeOH, and the reaction mixture was stirred at room temperature. After 16 h, the reaction mixture was concentrated in vacuo, and the crude product was purified by preparative RP-HPLC using an isocratic mobile phase (100% water). The desired product 3 was obtained as a yellow oil (1.69 g, 5.41 mmol, 84%). 1 H-NMR: (400MHz, methanol-d4): δ[ppm]=3.19(s,3H),3.68(t,1H),4.28(d,2H),4.71(s,2H),7.58(d,2H),7.86(d,2H). 13 C-NMR (101 MHz, methanol-d4): δ [ppm] = 42.13, 49.41, 53.32, 60.70, 65.97, 71.13, 82.44, 127.72, 132.86, 133.96, 134.78, 168.21. ESI-MS:232.2([M+]+, calculated value:232.3).

[0118] Example 1: Synthesis of derivatizing agent 5a [ka] To a solution of CuBr (16 mg, 0.10 mmol) and THPTA (44 mg, 0.10 mmol) in THF / water (2 mL, 1 / 1 (v / v)) under an argon atmosphere was added a solution of alkyne 3 (310 mg, 1.0 mmol) and azide 4 (101 mg, 0.33 mmol) in THF / water (2 mL, 1 / 1 (v / v)). The reaction mixture was stirred at room temperature for 24 h and then concentrated in vacuo. The crude product was purified by preparative RP-HPLC using a linear gradient of water / acetonitrile 100 / 0 → 0 / 100 in 60 min. The desired product 5a was obtained as a beige solid (90 mg, 0.15 mmol, 44%) after lyophilization. 1H-NMR:(400MHz,DMSO-d6):δ[ppm]=2.05(m,3H),2.91(m,5H),3.18(m,3H),3.76(m,5H),4.57(m ,5H),6.51(d,1H),6.82(s,1H),7.28(d,1H),7.75(d,2H),7.94(d,2H),8,27(m,1H),8.53(m,1H) ESI-MS:538.5([M+]+, calculated value:538.6).

[0119] Example 2: Coupling of derivatizing agent 5a with analyte (testosterone) - synthesis of conjugate 6a [ka] Hydrazide 5a (55 mg, 90.0 μmol) and testosterone (77 mg, 270 μmol) were dissolved in 1 ml of methanol / formic acid (99 / 1, v / v). The reaction mixture was stirred at room temperature for 16 h and then concentrated in vacuo. The crude product was purified by preparative RP-HPLC using a linear gradient of water / acetonitrile 100 / 0 → 0 / 100 in 60 min. The desired hydrazone 6a was obtained as a beige solid (35.6 mg, 40.0 μmol, 44%) after lyophilization. 1 H-NMR: (400MHz, methanol-d4): δ[ppm]=0.50-2.45(m,27H),2.91(m,5H),3.17(m,3H),3.76(s,4H),4.47(m,2H),4.59(m,2H) ),4.66(m,1H),5.60(s,1H),6.49(d,1H),6.81(s,2H),7.28(d,1H),7.72(d,2H),7.94(d,2H),8.28(s,1H),8.50(m,1H). ESI-MS:808.5([M+]+, calculated value:808.5).

[0120] Example 3: Synthesis of derivatizing agent 5b [ka] To a solution of CuBr (16 mg, 0.10 mmol) and THPTA (44 mg, 0.10 mmol) in THF / water (2 mL, 1 / 1 (v / v)) under an argon atmosphere was added a solution of alkyne 3 (310 mg, 1.0 mmol) and azide 4b (111 mg, 0.33 mmol) in THF / water (2 mL, 1 / 1 (v / v)). The reaction mixture was stirred at room temperature for 24 h and then concentrated in vacuo. The crude product was purified by preparative RP-HPLC using a linear gradient of water / acetonitrile 100 / 0 → 0 / 100 in 60 min. The desired product 5b was obtained as a beige solid (138 mg, 0.21 mmol, 64%) after lyophilization. ESI-MS:568.4([M+]+, calculated value:568.6).

