Pyridinium, quinolinium, acridinium, pyranium, benzopyranium or xanthonium reactive desorption and / or laser ablation ionization substrates and uses thereof

Abstract

The present disclosure relates to the use of a compound of formula I or a salt thereof as a reactive matrix for desorption and laser ablation ionization spectrometry. The disclosure further relates to a compound of formula II or a salt thereof, and uses of a compound of formula II or III or a salt thereof. # imgabs0 #

Description

[0001] This case is a divisional application for the invention patent application with the application date of March 6, 2019, application number 201980025247.X, and invention name "Pyridinium, quinolinium, acridinium, pyrylium, benzopyrylium or xanthene reactive desorption and / or laser ablation ionization matrix and its use". Technical Field

[0002] The present invention relates to organic chemical compounds and to the use of such compounds as reactive matrices for desorption and / or laser ablation ionization spectrometry. Background Art

[0003] Advanced mass spectrometry techniques have revolutionized the analysis of biomolecules. Koichi Tanaka was awarded the 2002 Nobel Prize in Chemistry for his development of soft laser desorption techniques for mass spectrometry. These developments laid the foundation for many of today's powerful mass spectrometry techniques that utilize laser desorption and / or ablative ionization, including one of the most widely used MS analysis techniques for large molecules: matrix-assisted laser desorption ionization (MALDI-MS).

[0004] MALDI relies primarily on the co-crystallization of an excess of auxiliary matrix with the analyte. The shallow layers of the resulting crystals are ablated by radiation from a short laser pulse, leading to partial ionization and detection of the sample. It is well established that an effective auxiliary matrix must exhibit strong light absorption at the wavelength of the pulsed laser (typically 337 nm for nitrogen, or 355 nm for Nd:YAG lasers). In addition, it needs to effectively co-crystallize with the analyte molecules after solvent evaporation and promote cationization (protonation) of the analyte in the gas phase. For this reason, the most commonly used positive ion mode MALDI-MS matrices such as α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) are acidic compounds that promote proton transfer, and they have high optical absorption in the 290-370 nm range to transfer laser energy to the analyte.

[0005] MALDI and similar techniques use laser energy to absorb the matrix to volatilize and ionize the sample molecules for subsequent mass spectrometer detection. The analyte is mixed with an excess of a suitable matrix material, which is typically a cinnamic acid, benzoic acid, or picolinic acid derivative. The laser is then used to irradiate the matrix / analyte mixture. The matrix typically has strong light absorption at the laser wavelength, thus facilitating surface desorption and ablation of the matrix / analyte crystals. The analyte molecules, now in the gas phase, are typically protonated by the acidic gas phase matrix molecules, and the resulting charged analyte molecules can then be accelerated to a mass spectrometer operating in positive ion mode. Many types of compounds can be analyzed with MALDI-MS, including but not limited to proteins, peptides, nucleotides, oligonucleotides, lipids, polysaccharides, and oligosaccharides, not to mention small molecules such as metabolites and neurotransmitters.

[0006] MALDI and similar techniques can even be used as imaging techniques (mass spectrometry imaging, MSI) to identify and map the distribution of molecules in tissue sections with near-cellular spatial resolution.

[0007] However, MALDI-MS and MSI have a number of disadvantages, including relative insensitivity to signal interference from certain compound classes and MALDI matrices in the low molecular weight region (100–600 Da). To address these issues, in situ chemical derivatization of primary amines such as neurotransmitters using pyrylium salts such as 2,4-diphenyl-pyrylium tetrafluoroborate (DPP-TFB) has been investigated. This work is described in Shariatgorji et al. Neuron 2014, 84, 697-707 and Shariatgorji et al. J. Am Soc. Mass Spectrom. 2015, 26(6), 934-939. DPP-TFB reacts selectively with primary amines to generate N-alkyl or N-aryl pyridinium derivatives under mild conditions. These charged derivatives have sufficient laser desorption and ionization efficiencies to enable the detection of endogenous small molecule primary amines such as various neurotransmitters. Furthermore, the DPP-TFB derivatives underwent self-assisted laser desorption ionization, i.e., no separate matrix molecule was required because the DPP-TFB salt itself served as the “reactive matrix”.

[0008] There remains a need for improved methods of ionizing molecules for mass spectrometry detection. Summary of the invention

[0009] The inventors of the present invention have identified a number of disadvantages of prior art methods of ionizing molecules by desorption and / or laser ablation for mass spectrometry.

[0010] In the prior art, diphenyl derivatives of pyrylium (DPP) have been used as reactive matrices for MALDI and MALDI-MSI containing primary amine groups. However, many small molecules, such as drugs and their metabolites, endogenous metabolites, neurotransmitters and their metabolites, biomarkers, toxins and natural compounds do not contain primary amines and are therefore not suitable for this technique. In addition, the use of DPP requires intensive incubation in a chamber saturated with 50% methanol. This increases the risk of sample diffusion in MALDI-MS and delocalization of target compounds in MALDI-MSI. In addition, pyrylium-based reactive matrices such as DPP lack effective means for identifying the generated ions.

[0011] It is an object of the present invention to provide an apparatus for desorption and / or laser ablation which overcomes or at least alleviates one or more of the above-mentioned disadvantages.

[0012] These objects are achieved by using the compounds of formula I according to the appended claims as reactive matrices for desorption and / or laser ablation ionization spectrometry.

[0013] The compound of formula I has the following structure:

[0014]

[0015] in Selected from the group consisting of:

[0016]

[0017] –Ar 1 is optionally substituted and selected from the group consisting of phenyl, biphenyl, terphenyl, C 10 –C 30 PAHs and C4–C 30 a monocyclic or polycyclic heteroaryl group;

[0018] –L– is selected from a bond, –(CH2) m – or any π-conjugated linker moiety;

[0019] –G 1 Selected from –H, –Me or –Ar 2 ;

[0020] -R is optional with one or more D, T or 13 C atoms are labeled C1–C 15 alkyl;

[0021] –G 2Selected from the group consisting of: –F, –Cl, –Br, –I, –NO2, –CN, –OR′, –OC(O)R′, –SR′, –S(O)R′, –S(O)(O)R′, –NR′R″ and –(CH2) n –Z–NH–NH2;

[0022] -R' and -R" are each independently selected from phenyl, tolyl and C1-C 15 alkyl;

[0023] m and n are each independently 0 to 15;

[0024] –Z– is selected from a bond, –CH2– or –C(O)–;

[0025] –G 3 Selected from –H, –Me or –Ar 3 ;

[0026] –Ar 2 and –Ar 3 Each of which is optionally substituted independently and is independently selected from the group consisting of phenyl, biphenyl, terphenyl, C 10 –C 30 PAHs and C4–C 30 a monocyclic or polycyclic heteroaryl group;

[0027] The condition is: when for When Ar 1 ,Ar 2 or Ar 3 At least one of them is substituted by chlorine, bromine, deuterium or tritium.

[0028] The compound of formula I as defined above has many advantageous features as the purpose of reactive matrix for desorption and / or laser ablation ionization mass spectrometry. Reactive matrix can target many substances that previously known reactive matrix cannot touch. Therefore, for example, in addition to primary amines, active matrix can also react with secondary amines, phenol hydroxides, aldehydes and ketones. Reactive matrix may not require any incubation, and the reaction occurs when reactive matrix is ​​added / spotted / sprayed onto the sample without any cooling or heating. All reactive matrices form charged derivatives when reacting with target molecules, thus increasing the mass spectrometry sensitivity to target molecules. In addition, other recognition means, such as specific isotope labels, can be introduced into reactive matrix.

[0029] The compound of formula I may have Ia or Ib,

[0030]

[0031] in Selected from the group consisting of:

[0032]

[0033] According to another aspect, the objects of the present invention are achieved by compounds of formula II according to the appended claims.

[0034] The compound of formula II has the following structure:

[0035]

[0036] in Selected from the group consisting of:

[0037]

[0038] –Ar 1 is optionally substituted and selected from the group consisting of biphenyl, terphenyl, C 10 –C 30 PAHs and C6–C 30 Polycyclic heteroaryl;

[0039] –L– is selected from a bond, –(CH2) m – or any π-conjugated linker moiety;

[0040] –G 1 Selected from –H, –Me or –Ar 2 ;

[0041] -R is optionally replaced by one or more D, T or 13 C atoms are labeled C1–C 15 alkyl;

[0042] –G 2 Selected from the group consisting of: –F, –Cl, –Br, –I, –NO2, –CN, –OR′, –OC(O)R′, –SR′, –S(O)R′, –S(O)(O)R′, –NR′R″ and –(CH2) n –Z–NH–NH2;

[0043] -R' and -R" are each independently selected from phenyl, tolyl and C1-C 15 alkyl;

[0044] m and n are each independently 0 to 15;

[0045] –Z– is selected from a bond, –CH2– or –C(O)–;

[0046] –G 3 Selected from –H, –Me or –Ar 3 ;

[0047] –Ar 2 and –Ar 3 Each of which is optionally substituted independently and is independently selected from the group consisting of phenyl, biphenyl, terphenyl, C 10 –C 30 PAHs and C4–C 30 Monocyclic or polycyclic heteroaryl;

[0048] The condition is: when for When –L–Ar 1 Located at the 4-position of the pyrylium ring; and

[0049] Ar 1 ,Ar 2 or Ar 3 At least one of them is substituted by chlorine, bromine, deuterium or tritium.

[0050] Being located at the 4-position of the pyrylium ring means being located at a ring position completely opposite to the oxygen of the pyrylium ring, regardless of whether the pyrylium ring is a monocyclic pyrylium, benzopyrylium or dibenzopyrylium ring system. The advantages of the compound of formula II are similar to the advantages of using the compound of formula I as a reactive matrix for desorption and / or laser ablation ionization spectrometry as described above. In addition, the compounds of formula I and / or formula II can be used for further spectroscopy or spectrometry methods, in which it may prove useful to label molecules with chromophores and / or to charge label molecules. These methods include ultraviolet spectroscopy and ultraviolet imaging, as well as other mass spectrometry methods, such as electrospray ionization.