[0121] Example 4: Coupling of derivatizing agent 5b with analyte (testosterone) - synthesis of conjugate 6b [ka] Hydrazide 5b (115 mg, 0.18 mmol) and testosterone (153 mg, 0.53 mmol) were dissolved in 1 ml of methanol / formic acid (99 / 1, v / v). The reaction mixture was stirred at room temperature for 16 h and then concentrated in vacuo. The crude product was purified by preparative RP-HPLC using a linear gradient of water / acetonitrile 100 / 0 → 0 / 100 in 60 min. The desired hydrazone 6b was obtained as a beige solid (54.0 mg, 58.8 μmol, 32%) after lyophilization. 1 H-NMR: (400MHz, methanol-d4): δ[ppm]=0.56-2.40(m,20H),2.89(s,3H),3.76(s,3H),3.83(m,1H),4.6 0(m,3H),6.52(m,1H),6.83(s,1H),7.28(d,1H),7.70(m,2H),7.95(m,2H),8.12(s,1H),8.47(m,2H). ESI-MS:838.5([M+]+, calculated value:838.5).

[0122] Comparative Example 2: Coupling of derivatizing agent 3 with analyte (testosterone) - synthesis of conjugate 6c [ka] Reaction of alkyne 3 and testosterone (153 mg, 0.53 mmol) gave conjugate 6c.

[0123] Example 5: Laser desorption / ionization mass spectrometry 5a Sample preparation and general measurement details Stock solutions of all relevant analytes 6a, 6b, 6c, and testosterone were individually prepared at a concentration of 1.0 mg / ml (100% acetonitrile; ACN) and further diluted to individual molar concentrations of 12 μM (80 / 10 = ACN / HO; abbreviated as 80% ACN). An analyte mixture was prepared from the molar concentrated stock solutions to obtain a molar concentration of each analyte of 3 μM (80% ACN). Further dilutions of this analyte mixture solution were prepared at 300 nM, 30 nM, and 3.0 nM (80% ACN).

[0124] For sinapinic acid matrix experiments, a matrix solution consisting of 10 mg / ml sinapinic acid in 50% ACN, 0.1% formic acid (FA) was freshly prepared.

[0125] The MALDI target plate used was a standard 96-well steel plate (Waters Corp.). Similarly, the SALDI-MS target plate is based on a steel plate but has a functional amorphous aC:H:Si:X (X = heteroatom modification) plasma-activated chemical vapor deposition (PACVD) surface coating as the top layer.

[0126] All of the following laser desorption / ionization mass spectrometry (LDI-MS) measurements were performed on a MALDI Synapt G2-Si (Waters Corp.) MALDI-Q-ToF mass spectrometer in positive ion mode. The laser repetition rate of the LDI-MS system was set to 1.0 kHz for MALDI experiments and 200 Hz for matrix-free SALDI experiments, utilizing a 355 nm Nd:YAG laser wavelength. Each analyte spot was measured by a random path across the analyte spot area, utilizing a 12 Hz plate movement during laser irradiation. The total acquisition time for each measurement was set to 30 s with a 0.5 s mass spectrum scan time. While the laser intensity can be varied on a relative scale up to 500, similar to a maximum output energy of 30 μJ, the optimal laser energy for sinapinic acid MALDI experiments was found to be 280 units, and the optimal laser energy for matrix-free SALDI experiments was increased to 380 units. Individual voltage settings are outlined in detail for each measurement.

[0127] The corresponding data analysis was performed using MassLynx 4.2 (SCN983, Waters Corp.) mass spectrometer software. All acquired mass spectral functions of each sample spot were accumulated over the entire analysis time of 30 seconds. Acquisition parameters for MALDI measurements: Polarity LDI+ Starting mass m / z200 End mass m / z1000 Acquisition time 30 seconds Cycle time: 0.514 seconds Scan time 0.500 seconds Inter-scan delay 0.014 seconds Data Format Continuum Analyzer Sensitivity Mode Maldi plate speed 12.0Hz Maldi laser firing rate 1000Hz Maldi Laser Energy 280.0 units Sample plate 0.0V Maldi extraction 10.0V Hexapole bias 10.0V Aperture 0 5.0V Hexapole RF amplitude 350V LM resolution 4.4 HM resolution 15.0 Acquisition parameters for each SALDI measurement: Polarity LDI+ Starting mass m / z 50 Ending mass m / z 1000 Acquisition time 30 seconds Cycle time: 0.514 seconds Scan time 0.500 seconds Inter-scan delay 0.014 seconds Data Format Center of Gravity Analyzer Sensitivity Mode Maldi plate speed 12.0Hz Maldi laser firing rate 200.0Hz Maldi Laser Energy 380.0 units Sample plate 0.0V Maldi extraction 10.0V Hexapole bias 10.0V Aperture 0 5.0V Cooling gas flow 50.0 LM resolution 4.4 HM resolution 15.0

[0128] 5b: Evaluation of derivatization reagents in matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) We first tested the suitability of the isolated derivatized testosterone conjugates 6a and 6b for potential matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) applications. The performance of the two chromophore-containing conjugates 6a and 6b was compared against native testosterone (T) and derivatized testosterone conjugate 6c (which serves as the gold standard in liquid chromatography-based MS applications but does not contain the LDI-suitable chromophore). Equimolar four-analyte mixtures (containing all four analytes 6a, 6b, 6c, and testosterone (T)) were prepared at different concentrations (3 μM, 300 nM, 30 nM, and 3 nM) and subsequently premixed (1:1) with a sinapinic acid matrix solution. Next, 1 μl of this analyte matrix solution was applied to a MALDI target steel plate by dry droplet preparation. The resulting analyte matrix crystals were measured by MALDI-MS in full-scan positive ion mode (m / z 200 to m / z 1000; see the Experimental Section for further details). As expected, analysis of the full-scan MALDI mass spectrum (see Figure 1) showed only very low signal intensities for the m / z corresponding to the native analyte ([TH]). + , protonated testosterone cation). In comparison, derivatized testosterone conjugates 6a and 6b were detected with significantly higher intensity (approximately 96-fold signal enhancement, see Figure 2). Compared to the state-of-the-art conjugate 6c, the novel reagent clearly produces detectable ions with m / z values ​​that are out of the low-molecular-weight matrix background (m / z = 200-700 in this experiment) and therefore less susceptible to matrix-based interferences. This solves a common problem with MALDI-MS (Anal Bioanal Chem 410, 4015-4038 (2018)).

[0129] Figure 2 shows the underivatized testosterone [TH] in the presence of a sinapinic acid MALDI matrix. + compared with the derivatized compound [6a] + , [6b] + and [6c] +A dilution series of equimolar (3 μM, 300 nM, 30 nM, and 3 nM) analyte mixtures consisting of 100 μM, ... and 100 μM of analyte is shown. The number of counts detected is displayed on a logarithmic scale relative to the corresponding molar concentration.

[0130] However, application of chromophore-bearing conjugates 6a and 6b to sinapinic acid did not result in signal enhancement at concentrations below 300 nM compared to the state-of-the-art conjugate 6c. This observation indicated that in the presence of an appropriate matrix, in this case sinapinic acid, energy transfer of the laser beam to the analyte of interest was mediated through the matrix rather than through the chromophore-containing derivatization reagent, which was crucial given the large excess of matrix molecules relative to the derivatized analyte. In summary, the chromophore-containing derivatization reagents described herein provided for MALDI-MS applications primarily offer the benefits of a "mass tag" that enables the detection of low-molecular-weight analytes in the absence of matrix-induced low-molecular-weight interferences.

[0131] 5c: Evaluation of derivatization reagents for laser desorption / ionization mass spectrometry (LDI-MS) on functionally coated SALDI plates As intended by the overall conceptual design, the isolated derivatized testosterone conjugates 6a and 6b were evaluated for laser desorption / ionization mass spectrometry (MALDI-MS) applications. The performance of the two chromophore-containing conjugates 6a and 6b was again compared against native testosterone (T) and the derivatized testosterone conjugate 6c, which does not contain the LDI-suitable chromophore. Equimolar four-analyte mixtures (containing all four analytes 6a, 6b, 6c, and testosterone (T)) with different concentrations (3000 nM, 300 nM, 30 nM, and 3 nM) were prepared and applied directly to a functionalized SALDI-MS target plate by dried droplet preparation. The resulting spots were measured by SALDI-MS in full-scan positive ion mode (m / z 50 to m / z 1000; see the Experimental Section for further details). As expected, analysis of the full scan LDI mass spectra (see example at 3 μM concentration in Figure 3) revealed that the m / z of derivatized testosterone-conjugates 6a and 6b was significantly higher than that of the native analyte ([TH] + , protonated testosterone cation), which corresponds to an approximately 15-fold signal enhancement at the 3 μM concentration.

[0132] Figure 4 shows the underivatized testosterone [TH] in the presence of a sinapinic acid MALDI matrix. + compared with the derivatized compound [6a] + , [6b] + and [6c] + A dilution series of equimolar (3 μM, 300 nM, 30 nM, and 3 nM) analyte mixtures consisting of 100 μM, ... and 100 μM of analyte is shown. The number of counts detected is displayed on a logarithmic scale relative to the corresponding molar concentration.