[0051] The compound of formula II may have formula IIa or IIb,

[0052]

[0053] in Selected from the group consisting of:

[0054]

[0055] According to another aspect, the objects of the present invention are achieved by using a compound of formula III according to the appended claims as a reactive matrix for desorption and / or laser ablation ionization spectrometry.

[0056] The compound of formula III has the following structure:

[0057]

[0058] in:

[0059] –G 1 Selected from –H, –Me or –Ar2 ;

[0060] -R is optionally replaced by one or more D, T or 13 C atoms are labeled C1–C 15 alkyl;

[0061] –G 2 Selected from the group consisting of: –F, –Cl, –Br, –I, –NO2, –CN, –OR′, –OC(O)R′, –SR′, –S(O)R′, –S(O)(O)R′ and –NR′R″ and –(CH2) n –Z–NH–NH2;

[0062] -R' and -R" are each independently selected from phenyl, tolyl and C1-C 15 alkyl;

[0063] m and n are each independently 0 to 15;

[0064] –Z– is selected from a bond, –CH2– or –C(O)–;

[0065] –G 3 Selected from –H, –Me or –Ar 3 ;and

[0066] –Ar 2 and –Ar 3 Each of which is optionally substituted independently and is independently selected from the group consisting of phenyl, biphenyl, terphenyl, C 10 –C 30 PAHs and C4–C 30 A monocyclic or polycyclic heteroaryl group.

[0067] The advantages of compounds of formula III are similar to the advantages of using compounds of formula I or II as reactive matrices for desorption and / or laser ablation ionization spectrometry as described above. However, compounds of formula III may also have the advantages of improved atom economy and / or ease of manufacture.

[0068] The compound of formula III may have formula IIIa or IIIb.

[0069]

[0070] The following considerations and explanations apply equally to the use of compounds of formula I, Ia, Ib, III, IIIa and / or as described above, just as they do with compounds of formula II, IIa and IIb as described above, unless otherwise indicated or conflicting with the definitions of formula I, Ia, Ib, II, IIa, IIb, III, IIIa and IIIb as described above.

[0071] Ar 1 ,Ar 2 and Ar 3 Each of the following may be optionally substituted independently by one or more substituents selected from the group consisting of: -D, -T, -F, -Cl, -Br, -I, -NO2, -CN, -R"', -OR"', -OC(O)R"', -SR"', -S(O)R"', -S(O)(O)R"' and -NR"'R"", wherein -R"' and -R"" are each independently selected from phenyl, tolyl and C1-C 15 Alkyl. Such substituents may affect the degree of conjugation of the Ar group in question, as well as its electron donating or electron withdrawing properties. In this way, the reactivity and desorption / ablation properties of the compound can be adjusted. For example, Ar 1 It may be optionally substituted with one or more -Ph, -Cl or -Br groups.

[0072] Ar 1 It may be selected from the group consisting of biphenyl, terphenyl, naphthalene, anthracene, phenanthrene and pyrene. These aromatic groups have been shown to provide compounds with sufficient desorption / ablation properties to serve as reactive matrices for desorption and / or laser ablation ionization spectrometry.

[0073] If -L- is a π-conjugated linker moiety, it may be selected from the group consisting of: -O-, -S-, -NH-, as well as All of these linkers are in Ar 1 Provide sufficient conjugation paths between the groups and the charged aromatic rings of the compound.

[0074] –L– may preferably be a bond, i.e. 1 direct bond between the group and the charged aromatic ring of the compound. This allows Ar 1 The conjugation between the radical and the charged aromatic ring of the compound does not increase the molecular weight of the compound.

[0075] –G 1 Can be –H. –G 1 The phenyl group may be optionally substituted with one or more -Br substituents. Due to the characteristic isotopic pattern and mass loss of the bromide substituents, this allows for easy identification of molecules derived from the compound.

[0076] In some embodiments, the compound of Formula I, Ia, Ib, II, IIa or IIb can be -R may optionally be labeled with one or more deuterium, tritium or carbon-13 atoms and may be selected from the group consisting of: methyl, ethyl, n-propyl and n-butyl, and -G 2Can be selected from the group consisting of: -F, -Cl, -Br and -I. Such compounds allow for the derivatization of nucleophilic target molecules by nucleophilic substitution at the alkylpyridine moiety.

[0077] In some embodiments Can be Z may be C=O, and n may be from 1 to 3. Such compounds allow the derivatization of carbonyl-containing target molecules, such as ketones or aldehydes, by forming a hydrazone between the carbonyl group of the target molecule and the hydrazine moiety of the compound.

[0078] In some embodiments, Can be And –G 3 It may be a phenyl group optionally substituted with one or more -Br substituents. Such compounds allow the derivatization of primary amine-containing target molecules by nucleophilic attack on the pyrylium ring followed by ring closure to provide pyridinium derivatives.

[0079] Other objects, advantages and novel features of the present invention will become apparent to those skilled in the art from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] For a more complete understanding of the present invention and further objects and advantages thereof, the detailed description given below should be read in conjunction with the accompanying drawings in which like reference numerals represent similar items in the various views and in which:

[0081] Figure 1a Schematic illustration of the derivatization of nucleophiles using 2-substituted N-alkylpyridinium based reactive substrates.

[0082] Figure 1b Derivatization of carbonyl-containing compounds using hydrazine-based reactive substrates is schematically illustrated.

[0083] Figure 1c Derivatization of primary amines using pyridinium-based reactive substrates is schematically illustrated.

[0084] Figure 2a The general synthetic approach to produce 2-substituted N-alkylpyridinium-based reactive substrates is schematically illustrated.

[0085] Figure 2b This schematically illustrates how Figure 2a The general synthetic method shown produces a series of 2-substituted N-alkylpyridinium compounds.

[0086] Figure 3 The general synthetic method for producing N-substituted pyridine reactive substrates bearing hydrazine reactive moieties is schematically illustrated.

[0087] Figure 4 is a MALDI mass spectrum showing the detection of the hydrazone derivative of 11-deoxycorticosterone without the addition of any additional auxiliary matrix.

[0088] Figure 5 is a series of images (a)-(f) showing MALDI-MSI analysis of fluvoxamine in mouse brain tissue sections. DETAILED DESCRIPTION

[0089] The reactive matrix of the present invention can facilitate mass spectrometry analysis, such as MALDI-MS (I) and DESI-MS (I) analysis of a variety of compounds, including drugs and their metabolites, endogenous neurotransmitters, amino acids, metabolites, steroids, toxins and environmental pollutants, food additives, food ingredients and any other small molecules containing primary and secondary amines, phenol hydroxides and carbonyl functional groups. The reactive matrix of the present invention is an important tool for rapid monitoring / molecular imaging of the above compounds in the following fields, such as: general biology, general medicine, neuroscience, cancer research, drug development, toxicology, environmental science and food chemistry.

[0090] Reactive matrices are suitable for a wide range of mass spectrometry (MS) and mass spectrometry imaging (MSI) techniques using desorption and / or laser ablation. These techniques include, but are not limited to:

[0091] Matrix-assisted laser desorption / ionization (MALDI-MS, MALDI-MSI);

[0092] Desorption electrospray ionization (DESI-MS, DESI-MSI);

[0093] matrix-assisted laser desorption electrospray ionization (MALDESI-MS, MALDESI-MSI);

[0094] nanospray desorption electrospray ionization (nano-DESI-MS, nano-DESI-MSI);

[0095] Secondary ion mass spectrometry (SIMS, SIMS-MSI);

[0096] Matrix-enhanced secondary ion mass spectrometry (ME-SIM, ME-SIMS-MSI); and

[0097] Laser ablation electrospray ionization (LAESI-MS, LAESI-MSI).

[0098] Reactive matrices can also be used in other forms of spectroscopy or mass spectrometry. For example, they can be used to label molecules with chromophores prior to UV spectroscopy or UV imaging. They can also be used to provide a permanent charge ("charge labeling") to molecules prior to mass spectrometry using electrospray ionization (ESI), and can therefore be used in liquid extraction surface analysis (LESA) including ESI or MALDI.

[0099] Reactive matrices are compounds that react with one or more target analyte molecules and facilitate mass spectrometric analysis of the target molecules. In some cases, the use of a reactive matrix can completely avoid the need to use another matrix to assist in ionization. However, in some cases, a reactive matrix can also be used with a conventional non-reactive matrix to improve the detection of target molecules.

[0100] The reactive matrix disclosed herein comprises at least two functional domains: a highly reactive domain that facilitates covalent analyte charge labeling by derivatization; and a conjugated chromophore domain to facilitate laser-assisted desorption and / or ablation. However, in some cases where the reactive domain is part of a polycyclic ring system, the reactive domain and the conjugated chromophore domain can be integrated into the same ring system. The reactive matrix can also comprise a recognition domain; that is, a suitable isotope or element having a unique isotopic pattern and mass defect to facilitate mass spectrometric identification of compounds derived from the reactive matrix.

[0101] Reaction Domain

[0102] The reactive domain of the reactive matrix can be an N-alkylpyridinium moiety with a suitable leaving group, an N-substituted pyridine or pyrylium moiety with a hydrazine moiety. All of these reactive domains have the advantage of providing a permanent positive charge to the analyte after derivatization due to the presence of a nitrogen-substituted pyridine moiety after derivatization. This greatly improves the sensitivity of the analyte for positive ion mass spectrometry, especially for analytes that are difficult to protonate.

[0103] Reactive N-alkylpyridinium moieties with suitable leaving groups can react selectively with molecules containing any of a number of common nucleophilic functional groups, including phenolic hydroxyls and primary and secondary amines. Figure 1a A reaction scheme demonstrating this derivatization reaction is shown. The reaction does not require any extended incubation time to occur. The leaving group of the N-alkylpyridine (G 2 The group) can be any suitable leaving group for a nucleophilic aromatic substitution. Such leaving groups include –F, –Cl, –Br, –I, –NO2, –CN, –OR′, –OC(O)R′, –SR′, –S(O)R′, –S(O)(O)R′, and –NR′R″, wherein R′ and R″ are each independently phenyl or C1-C 15The leaving group can be appropriately located at the 2- or 4-position of the pyridine ring, which is considered a nucleophilic aromatic substitution (S N Ar) is the most reactive position. However, the placement of the leaving group at the 3-position has been shown to also provide sufficient reactivity, at least in some cases. N-alkyl groups are described below.