[0133] This signal enhancement was particularly beneficial for sensitive measurements at low concentrations. As an example, natural testosterone was undetectable at 3 nM, whereas reagents 5a and 5b ([6a]) were detectable at this low concentration. + and [6b] +The use of LDI-compatible derivatization reagents 5a and 5b resulted in the detection of the corresponding testosterone conjugates ([6a] + and [6b] + It is important to note that this served to increase the signal of the conjugate (as ) compared to the state-of-the-art conjugate 6c, which lacks an LDI-compatible chromophore. + So, the absolute number of detected counts is [6a] + and [6b] + More importantly, the S / N ratio was 1.4 and 2.1 times higher at 3 nM [6a]. + (S / N=304) and [6b] + Regarding (S / N=39) [6c] + The S / N ratio was significantly improved compared to that of the conventional method (S / N = 2). This finding supported the basic research hypothesis that the use of permanently charged derivatization reagents carrying LDI-compatible chromophores helps improve the energy transfer of the laser beam to the derivatized analytes of interest, thereby increasing the desorption / ionization efficiency and ultimately resulting in enhanced sensitivity (J Am Soc Mass Spectrom 2007, 18, 9, 1582-1590). As a result, the LDI-compatible derivatization reagents 5a and 5b described herein offered technical advantages for achieving highly sensitive LDI-MS applications.

[0134] Example 6 Generalization of Chromophores The tested derivatization reagents 5a–5c were characterized by UV spectroscopy. The sinapinic acid-derived reagents 5a and 5b exhibited absorption maxima at 307 nm and 315 nm, respectively, which are still comparable to routinely used sinapinic acid. Because the absorption maxima of these two compounds are relatively close (<40 nm) to the wavelength of laser light (355 nm), efficient energy transfer is expected. This clearly demonstrates that 5a and 5b are suitable for LDI applications due to their ability to absorb the energy of the laser beam (typically 355 nm (J Mass Spectrom. 2021;56:4664)), which is considered an essential factor for MALDI matrices (Chem. Rev. 2003, 103, 2,395–426). Reagent 5c has a local absorption maximum at 240 nm, which is more than 100 nm lower than the wavelength of the routinely used Nd:YAG laser. Based on our understanding and available data, this results in less spectral overlap with the laser beam compared to 5a and 5b, resulting in lower desorption / ionization efficiency.

[0135] Other small molecules (i.e., 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid) with similar local absorption maxima but different chemical structures (vs. sinapinic acid) are also known to be suitable for the (MA)LDI process (see Table 1). Following this data, it was concluded that the presented concept is not limited to structures 5a and 5b but is applicable to any other derivatization reagent equipped with chromophore C, a suitable LDI chromophore. [Table 1]

[0136] References Higashi et al.(2016)J.of Pharmaceutical and Biomedical Analysis 130 p.181-190 International Publication No. 2011 / 091436 International Publication No. 2011 / 091436 Rahimoff et al.(2017)J.Am.Chem.Soc.139(30),p.10359-10364 J.Sep.Sci.2016,39,102-114;Biomed.Chromatogr.2011;25:1-10 Trends in Analytical Chemistry 143(2021)116399; J Mass Spectrom.2021;56:e4731 J.Sep.Sci.2016,39,102-114 Biomed.Chromatogr.2011;25:1-10 Anal.Chem.2020,92,6224-6228 Chem Asian J.2021,16,868-878 Crit Rev Anal Chem.2021 Dec 30;1-17 Mass Spec Rev.2019;38:3-21 Scientific Reports | 5:17853,ChemBioChem 2021,22,1430-143 International Journal of Mass Spectrometry 353(2013)54-59 Anal.Chem.2014,86,21,10724-10731 T.Higashi J Steroid Biochem Mol Biol.2016 Sep;l62:57-69 H.Ban et al J.Am.Chem.Soc.,2010,132(5),pp 1523-1525 Anal.Bioanal.Chem.2011,399,2597,DOI 10.1007 / s00216-010-4063-3 Anal Bioanal Chem.2018,410,4015-4038 J Am Soc Mass Spectrom 2007,18,9,1582-1590

Claims

1. A derivatization agent, preferably for analytes intended to be analyzed by laser desorption / ionization mass spectrometry (LDI-MS), comprising a structural element of formula (I), C-L1-Z-(L2) ｐ -X(I) During the ceremony, C is a chromophore having an absorption maximum in the range of 280-400 nm; Z is a charge unit containing at least one permanently charged moiety; X is a reactive group; L1 and L2 are each a linker unit; A derivatizing agent wherein p is either 0 or 1.