[0104] like Figure 1b As shown, nitrogen-substituted pyridines with reactive hydrazine moieties can selectively react with any analyte with a carbonyl functional group. The reaction proceeds very quickly. The hydrazine moiety is bonded to the N position or another position of the pyridine ring, such as the 2 or 4 position, via a linker. The linker can suitably be an alkyl chain, optionally comprising a carbonyl group closest to the hydrazine moiety. Alternatively, the linker can be a direct bond between the N-substituted pyridine and the nitrogen of the hydrazine moiety. If the hydrazine moiety is bonded to the carbon of the pyridine ring (via a linker), the N position of the pyridine ring is alkylated to provide a permanently charged N-alkyl pyridine.

[0105] The N-alkyl group of the pyridine reactive domain can be any suitable group, such as C1–C 15 For example, by having a rich 2 H. 3 H. 13 C or 14 C, the N-alkyl group can be isotopically enriched. This facilitates the identification of analytes derived by mass spectrometry. The N-alkyl group can be a chiral group, allowing stereoisomerization between analytes. When the reaction domain is a 2-substituted N-alkylpyridinium, in order to be able to perform a nucleophilic substitution reaction at the 2-position of the pyridine ring, the N-alkyl group is preferably non-bulky. For example, the N-alkyl group can be a methyl group.

[0106] The pyrylium reactive moiety can selectively react with analytes having primary amine functional groups to produce Figure 1c N-Alkylpyridine derivatives are shown. This reaction may require considerable incubation times, depending on the exact nature of the pyrylium moiety and the analyte.

[0107] Conjugated chromophore domain

[0108] The conjugated chromophore domain of the reactive matrix comprises Ar selected from the group consisting of 1 Groups: phenyl, biphenyl, terphenyl, C 10 –C 30 PAHs and C4–C 30Monocyclic or polycyclic heteroaryl. Polycyclic aromatic hydrocarbons refer to carbocyclic aromatic ring systems containing two or more fused rings. Suitable polycyclic aromatic hydrocarbons are listed, for example, as structure numbers 4-521 in NIST Special Publication 922 (June 2011 revised edition) "Polycyclic Aromatic Hydrocarbon Structure Index", which is incorporated herein by reference. The polycyclic aromatic hydrocarbons can be selected, for example, from the group consisting of naphthalene, fluorine, anthracene, phenanthrene, pyrene, tetracene, Perylene, corannulene, coronene, and pyranthracene. 30 Monocyclic or polycyclic heteroaryl refers to any heteroaryl ring system from 5-membered ring and above (i.e., furan, thiophene, pyrrole), including any heteroaryl analogs of polycyclic aromatic hydrocarbons as defined above. The heteroaryl ring may contain any combination of carbon and heteroatoms, including but not limited to oxygen, sulfur and nitrogen atoms. The conjugated chromophore domain preferably exhibits strong optical absorption at the wavelength of the pulsed laser (typically 337nm for nitrogen, or 355nm for Nd:YAG lasers). For example, a conjugated chromophore domain having an anthracene or substituted anthracene moiety provides a reactive matrix with a suitable absorptivity without excess molar mass.

[0109] Ar 1 The group may be provided with one or more substituents to adjust the properties of the reactive substrate. Suitable substituents may include, for example, -D, -T, -F, -Cl, -Br, -I, -NO2, -CN, -R"', -OR"', -OC(O)R"', -SR"', -S(O)R"', -S(O)(O)R"' and -NR"'R"", wherein -R"' and -R"" are each independently selected from phenyl, tolyl and C1-C 15 alkyl.

[0110] "Conjugated chromophore domain" means that the chromophore contains a conjugated π-system, not necessarily that the chromophore is conjugated to the reactive domain. However, in order to provide suitable absorbance, the conjugated system may extend to the pyridinium / pyrylium ring of the derivatized analyte. This is in the case of Ar 1 This is particularly preferred when the group itself does not have a sufficiently extended π-system to absorb the above wavelengths. This is achieved by using a π-conjugated linker moiety to connect Ar 1 The π-conjugated linker moiety refers to the 1 The Ar 1 A direct bond between the group and the pyridinium / pyrylium moiety, or in the case of a direct connection of Ar 1 The path from the group to the pyridinium / pyrylium moiety contains only sp and / or sp 2 Such a moiety may be, as described above, Ar 1A direct bond between the group and the pyridinium / pyrylium moiety, or it may be a moiety selected from the group consisting of: -O-, -S-, The π-conjugated linker moiety may include sp 3 The side groups of the hybrid atoms are not directly bonded to Ar. 1 The π-conjugated linker moiety may contain chiral side groups to facilitate stereoisomerization between analytes. 1 The π-conjugated linker moiety can also allow axial chirality (atropization) by limiting the rotation of the group relative to the pyridinium / pyrylium moiety. This is especially true if the π-conjugated linker moiety is a direct bond. However, if Ar 1 If the group itself has suitable absorption, then it is not necessarily advantageous to extend the conjugated system into the pyridinium / pyrylium ring, and in this case, a non-conjugated linker can be used, such as an alkyl chain or a sp-containing 3 Any other chain of hybridized carbon atoms connecting the pyridinium / pyrylium ring to Ar 1 Group.

[0111] You can choose the appropriate G 1 Groups are used to adjust the chromophore and reaction properties of reactive substrates. 1 Can be selected from –H, –Me or –Ar 2 , where –Ar 2 is optionally substituted and selected from the group consisting of phenyl, biphenyl, terphenyl, C 10 –C 30 PAHs and C4–C 30 Monocyclic or polycyclic heteroaryl. If G 1 For aryl (Ar 2 ), this can further extend the π conjugation to G 1 groups, thereby changing the absorbance of the reactive matrix. 1 Ar 2 When the group is 2 The group provides one or more substituents that can further tune the reactivity and chromophore properties of the reactive substrate. Suitable substituents may include -D, -T, -F, -Cl, -Br, -I, -NO2, -CN, -R"', -OR"', -OC(O)R"', -SR"', -S(O)R"', -S(O)(O)R"', and -NR"'R"", wherein -R"' and -R"" are each independently selected from phenyl, tolyl, and C1-C 15 alkyl.

[0112] When the reaction domain is the pyrylium moiety, a suitable G may be selected. 3The chromophore and reaction properties of the reactive matrix can be adjusted by using the following groups. 3 The groups can be independently selected from 1 Same substituents.

[0113] Integrated reactive domain and conjugated chromophore domain

[0114] In some cases, where the reactive domain is part of a polycyclic ring system, a separate conjugated chromophore domain may not be required because the reactive domain itself has sufficiently good chromophore properties, i.e., the reactive domain and the conjugated chromophore domain may be integrated into the same ring system. For example, this may be the case where the reactive domain is a fused N-alkylpyridinium moiety, such as an N-alkylacridine moiety or an N-alkylquinoline moiety. Such an integrated system may provide improved atom economy.

[0115] Identify the domain

[0116] The reactive matrix can be provided with a recognition domain that facilitates mass spectrometric recognition of analytes that are charge labeled with the reactive matrix. This can be achieved, for example, by providing chlorine or bromine substituents on the reactive matrix, since these atoms provide unique mass spectrometric isotope patterns and mass defects. Alternatively, the reactive matrix can be prepared by using deuterium, tritium, or carbon isotopes such as 13 C or 14 By labeled, isotopically enriched, or substituted with a specific isotope, it is meant that the specific isotope (e.g., 2 H. 3 H. 13 C or 14 C) occurs at a position in a molecule in excess of the natural abundance at that position. For example, "recognizing" isotopes (e.g. 2 H. 3 H. 13 C or 14 C) with the most abundant naturally occurring isotope at the relevant position (e.g. 1 H or 12 C) may be in a ratio of at least 1:10, such as at least 1:1 or at least 10:1.

[0117] The reactive matrix has a permanent positive charge and can therefore be formed into a salt with one or more anionic counterions. Such salts can, for example, comprise one or more anions selected from the non-exhaustive group consisting of chloride, bromide, iodide, toluenesulfonate, methanesulfonate, tetrafluoroborate, perfluorobutanesulfonate, benzenesulfonate, hexafluorophosphate and trifluoromethanesulfonate.

[0118] Application of reactive matrices in desorption and / or laser ablation ionization mass spectrometry

[0119] When preparing samples for mass spectrometry analysis or imaging, reactive matrix materials do not need to be incubated with the sample in most cases. When the reagents are added / spotted / sprayed onto the sample, the reaction occurs simultaneously without any cooling or heating. Depending on the nature of the reaction domain, reactive matrices target substances with primary amine, secondary amine, phenol hydroxide, aldehyde or ketone functional groups. All reactive matrices carry a permanent positive charge after reaction with the functional groups of the analyte. This allows for highly sensitive detection and analysis of the derivatized analytes.

[0120] Example

[0121] Example 1: Synthesis of 2-Substituted N-Alkylpyridinium Reactive Substrates

[0122] The 2-substituted N -alkylpyridinium active substrates were synthesized via a modular synthetic route, e.g. Figure 2a Schematically shown. The two domains (chromophore domain and reactive domain) are fused in step A using a Suzuki-Miyara cross-coupling reaction. Thus, treatment of appropriate 2-substituted bromopyridine derivatives with a series of arylboronic acids under palladium catalysis provides precursor compounds in moderate to excellent yields. These precursors can be regioselectively brominated with N-bromosuccinimide in an optional step B to introduce the recognition domain. The precursor is then alkylated under thermal conditions in step C to yield the desired reactive substrate. The selection of reactive substrates generated by this method is as follows Figure 2b As shown. Notably, the final compounds were easily isolated by simple filtration, which eliminated the need for potentially problematic purification steps. The compounds were found to be stable after storage under vacuum for several months, but degraded slowly when stored in solution.

[0123] General Procedure for the Synthesis of 2-Fluoropyridine Using 2-Fluoro-4-phenylpyridine as an Example A

[0124] All reagents were purchased at the highest commercial quality and used without further purification. Unless otherwise stated, yields refer to isolated, homogeneous and spectrally pure material. The crude reaction mixture was purified by silica gel chromatography (E. Merck silica gel, particle size 0.043-0.063 mm). Thin layer chromatography was performed using E. Merck silica plates (60F-254) with UV light (254 nm) as the imaging agent. Recording at 400 MHz 1 H NMR spectra, recorded at 100 MHz 13 C{ 1 H} NMR spectrum. 1 H NMR and 13 C{ 1 The chemical shift of the H}NMR spectrum is determined by the residual solvent signal ( 1H, 7.26 ppm CDCl3, 2.05 ppm acetone-d6, 1.95 ppm CD3CN; 13 C 77.16ppm CDCl3, 29.9ppm acetone-d6, 1.39ppm CD3CN) reference tetramethylsilane. LC / MS analysis was performed on a C18 column (50×3.0mm, particle size 2.6μm, pore size ), electrospray ionization source and a single quadrupole detector. Accurate mass values ​​were determined on a mass spectrometer equipped with an electrospray or MALDI ion source and a TOF or FTICR analyzer.

[0125] To a stirred solution of 4-bromo-2-fluoropyridine (528 mg, 3 mmol), phenylboronic acid (399 mg, 3.3 mmol), Pd(PPh3)4 (69 mg, 0.06 mmol) in toluene / ethanol (4:1, 25 ml) was added a solution of Na2CO3 (1.27 g, 12 mmol) in water (10 ml). The resulting yellow solution was stirred at 80°C for 2 hours, during which time the solution turned black. After cooling to room temperature, the reaction mixture was extracted with EtOAc (3 x 50 ml). The combined organic layers were washed with brine (50 ml), dried over magnesium sulfate, filtered and concentrated in vacuo. The crude product was purified by hot filtration from methanol and then by silica gel chromatography (5% EtOAc in n-pentane) to give the title compound (490 mg, 2.8 mmol, 94%) as a pale yellow solid.

[0126] Optional General Procedure B for Bromination of 2-Fluoropyridine Using 4-(10-Bromoanthracen-9-yl)-2-Fluoropyridine as an Example

[0127] To the CHCl3 (35ml) solution of 4-(anthracene-9-yl)-2-fluoropyridine (715mg, 2.75mmol) stirred, N-bromosuccinimide (1.08g, 6.07mmol) was added, and the resulting solution was refluxed for 48 hours. After cooling to room temperature, the mixture was diluted with water (25ml) and extracted with CH2Cl2 (3x 25ml). The combined organic layer was dried over magnesium sulfate, filtered and concentrated in vacuo. The title compound (784mg, 85%) was obtained as a yellow solid by flash column chromatography (SiO2, EtOAc: toluene: pentane 5:15:80 (v / v / v)).

[0128] General Procedure for the Preparation of N-Alkylaryl Fluoropyridinium Salts Exemplified by 2-Fluoro-1-methyl-4-phenylpyridinium-1-iodide C

[0129] 2-Fluoro-4-phenylpyridine (200 mg, 1.15 mmol) was dissolved in MeI (2 mL) and the resulting solution was heated in a sealed vial at 60 ° C for 72 hours. The resulting precipitate was collected by filtration, washed with ether (3×5 ml) and acetone (2 ml), and dried in vacuo to give the title compound (273 mg, 0.87 mmol, 75%) as a yellow solid. These compounds react easily with any nucleophilic solvent and should be treated accordingly.

[0130] The following compounds illustrate reactive matrices that can be prepared by the general method described above:

[0131]

[0132] 2-Fluoro-1-methyl-4-phenylpyridin-1-ium iodide (2)

[0133] Prepared according to general procedures A and C starting from 2-fluoro-4-bromopyridine. 1 H NMR (400MHz, CDCl3 / CD6CO): δ9.25–9.11(m,1H),8.42–8.33(m,2H),8.11–8.00(m,2H),7.72–7.57(m,3H),4.48(d,J=3.7Hz,3H).

[0134] 13 C NMR (100MHz, CDCl3 / CD6CO): δ 161.7 (d, J = 11.4Hz), 144.6 (d, J = 7.2Hz), 132.8, 129.7, 128.1, 121.1 (d, J = 3.0Hz), 110.2 (d, J = 21.5Hz), 41.2 (d, J = 5.2Hz).

[0135] Exact mass (MALDI, m / z): C 12 H 11 FN([M + ]) Calculated value: 188.0870, found value m / z 188.0877.

[0136]

[0137] 2-Fluoro-1-methyl-4-(naphthalen-2-yl)pyridin-1-ium iodide (3)

[0138] Prepared according to general procedures A and C starting from 2-fluoro-4-bromopyridine. 1H NMR (400MHz, CDCl3 / CD6CO): δ8.90(dd,J=6.7,4.9Hz,1H),8.62(d,J=2.0Hz,1H),8.36(dd,J=6.7,2.1Hz,1H),8.24(dd,J=5 .7,2.1Hz,1H),8-18–8.14(m,2H),8.10–8.02(m,1H),8.00(dt,J=8.7,2.2Hz,1H),7.82–7.66(m,2H),4.36(d,J=3.5Hz,3H).

[0139] 13 C NMR (100MHz, CDCl3 / CD6CO): δ161.6 (d, J = 11.7Hz), 143.6 (d, J = 6.8Hz), 135.2, 133.0, 130.0, 129 .9, 129.4, 128.7, 127.9, 127.7, 123.6, 121.4 (d, J = 2.8), 111.3 (d, J = 21.9Hz), 41.5 (d, J = 5.0Hz).

[0140] Exact mass (MALDI, m / z): C 16 H 13 FN([M + ]) Calculated value: 238.1027, found value m / z 238.1028.

[0141]

[0142] 5-(Anthracene-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide (4)

[0143] Prepared according to general procedures A and C starting from 2-fluoro-4-bromopyridine. 1 H NMR (400MHz, CDCl3 / CD6CO): δ8.77(s,1H),8.64(ddd,J=8.5,5.6,2.3Hz,1H),8.59(dd,J=4.2,2.2Hz,1 H), 8.18 (dt, J = 8.2, 1.3Hz, 2H), 8.05 (dd, J = 8.7, 4.0Hz, 1H), 7.61–7.52 (m, 6H), 4.29 (d, J = 3.8Hz, 3H).

[0144] 13C NMR (100MHz, CDCl3 / CD6CO): δ154.4(d,J=11.4Hz), 145.8(d,J=6.8Hz), 136.0(d,J=3.8Hz), 13 7.6, 130.8, 130.4, 129.5, 127.9, 126.6, 126.4, 125.4, 115.5 (d, J = 20.3Hz), 42.9 (d, J = 5.1Hz).

[0145] Exact mass (MALDI, m / z): C 20 H 15 FN([M + ]) Calculated value: 288.1183, found value m / z 288.1179.

[0146]

[0147] 2-Fluoro-1-methyl-4-(phenanthren-9-yl)pyridin-1-ium iodide (5)

[0148] Prepared according to general procedures A and C starting from 2-fluoro-4-bromopyridine.

[0149] 1 H NMR (400MHz, CDCl3 / CD6CO): δ8.85–8.79(m,1H),8.78–8.69(m,2H),8.05–8.02(m,2H),8.00(dd,J=5.0,1.9H z,1H),7.96(s,1H),7.88(dd,J=8.2,1.2Hz,1H),7.84–7.77(m,2H),7.74–7.66(m,2H),4.30(d,J=3.6Hz,3H).

[0150] 13 C NMR (100MHz, CDCl3 / CD6CO): δ163.4(d,J=11.5Hz), 143.9(d,J=6.7Hz), 132.3(d,J=1.9Hz), 131.7,131.2,130.9,130.8,13 0.1, 129.7, 128.6, 128.5, 128.4, 128.3, 125.8 (d, J = 3.2Hz), 125.6, 124.3, 123.5, 115.5 (d, J = 20.5Hz), 42.3 (d, J = 5.0Hz).

[0151] Exact mass (MALDI, m / z): C 20 H 16 FN([M +]) Calculated value: 288.1183 Found m / z 288.1177.

[0152]

[0153] 4-([1,1':3',1"-terphenyl]-5'-yl)-2-fluoro-1-methylpyridin-1-ium iodide (6)

[0154] Prepared according to general procedures A and C starting from 2-fluoro-4-bromopyridine. 1 H NMR (400MHz, CDCl3 / CD6CO): δ8.71–8.68(m,1H),8.31(dd,J=6.7,2.0Hz,1H),8.26(dd,J=5.7,2.0Hz,1H),8.13–8 .10(m,1H),8.10(d,J=1.7Hz,2H),7.80–7.76(m,4H),7.55–7.49(m,4H),7.49–7.41(m,2H),4.23(d,J=3.6Hz,3H).

[0155] 13 C NMR (100MHz, CDCl3 / CD6CO): δ162.0(d,J=11.5Hz), 144.1(d,J=7.0Hz), 143.8,139.9,134.7(d,J=2.2 Hz), 130.7, 129.6, 128.9, 127.9, 126.1, 122.2 (d, J = 3.1Hz), 111.4 (d, J = 20.3Hz), 41.9 (d, J = 5.1Hz).

[0156] Exact mass (MALDI, m / z): C 24 H 20 FN([M + ]) Calculated: 340.1496, found m / z 340.1496

[0157]

[0158] 2-Fluoro-1-methyl-4-(10-phenylanthracen-9-yl)pyridin-1-ium iodide (7)

[0159] Prepared according to general procedures A and C starting from 2-fluoro-4-bromopyridine. 1H NMR (400MHz, CDCl3 / CD6CO): δ8.87–8.84(m,1H),7.98–7.94(m,2H),7.70(dd,J=8.7,1.4,0 .8Hz,2H),7.68–7.56(m,5H),7.55–7.51(m,2H),7.48–7.39(m,4H),4.38(d,J=3.7Hz,3H).