2. 2. The derivatization agent of claim 1, wherein the absorption maximum of chromophore C is an adsorption maximum determined by UV / VIS spectroscopy.

3. the chromophore C has the structure of formula (C), 【Chemistry 1】 In the formula, R １ , R ２ , R ３ , R ４ , R ５ represents a hydrogen atom; a hydroxyl group; NR ｘ R ｙ Group [R ｘ and R ｙ are independently a hydrogen atom or a C1-C5 alkyl group; independently selected from the group consisting of C1-C5 alkyl, C5-C10 (hetero)aryl and —O—C1-C3 alkoxy groups; R ６ is none or -CR ７ =CR ８ - group, and R ７ is a hydrogen atom or a C1-C3 alkyl group, and R ８ is selected from the group consisting of a hydrogen atom, a C1-C5 alkyl group, and an electron-withdrawing group; and the dotted line represents a bond to the linker, which is preferably a single bond.

4. The linker L1 is (C1-C5 alkylene-O-) ｍ a group wherein m is an integer ranging from 1 to 10, a C1-C20 alkylene group, a C1-C20 alkylene group-heteroaryl group, and a (C1-C5 alkylene)-O-(C1-C5 alkylene) group, and optionally a (hetero)aryl group, N ２ , NO, NO ２ , S ２ , SO, SO ２ , CO, and CO ２ The derivatizing agent of any one of claims 1 to 3, which is linked to or crossed by a unit selected from the group consisting of:

5. The derivatizing agent according to any one of claims 1 to 4, wherein the charged unit Z is positively or negatively charged.

6. The derivatizing agent according to any one of claims 1 to 5, wherein the linker L2 comprises 1 to 10 C atoms and optionally one or more heteroatoms.

7. 7. The derivatizing agent according to any one of claims 1 to 6, wherein the reactive group X is selected from the group consisting of a carbonyl-reactive unit, a diene-reactive unit, a hydroxyl-reactive unit, an amino-reactive unit, an imine-reactive unit, a thiol-reactive unit, a diol-reactive unit, a phenol-reactive unit, an epoxide-reactive unit, a disulfide-reactive unit, and an azide-reactive unit.

8. A kit comprising the derivatizing agent according to any one of claims 1 to 7.

9. 8. Use of a derivatizing agent according to any one of claims 1 to 7 for the mass spectrometric determination of analyte molecules, wherein said mass spectrometric determination is laser desorption / ionization mass spectrometry (LDI-MS), preferably (MA)LDI-MS, more preferably (MA)LDI-MS / MS or (SA)LDI-MS.

10. A conjugate of a derivatization agent and an analyte according to any one of claims 1 to 7, having the structure of formula (II): __________________________________ ｐ ________________________________ wherein C, L1, L2, p, Z and N are as defined in any one of claims 1 to 7; Xa is the remainder of a reactive group X as defined in any one of claims 1 to 7; A is the analyte, and Ya is the remainder of a reactive group Y attached to the analyte A that has reacted with the reactive group X of the derivatizing agent to form a covalent bond between Xa and Ya.

11. 11. The conjugate of claim 10, 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, and metabolites of such substances.

12. 12. The conjugate of claim 10 or 11, wherein the reactive group Y is selected from the group consisting of a carbonyl group, a diene group, a hydroxyl group, an amine group, an imine group, a thiol group, a diol group, a phenol group, an epoxide group, a disulfide group and an azide group.

13. Molecular weight in the range of ≧500 g / mol and / or M in the mass spectrum at m / z ≧500 ＋ 13. The conjugate of claim 10, comprising a neutral loss unit C-L1-Z having a peak in the mass spectrum of m / z ≥ 300 and / or a molecular weight of ≥ 300 g / mol and / or a peak in the mass spectrum of m / z ≥ 300.

14. 1. A method for mass spectrometric determination of an analyte molecule, comprising: (a) providing an analyte of interest; (b) providing a derivatizing agent comprising the structure of formula (I) as defined in any one of claims 1 to 7; (c) reacting the analyte provided according to (a) with the derivatization agent provided according to (b), thereby forming a preferably covalent conjugate of the analyte and the derivatization agent; and (d) subjecting the conjugate formed in (c) to mass spectrometry, wherein the mass spectrometry is preferably laser desorption / ionization mass spectrometry (LDI-MS), more preferably (MA)LDI-MS, more preferably (MA)LDI-MS / MS, or (SA)LDI-MS, more preferably (SA)LDI-MS / MS. A method comprising:

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