[0160] 13 C NMR (100MHz, CDCl3 / CD6CO): δ164.1(d,J=11.5Hz),144.8(d,J=6.8Hz),141.3,138.3,131.3,130.0,129.3,1 29.2, 128.9, 128.7, 128.0 (d, J = 3.5Hz), 127.9, 127.8, 126.4, 125.2, 117.0 (d, J = 20.9Hz), 42.4 (d, J = 5.1Hz).

[0161] Exact mass (MALDI, m / z): C 26 H 19 FN([M + ]) Calculated: 364.1496, found m / z 364.1486

[0162]

[0163] 4-(10-Bromoanthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium (8)

[0164] Prepared according to general procedures A, B and C starting from 2-fluoro-4-bromopyridine. 1 H NMR (400MHz, CDCl3 / CD6CO): δ9.01(t,J=5.6Hz,1H),8.53(dd,J=8.9,1.0Hz,2H),7.78(dd,J=6.4,1.7Hz,1H),7.69(dd,J=4.3,1. 7Hz, 1H), 7.56 (ddd, J=9.0, 6.4, 1.2Hz, 2H), 7.50 (dd, J=8.7, 1.1Hz, 2H), 7.42 (ddd, J=8.8, 6.4, 1.1Hz, 2H), 4.36 (d, J= 3.7Hz, 3H).

[0165] 13C NMR (100MHz, CDCl3 / CD6CO): δ161.0(d,J=11.5Hz), 143.7(d,J=6.5Hz), 129.2, 128.7(d,J=1.6Hz), 128.6 , 127.5, 127.2, 127.1, 126.5 (d, J = 3.4Hz), 125.4, 124.6, 124.5, 116.6 (d, J = 16.9Hz), 41.40 (d, J = 5.0Hz).

[0166] Exact mass (MALDI, m / z): C 20 H 14 BrFN([M + ]) Calculated value: 366.0288, found value m / z 366.0284.

[0167]

[0168] 4-(Anthracene-9-yl)-2-fluoro-1-ethylpyridin-1-ium (9)

[0169] Prepared according to general procedures A and C starting from 2-fluoro-4-bromopyridine and using ethyl iodide as alkylating agent. 1 H NMR (400MHz, CDCl3 / CD6CO): 9.32 (dd, J=6.4, 4.8Hz, 1H), 8.67 (s, 1H), 8.12–8.07 (m, 2H), 7.96 (dd, J=6.4, 1.7Hz, 1H), 7.79(dd,J=4.8,1.7Hz,1H),7.64–7.61(m,2H),7.55–7.45(m,4H),4.92(qd,J=7.4,2.4Hz,2H),1.81(t,J=7.3Hz,3H).

[0170] 13 C NMR (100MHz, CDCl3 / CD6CO): δ163.3(d,J=11.3Hz),143.5(d,J=6.5Hz),130.9,130.5,129.0,128.7,128.1(d,J =1.7Hz), 127.9 (d, J = 3.0Hz), 127.8, 125.9, 124.4, 117.3 (d, J = 19.5Hz), 51.8 (d, J = 4.3Hz), 14.8 (d, J = 1.0Hz).

[0171] Exact mass (MALDI, m / z): C 21 H 17 FN([M +]) Calculated value: 302.1340, found value m / z 302.1334.

[0172]

[0173] 4-(Anthracene-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide (10)

[0174] Prepared according to general procedures A and C starting from 2-fluoro-4-bromopyridine. 1 H NMR (400MHz, CDCl3 / CD6CO): δ8.89(t,J=5.5Hz,1H),8.72(s,1H),8.15–8.13(m,2H ),7.92–7.90(m,1H),7.87–7.85(m,1H),7.61–7.46(m,6H),4.39(d,J=3.7Hz,3H).

[0175] 13 C NMR (100MHz, CDCl3 / CD6CO): δ163.7 (d, J = 11.4Hz), 144.6 (d, J = 6.8Hz), 131.2, 130.7, 129. 4, 129.1, 128.0, 127.7 (d, J = 3.2Hz), 126.2, 124.8, 117.6 (d, J = 20.9Hz), 42.4 (d, J = 5.1Hz).

[0176] Exact mass (MALDI, m / z): C 20 H 15 FN([M + ]) Calculated value: 288.1183, found value m / z 288.1184.

[0177]

[0178] 4-(Anthracene-9-yl)-2-fluoro-1-methylpyridin-1-ium trifluoromethanesulfonate (11)

[0179] Prepared according to general procedures A and C starting from 2-fluoro-4-bromopyridine and using methyl triflate as methylating agent.

[0180] 1H NMR (400MHz, CDCl3 / CD6CO): δ8.81–8.73(m,1H),8.72(s,1H),8.15–8.02(m,2H),7.84(dd ,J=6.4,1.7Hz,1H),7.78(dd,J=4.6,1.7Hz,1H),7.62–7.43(m,6H),4.38(d,J=3.7Hz,3H).

[0181] 13 C NMR (100MHz, CDCl3 / CD6CO): δ163.7(d,J=11.4Hz), 144.6(d,J=6.8Hz), 131.0,13 0.6, 129.1, 128.9, 128.4, (d, J = 3.2Hz), 126.1, 124.4, 117.2, 42.1 (d, J = 5.1Hz).

[0182] Exact mass (MALDI, m / z): C 20 H 15 FN([M + ]) Calculated value: 288.1183, found value m / z 288.1176.

[0183] Example 2: Determination of MALDI-MS sensitivity of derivatized model compounds

[0184] In order to evaluate and compare the efficiency of synthetic substances as reactive matrices for high-sensitivity MALDI-MSI analysis, small molecules with different functional groups were found in the cortical region of rat control brain tissue sections. On the same tissue sections, a rectangular region of interest was selected in the striatum region of each brain. MALDI-MSI analysis was performed on all model substances and selected striatum regions with different synthetic reactive matrices. The signal-to-noise ratio (S / N) values ​​of all derivative compounds were calculated using internally developed software (Kallback, P., Nilsson, A., Shariatgorji, M. & Andren, PEmsIQuant--Quantitation Software for Mass Spectrometry Imaging Enabling Fast Access, Visualization, and Analysis of Large Data Sets. Anal Chem 88, 4346-4353 (2016)). The results are shown in Table 1 below.

[0185] Table 1

[0186]

[0187] ND = Not Detected (S / N ratio <3) * = 10-fold higher than the concentration of the analyte used

[0188] DA = dopamine; PEA = phenylethylamine; EPI = epinephrine; HD = hordenine

[0189] From the S / N values, it can be concluded that the ethylated and methylated iodide salts of 4-(anthracen-9-yl)-2-fluoro-1-alkylpyridin-1-ium (entries 9 and 10) produced the best S / N values ​​for the model compounds tested. The traditional non-reactive matrices CHCA and DHB (entries 13 and 12, respectively), which are the most widely used positive ion mode non-reactive / non-selective MALDI-MS auxiliary matrices for small molecules, were compared to the newly synthesized reactive matrices. The data in Table 1 show that the sensitivity is significantly improved when the synthesized reactive matrix is ​​used compared to the traditional non-reactive matrix, from at least 10 times for the detection of phenylethylamine to about 500 times for the analysis of dopamine.

[0190] Example 3: Synthesis of 4-(anthracen-9-yl)-1-(2-hydrazino-2-oxoethyl)pyridin-1-ium

[0191] Figure 3 A general method for synthesizing reactive substrates having a reactive hydrazine moiety attached to the N-pyridine position is shown. The procedure is exemplified by the synthesis of 4-(anthracen-9-yl)-1-(2-hydrazinyl-2-oxoethyl)pyridin-1-ium, as described below. However, it is noted that Boc-hydrazine can react with acyl halides or alkyl halides other than chloroacetyl chloride, thereby being able to provide reactive substrates having a hydrazine moiety attached to the nitrogen of the pyridine ring.

[0192] tert-Butyl 2-(2-chloroacetyl)hydrazine-1-carboxylate

[0193] To a stirred and cooled (0°C) solution of tert-butyl hydrazinecarboxylate (1.98 g, 15 mmol, 2 eq) in DCM (20 ml) and pyridine (0.79 g, 10 mmol, 1.5 eq) was added a solution of chloroacetyl chloride (1.12 g, 10 mmol, 1.5 eq) in DCM (5 ml). The reaction mixture was stirred for 6 hours and washed three times with diluted HCl (50 ml). The organic phase was dried over Na2SO4. The solvent was removed under reduced pressure to give the title compound (1.16 g, 55%) as a clear oil.

[0194] 4-(Anthracene-9-yl)-1-(2-(2-(tert-butoxycarbonyl)hydrazine)-2-oxoethyl)pyridin-1-ium chloride

[0195] A solution of 4-(anthracen-9-yl)pyridine (0.33 g, 1.29 mmol, 1 eq., produced by General Method A above), tert-butyl 2-(2-chloroacetyl)hydrazine-1-carboxylate (0.54 g, 2.58 mmol, 2 eq.) in chloroform (2 ml) was heated at 120° C. for 30 minutes. The reaction mixture was left to crystallize, the solid product was filtered and washed with ethyl acetate. It was purified by vapor diffusion crystallization (EtOH and diethyl ether). The yield of the yellow product (0.44 g, 81%).

[0196] 4-(Anthracene-9-yl)-1-(2-hydrazino-2-oxoethyl)pyridin-1-ium

[0197]

[0198] To a stirred solution of 4-(anthracen-9-yl)-1-(2-(2-(tert-butoxycarbonyl)hydrazine)-2-oxoethyl)pyridin-1-ium chloride (50 mg, 0.12 mmol) in 1 ml of 10% MeOH in DCM was added dropwise 1 ml of 4M HCl in 1,4-dioxane under nitrogen. The reaction mixture was stirred at room temperature overnight under nitrogen. The precipitate formed was filtered, washed with EtOAc and dried. Yield of yellow powder (36 mg, 94%). 1 H NMR(400MHz, DMSO-d6)δ9.35(d,J=6.0Hz,2H),8.90(s,1H),8.42(d,J=6.3Hz, 2H), 8.25 (dd, J = 8.3, 1.3Hz, 2H), 7.60 (m, 2H) 7.55, 7.41 (d, 2H), 5.88 (s, 2H). 13 C NMR (101MHz, DMSO-d6) δ164.5,157.2,147.0,131.0,130.8,130.3,129.8,129.4,128.7,127.9,126.3,124.9,60.1.

[0199] Example 4: Use of 4-(anthracen-9-yl)-1-(2-hydrazino-2-oxoethyl)pyridin-1-ium as a reactive matrix for MALDI-MS analysis of 11-deoxycorticosterone

[0200] The synthesized 4-(anthracen-9-yl)-1-(2-hydrazino-2-oxoethyl)pyridin-1-ium reactive matrix was added to a sample of 11-deoxycorticosterone on a metal plate, and the resulting derivative was directly analyzed by MALDI-MS operated in positive ion mode. The final MALDI spectrum is shown in Figure 4As shown. At m / z = 640.6, the peak corresponding to the reactive matrix bound to 11-deoxycorticosterone in the hydrazone form can be clearly observed. In order to obtain the MALDI spectrum, no additional auxiliary MALDI matrix material is required.

[0201] In control experiments without reactive matrix and with the conventional auxiliary matrix substance DHB, no signal for 11-deoxycorticosterone was observed at relevant sample concentrations.

[0202] Example 5: Synthesis and MALDI-MS Utility of Further Pyridinium Reactive Matrices

[0203] The following compounds illustrate additional reactive substrates that can be prepared by General Methods A and / or C and the methods described below.

[0204]

[0205] 4-(Dibenzo[b,d]thiophen-4-yl)-2-fluoro-1-methylpyridin-1-ium iodide (14)

[0206] Prepared according to general procedures A and C starting from 2-fluoro-4-bromopyridine. 1 H NMR (400MHz, CD3CN / CD3Cl) δ8.70(dd,J=6.6,5.0Hz,1H),8.50(dd,J=7.7,1.3Hz,1H),8.38–8.30(m,1H),8.28(dd,J=6.6,2.0Hz,1H),8.19( dd,J=5.4,2.0Hz,1H),8.02–7.93(m,1H),7.80(dd,J=7.5,1.4Hz,1H),7.75(dd,J=7.7,7.6Hz,1H),7.62–7.56(m,2H),4.25(d,J=3.6Hz,3H).

[0207] 13 C NMR (101MHz, CD3CN / CD3Cl) δ161.0(d,J=11.2Hz),143.7(d,J=7.1Hz),137.4,137.1,136.7,134.0,128.6(d,J=2.0 Hz), 127.8, 127.3, 125.2, 124.7, 124.5, 122.1 (d, J = 3.6Hz), 122.0, 121.6, 111.8 (d, J = 21.4Hz), 40.8 (d, J = 5.8Hz).

[0208] MS (m / z) C 18 H 13 FNS +([M + ]) Calculated value: 294.1 Found m / z 294.1.

[0209]

[0210] 2-Fluoro-1-methyl-4-(naphthalen-1-yloxy)pyridin-1-ium iodide (15)

[0211] To a solution of 2,4-difluoropyridine (3.5 mmol) in DMF (4 mL) was added 1-naphthol (1.5 eq.) and K2CO3 (3 eq.). The resulting solution was stirred at room temperature for 18 h and then extracted with EtOAc and brine. The intermediate 2-fluoro-4-(naphthalene-1-yloxy)pyridine was isolated by silica gel chromatography eluting with 5% EtOAc in n-hexane. The title compound was prepared starting from 2-fluoro-4-(naphthalene-1-yloxy)pyridine according to General Procedure C. 1 H NMR (400MHz, CD3CN / CD3Cl) δ8.45(ddd,J=7.2,5.6,1.3Hz,1H),8.11(d,J=8.9Hz,1H),8.05–7.99(m,1H),7.99–7.90(m,1 H),7.76(d,J=2.5Hz,1H),7.66–7.55(m,2H),7.38–7.32(m,2H),7.20(dd,J=6.3,2.6Hz,1H),4.06(d,J=3.5,1.3Hz,3H).

[0212] 13 C NMR (101MHz, CD3CN / CD3Cl) δ175.1 (d, J = 12.5Hz), 161.7 (d, J = 273.1Hz), 150.8, 146.4 (d, J = 5.4Hz), 135.1, 133. 3, 132.7, 129.1, 129.0, 128.6, 128.0, 120.3, 119.3, 113.6 (d, J = 2.5Hz), 101.5 (d, J = 25.4Hz), 41.7 (d, J = 5.0Hz).

[0213] MS (m / z) C 16 H 13 FNO+([M + ]) Calculated value: 254.1 Found m / z 254.2.

[0214]

[0215] 2-(Anthracene-9-yl)-4-fluoro-1-methylpyridin-1-ium iodide (16)

[0216] Prepared according to general procedures A and C starting from 4-fluoro-2-bromopyridine. 1 H NMR(400MHz,CD3CN)δ9.26(dd,J=7.1,5.5Hz,1H),8.96(s,1H),8.30–8.22(m,2H),8.09(td,J=6.9,3.0Hz ,1H),7.95(dd,J=7.0,3.0Hz,1H),7.68–7.55(m,4H),7.44(dq,J=8.0,1.0Hz,2H),3.78(d,J=1.0Hz,3H).

[0217] 13 C NMR(101MHz,CD3CN)δ173.3(d,J=280.8Hz),159.0(d,J=14.1Hz),153.3(d,J=13.3Hz),132.9,13 1.9, 130.3, 130.2, 129.7, 127.2, 124.7, 123.5, 121.5 (d, J = 22.1Hz), 117.2 (d, J = 22.3Hz), 46.8.

[0218] MS (m / z) C 20 H 15 FN + ([M + ]) Calculated value: 288.1 Found m / z 288.1.

[0219]

[0220] 4-(Anthracene-9-yl)-2-iodo-1-methylpyridin-1-ium chloride (17)

[0221] Prepared according to general procedures A and C starting from 4-fluoro-2-chloropyridine. 1 H NMR (400MHz, DMSO-d6) δ9.46 (d, J = 6.3Hz, 1H), 8.91–8.82 (m, 2H), 8.29–8.18 (m, 3H), 7.65–7.59 (m, 2H), 7.59–7.52 (m, 4H), 4.54 (s, 3H).

[0222] MS (m / z) C 20 H 15 IN + ([M + ]) Calculated value: 396.0 Found m / z 396.0.

[0223]

[0224] 4-(Anthracene-9-ylmethyl)-2-fluoro-1-methylpyridin-1-ium iodide (18)

[0225] Prepared according to general procedures A and C starting from 2-fluoropyridin-4-ylboronic acid and 9-(bromomethyl)anthracene. 1 H NMR(400MHz,CD3CN)δ8.67(s,1H),8.33(dd,J=6.6,4.8Hz,1H),8.21–8.11(m, 4H),7.64–7.51(m,5H),7.47–7.42(m,1H),5.35(s,2H),4.04(d,J=3.6Hz,3H).

[0226] 13 C NMR(101MHz,CD3CN)δ169.2(d,J=11.2Hz),158.5(d,J=280.5Hz),144.4(d,J=6.6Hz),132.7,131.3,130.4,129 .3,128.1,128.0,126.4,124.8,124.7(d,J=3.2Hz), 114.2(d,J=20.5Hz), 42.1(d,J=5.4Hz), 34.4(d,J=1.8Hz).

[0227] MS (m / z) C 21 H 17 FN + ([M + ]) Calculated value: 302.1 Found m / z 302.2.

[0228]

[0229] 2-Iodo-1-methylquinolinium chloride (19)

[0230] Prepared according to general procedure C starting from 2-chloroquinoline. 1 H NMR(400MHz DMSO-d6)δ7.90(d,J=9.4Hz,1H),7.72(dd,J=7.7,1.6Hz,1H),7.63(ddd,J=8.7,7.1,1.6Hz,1H ),7.53(d,J=8.5Hz,1H),7.27(ddd,J=7.7,7.1,1.1Hz,1H),6.61(d,J=9.5Hz,1H),3.61(s,3H);

[0231] 13C NMR (101MHz, DMSO-d6) δ161.1,139.7,139.2,130.8,128.7,121.9,121.1,120.1,114.6,29.0.

[0232] MS (m / z) C 10 H9IN + ([M + ]) Calculated value: 270.0 Found m / z 270.0.

[0233]

[0234] 2-Chloro-1-methylquinolin-1-ium trifluoromethanesulfonate (20)

[0235] Prepared according to general procedure C starting from 2-chloroquinoline and methyl triflate. 1 H NMR (400MHz, DMSO-d6) δ7.89(d,J=9.5Hz,1H),7.71(dd,J=7.7,1.6Hz,1H),7.62(ddd,J=8.6,7.1,1.6Hz ,1H),7.52(dd,J=8.5,0.9Hz,1H),7.26(ddd,J=7.9,7.2,1.1Hz,1H),6.60(d,J=9.4Hz,1H),3.61(s,3H).

[0236] 13 C NMR (101MHz, DMSO-d6) δ161.2,139.8,139.3,130.9,128.8,122.0,121.1,120.2,114.7,29.1.

[0237] MS (m / z) C 10 H9ClN + ([M + ]) Calculated value: 178.0 Found m / z 178.0.

[0238]

[0239] 9-Chloro-10-methylacridin-10-ium trifluoromethanesulfonate (21)

[0240] Prepared according to general procedure C starting from 9-chloroacridine and methyl triflate. 1H NMR (400MHz, (CD3)2CO) δ9.01 (ddd, J=8.7, 1.5, 0.6Hz, 2H), 8.97 (dt, J=9.3, 0.8Hz, 2H ),8.60(ddd,J=9.3,6.8,1.5Hz,2H),8.23(ddd,J=8.7,6.8,0.9Hz,2H),5.12(s,3H).; 13 C NMR (101MHz, DMSO-d6 / (CD3)2CO) δ177.3,143.2,134.4,127.3,122.6,121.6,116.5,34.0.

[0241] MS (m / z) C 14 H 11 C1N + ([M + ]) Calculated value: 228.1 Found m / z 228.1.

[0242]

[0243] 4-(Anthracene-9-ylethynyl)-2-fluoro-1-methylpyridin-1-ium iodide (22)

[0244] 9-(Propan-1-yn-1-yl)anthracene (1 eq.), CuI (0.11 eq.), Pd(PPh3)2Cl2 (0.1 eq.), 4-bromo-2-fluoropyridine (2.7 eq.) were dissolved in dry TEA (15 mL) and the reaction mixture was stirred at 110°C overnight. After cooling to room temperature, 100 mL of saturated NH4Cl solution was added and the aqueous phase was extracted three times with 100 mL of ether. The combined organic phases were dried over Na2SO4 and concentrated under vacuum. The crude mixture was purified by column chromatography (silica gel, pentane + 5% toluene and 2-5% EtOAc) to give the intermediate 4-(anthracen-9-ylethynyl)-2-fluoropyridine. The title compound was prepared according to General Procedure C starting from 4-(anthracen-9-ylethynyl)-2-fluoropyridine. 1 H NMR (400MHz, DMSO-d6) δ9.01(dd,J=6.6,5.1Hz,1H),8.97(s,1H),8.76(dd,J=5.6,1.8Hz,1H),8.72(dd,J=8.7,1.0Hz,2H),8.48(dd,J=6 .5,1.8Hz,1H),8.27(dd,J=8.4,1.1Hz,2H),7.82(ddd,J=8.7,6.6,1.3Hz,2H),7.71(ddd,J=8.0,6.6,1.2Hz,2H),4.21(d,J=3.7Hz,3H).

[0245] MS (m / z) C 22 H 15 FN + ([M + ]) Calculated value: 312.1 Found m / z 312.1.

[0246]

[0247] 3-(Anthracene-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide (23)

[0248] Prepared according to general procedures A and C starting from 2-fluoro-3-bromopyridine. 1 H NMR(400MHz,DMSO-d6)δ9.25–9.17(m,1H),8.94(s,1H),8.93–8.85(m,1H),8.30–8.22(m,3H) ,7.71(d,J=8.7Hz,2H),7.64(ddd,J=8.3,6.6,1.6Hz,2H),7.58(ddd,J=8.3,6.6,1.6Hz,2H).

[0249] MS (m / z) C 20 H 15 FN + ([M + ]) Calculated value: 288.1 Found m / z 288.2.

[0250]

[0251] 3-(Anthracene-9-yl)-5-fluoro-1-methylpyridin-1-ium iodide (24)

[0252] Prepared according to general procedures A and C starting from 5-fluoro-3-bromopyridine. 1 H NMR (400MHz, DMSO-d6) δ9.66 (ddd, J=3.9, 2.4, 1.1Hz, 1H), 9.22 (dd, J=1.30, 1.26Hz, 1H), 8.95 (ddd, J=8.2, 2.5, 1.4Hz, 1H), 8.91 (s, 1H), 8.26 (ddd, J=8.4, 1.4, 0.7Hz, 2H), 7.70–7.60 (m, 4H), 7.56 (ddd, J=8.7, 6.6, 1.4Hz, 2H), 4.45 (s, 3H).

[0253] 13C NMR (101MHz, CDCl3 / CD3CN) δ161.1(d,J=257.2Hz),144.6,141.6(d,J=7.6Hz),136.2(d,J=1 7.6Hz), 135.7 (d, J = 36.8Hz), 131.7, 130.7, 130.6, 129.5, 128.1, 126.6, 126.5, 125.7, 50.0.

[0254] MS (m / z) C 20 H 15 FN + ([M + ]) Calculated value: 288.1 Found m / z 288.2.

[0255]

[0256] 2-(Anthracene-9-yl)-6-fluoro-1-methylpyridin-1-ium trifluoromethanesulfonate (25)

[0257] Prepared according to general procedures A and C starting from 2-fluoro-6-bromopyridine. 1 H NMR (400MHz, CD3CN) δ8.95(s,1H),8.82(dd,J=7.5,7.0Hz,1H),8.27(d,J=7.7Hz,2H),8.07(dd,J=8 .9, 4.5Hz, 1H), 8.02–7.94 (m, 1H), 7.70–7.56 (m, 4H), 7.47 (d, J = 8.1Hz, 2H), 3.67 (d, J = 3.9Hz, 3H).

[0258] 13 C NMR(101MHz,CD3CN)δ161.8(d,J=281.4Hz),153.8(d,J=8.4Hz),151.5(d,J=12.8Hz),133.0,13 2.0, 131.0, 130.3, 129.6, 129.4, 129.3, 127.3, 124.8, 115.5 (d, J = 21.6Hz), 39.0 (d, J = 6.6Hz).

[0259] MS (m / z) C 20 H 15 FN + ([M + ]) Calculated value: 288.1 Found m / z 288.2.

[0260]

[0261] (E)-4-(2-(Anthracen-9-yl)vinyl)-2-fluoro-1-methylpyridin-1-ium iodide (26)

[0262] To an oven dried glass vessel was added Pd2(dba)3 (0.01 eq), Cy3P (0.04 eq) and 4-(anthracen-9-ylethynyl)-2-fluoropyridine (1 eq). The vessel was sealed and flushed three times with N2 before adding 0.2 mL of 1,4-dioxane. After the mixture was stirred at room temperature for 15 minutes, 25% aqueous formic acid (3 eq) was added and the reaction was heated to 80°C. After 2 hours, the solvent was removed under vacuum and the residue was purified by column chromatography (pentane, 5% toluene and 2-5% EtOAc) to give the intermediate (E)-4-(2-(anthracen-9-yl)vinyl)-2-fluoro-pyridine. The title compound was prepared starting from (E)-4-(2-(anthracen-9-yl)vinyl)-2-fluoro-pyridine according to General Procedures A and C. 1 H NMR(400MHz, DMSO-d6)δ9.03(d,J=16.4Hz,1H),8.91(dd,J=6.6,5.1Hz,1H),8.74(s,1H),8.56(dd,J=6.2,1.8H z,1H),8.41–8.35(m,3H),8.23–8.15(m,2H),7.67–7.56(m,4H),7.36(d,J=16.5Hz,1H),4.19(d,J=3.6Hz,3H).

[0263] MS (m / z) C 22 H 17 FN + ([M + ]) Calculated value: 314.1 Found m / z 314.2.

[0264]

[0265] 2-Fluoro-1-methyl-4-(pyren-1-yl)pyridin-1-ium iodide (27)

[0266] Prepared according to general procedures A and C starting from 4-fluoro-2-bromopyridine. 1 H NMR (400MHz, DMSO-d6) δ9.09 (dd, J=6.5, 5.0Hz, 1H), 8.58 (dd, J=5.5, 1.9Hz, 1H), 8.52 (d, J= 8.1Hz,1H),8.48–8.42(m,2H),8.41–8.30(m,4H),8.23–8.17(m,3H),4.32(d,J=3.7Hz,3H).

[0267] MS (m / z) C 22 H 15 FN + ([M + ]) Calculated value: 312.1 Found m / z 312.2.

[0268]

[0269] 4-(Anthracene-9-yl)-2-hydrazino-1-methylpyridin-1-ium(28)

[0270] To a solution of 4-(anthracen-9-yl)-2-fluoro-1-methyl-pyridinium iodide (10 mg, 0.036 mmol) in acetonitrile was added tert-butyl carbazate (5 mg, 0.036 mmol). The resulting mixture was stirred for 1 hour and then washed with water and ethyl acetate. The combined organic layers were then evaporated under reduced pressure to give the crude protected intermediate. The residue was dissolved in 0.2 mL of 4M HCl in dioxane and stirred at room temperature for 48 hours. The mixture was then extracted with ethyl acetate and water. The combined aqueous layers were evaporated under reduced pressure to give a yellow solid (3 mg, 40%) 1 H NMR(400MHz,DMSO-d6)δ8.69(s,1H),8.23–8.06(m,2H),7.91(d,J=6.8Hz,1H),7.74(dq,J=8.4,1.0Hz,2H ),7.63–7.39(m,4H),6.41(dd,J=1.9,0.6Hz,1H),6.26(dd,J=6.8,1.9Hz,1H),3.59(s,3H),3.56(s,4H).

[0271] 13 C NMR (101MHz, DMSO-d6) δ162.1,150.7,140.4,133.6,131.2,129.1,128.6,127.7,126.8,125.9,121.1,108.9,37.28.

[0272] MS (m / z) C 20 H 18 N3 + ([M] + ) Calculated value m / z 300.2, found value m / z 300.2

[0273] To evaluate the efficiency of the synthesized substances as reactive matrices for high-sensitivity MALDI-MSI analysis, small molecules with primary amine and / or phenolic groups were used as test substances. Two concentrations (0.1 mg / ml and 0.01 mg / ml) of 5-hydroxytryptamine (5-HT), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), 3-O-methyldopa (3-OMD) and γ-aminobutyric acid (GABA) were deposited (0.5 μl) on a MALDI metal target. The reactive matrix solution was prepared by dissolving the synthesized compounds in 70% acetonitrile (4.4 mM). An automatic pneumatic sprayer (TM-Sprayer, HTX Technology) was used to spray 5 ml of heated reagent on the spot standard. The nozzle temperature was set to 80 °C, and the reagent was sprayed in 30 times at a flow rate of 80 μl / min. MSI data were acquired on a MALDI-FT-ICR (Solarix XR 7T-2Ω, Bruker The spectra were obtained by raster scanning the spots at a spatial resolution of 250 μm on a Daltonics instrument in positive ionization mode. The signal-to-noise (S / N) values ​​of all the derivative compounds were extracted from the average spectrum of each spot using in-house developed software (msIQuant). The results are shown in Table 2 below.

[0274] Table 2

[0275]

[0276] Y = S / N greater than 3; N = S / N less than 3.

[0277] As can be seen in Table 2, all of the synthesized reactive matrices effectively promoted the ionization and desorption of a range of analytes.

[0278] Example 6: Use of bromo-pyrylium reactive matrix for MALDI-MSI detection of drugs in brain tissue sections

[0279] Charge labeling by pyrylium derivatization has been shown to improve the sensitivity of MALDI and DESI-MSI analysis of small molecule primary amines. Since this derivatization only adds carbon and hydrogen to the target compound, the isotopic pattern of the resulting product is indistinguishable from those species that have not been underivatized but are ionized, desorbed, and detected non-selectively. By adding bromine to the substituted pyrylium ion, this results in a unique isotopic pattern and mass loss of the derivatized species that distinguishes them from the underivatized compound.

[0280] Materials and reagents

[0281] Unless otherwise stated, tetrafluoroborate salts of 2,4,6-triphenylpyrylium (TPP) and 2-(4-bromo-phenyl)-4,6-diphenyl-pyrylium (Br-TPP), triethylamine (TEA) and all other chemicals were purchased from Sigma-Aldrich (Stockholm, Sweden) and used without further purification. Water, methanol and trifluoroacetic acid (TFA) were obtained from Merck (Hohenbrunn, Germany).

[0282] Animal experiments

[0283] All animals were housed in an air-conditioned room at 20 °C and 53% humidity (dark / light cycle of 12 h) with ad libitum access to food and water. The experiments were performed in accordance with the European Communities Council Directive of 24 November 1986 on the ethical use of animals (86 / 609 / EEC) and were approved by the local ethics committee of the Karolinska Institute (N350 / 08 and N40 / 13).

[0284] Adult male C57BL / 6 mice (3 months old, Charles River Laboratories, Germany) was used for fluvoxamine experiments. Fluvoxamine was dissolved in 0.25% Tween 80 / saline and injected intravenously at a dose of 40 mg / kg. Mice were killed by cervical dislocation 30 minutes after fluvoxamine administration. Vehicle controls were obtained by injecting mice with the same volume of saline administered in the experimental treatment. All brains were immediately removed, snap frozen, and stored at -80°C until further analysis.

[0285] Tissue preparation and derivatization

[0286] Frozen brain tissue was sectioned at 14 μm thickness using a cryostat microtome (Leica CM3050S; Leica Microsystems, Welzlar, Germany), thaw-mounted on conductive indium tin oxide (ITO) slides (Bruker Daltonics), and stored at −80° C. The sections were gently dried under a stream of nitrogen and allowed to dry at room temperature for 15 min before being optically imaged using an optical scanner (Epson perfection V500).

[0287] The brain samples were then coated with TPP and Br-TPP. The derivatization solution was prepared by dissolving 8 mg of TPP or Br-TPP in 6 ml of 80% methanol containing 3.5 μL TEA. An automatic pneumatic sprayer (TM-Sprayer; HTX Technologies, Carrboro, NC) was used to spray the warm reagent on the tissue sections. The nozzle temperature was set to 80°C, and the reagent was sprayed 30 times on the tissue sections at a flow rate of 80 μL / min at a linear speed of 110 cm / min. Before analysis, the samples were incubated in a chamber saturated with 50% methanol solution vapor for 15 minutes (dried once every 5 minutes with a nitrogen stream).

[0288] Mass spectrometry

[0289] All MALDI-MSI experiments were performed using a MALDI-TOF / TOF (Ultraflextreme, Bruker Daltonics, Bremen, Germany) mass spectrometer with a Smartbeam II 2kHz laser in positive ion mode. The laser power was optimized at the beginning of each run and then kept constant during the MALDI-MSI experiment. Fleximaging v 4.0 build 32 (Bruker Daltonics, Billerica, MA) was used for normalization and visualization of MALDI-MSI data.

[0290] result

[0291] TPP and its brominated derivative Br-TPP were used to map the localization of fluvoxamine in mouse brain tissue sections. Figure 5 . Figure 5 The relative abundance and spatial distribution of TPP (a, b) and Br-TPP (d, e)-derivatized fluvoxamine (m / z 609.3 and 687.2) in brain tissue sections of the control (a, d) and drug-treated (b, e) groups without any auxiliary matrix are shown in MALDI-MSI. Figure 5 Also shown are the average spectra of tissue sections treated with fluvoxamine and treated with TPP (c) and Br-TPP (f). Data are displayed using a rainbow scale and normalized to total ion counts. Scale bar, 5 mm; spatial resolution = 100 μm.

[0292] Fluvoxamine is a selective serotonin reuptake inhibitor (SSRI) that was originally developed as an antidepressant but is commonly used to treat anxiety disorders. The primary amine functional group of fluvoxamine reacts selectively with TPP and Br-TPP. Brain tissue sections from control and fluvoxamine-treated mice were derivatized with non-brominated TPP (a, b) and brominated Br-TPP (d, e). No interfering signals were detected in control brain tissue sections using TPP (a) and Br-TPP (d), while derivatized fluvoxamine was detected at m / z 609.3 using TPP (b) and at m / z 687.2 using Br-TPP (e) in the absence of any co-matrix. The distribution of fluvoxamine was found to be similar using both derivatization agents, mainly localized in the cortex and cerebellum. The isotope pattern of TPP-derivatized fluvoxamine represents a typical non-labeled compound (c), while Br-TPP-derivatized fluvoxamine containing bromine shows a specific isotope pattern that can be easily distinguished from the signals corresponding to the non-derivatized substances.

Claims

1. Use of a compound of formula I or a salt thereof as a reactive matrix for desorption and / or laser ablation ionization spectrometry: in: Selected from the group consisting of: –Ar 1 Selected from the group consisting of: biphenyl, terphenyl, C selected from naphthalene, fluorene, anthracene, phenanthrene, pyrene 10 -C 30 PAHs, and C6-C 30 A heteroaryl analog of a polycyclic aromatic hydrocarbon, and substituted by a substituent selected from the group consisting of: -H, -D, -T, -F, -Cl, -Br, -I, -NO2, -CN, -R"', -OR"', -OC(O)R″′, -SR″′, -S(O)R″′, -S(O)(O)R″′ and -NR″′R″″, wherein -R″′ and -R″″ are each independently selected from phenyl, tolyl and C1-C 15 alkyl; –L– is selected from a bond, –(CH2) m – or –O–, –S–, –NH–, In the group composed of; –G 1 Selected from –H, –Me or –Ar 2 ; -R is optionally replaced by one or more D, T or 13 C atom labeled C1-C 15 alkyl; –G 2 Selected from the group consisting of: –F, –Cl, –Br, –I and –(CH2) n –Z–NH–NH2; m and n are each independently 0 to 15; -Z- is selected from a bond, -CH2- or -C(O)-; and –Ar 2 Selected from the group consisting of phenyl, biphenyl, terphenyl, C 10 -C 30 PAHs and C4-C 30 The invention relates to a monocyclic or polycyclic heteroaryl group, and is substituted by a substituent selected from the group consisting of -H, -D, -T, -F, -Cl, -Br, -I, -NO2, -CN, -R"', -OR"', -SR"', -S(O)(O)R"' and -NR"'R"", wherein -R"' and -R"" are each independently selected from the group consisting of phenyl, tolyl and C1-C 15 alkyl.

2. The use according to claim 1, wherein the compound of formula I has formula Ia or Ib, And among them Selected from the group consisting of:

3. The use according to claim 1, wherein -L- is a bond.

4. The use according to claim 1, wherein -G 1 is –H.

5. The use according to any one of claims 1 to 4, wherein the compound of formula I, Ia or Ib is in - R is optionally labeled with one or more deuterium, tritium or carbon-13 atoms and is selected from the group consisting of: methyl, ethyl, n-propyl and n-butyl; and –G 2 Selected from the group consisting of: –F, –Cl, –Br and –I.

6. The use according to any one of claims 1 to 4, wherein for Z is C═O, and wherein n is 1-3.

7. A compound of formula II or a salt thereof: in: Selected from the group consisting of: –Ar 1 Selected from the group consisting of: biphenyl, terphenyl, C selected from naphthalene, fluorene, anthracene, phenanthrene, pyrene 10 -C 30 PAHs, and C6-C 30 A heteroaryl analog of a polycyclic aromatic hydrocarbon, and substituted by a substituent selected from the group consisting of: -H, -D, -T, -F, -Cl, -Br, -I, -NO2, -CN, -R"', -OR"', –OC(O)R″′, –SR″′, –S(O)R″′, –S(O)(O)R″′, and –NR″′R″″, where –R″′ and - R"" are each independently selected from phenyl, tolyl and C1-C 15 alkyl; –L– is selected from a bond, –(CH2) m – or –O–, –S–, –NH–, In the group composed of; –G 1 Selected from –H, –Me or –Ar 2 ; -R is optionally replaced by one or more D, T or 13 C atom labeled C1-C 15 alkyl; –G 2 Selected from the group consisting of: –F, –Cl, –Br, –I and –(CH2) n –Z–NH–NH2; m and n are each independently 0 to 15; -Z- is selected from a bond, -CH2- or -C(O)-; and –Ar 2 Selected from the group consisting of phenyl, biphenyl, terphenyl, C 10 -C 30 PAHs and C4-C 30 The invention relates to a monocyclic or polycyclic heteroaryl group, and is substituted by a substituent selected from the group consisting of -H, -D, -T, -F, -Cl, -Br, -I, -NO2, -CN, -R"', -OR"', -SR"', -S(O)(O)R"' and -NR"'R"", wherein -R"' and -R"" are each independently selected from the group consisting of phenyl, tolyl and C1-C 15 alkyl.

8. The compound according to claim 7, wherein the compound of formula II has formula IIa or IIb, And among them Selected from the group consisting of:

9. The compound according to claim 7, wherein -G 1 is –H.

10. A compound according to any one of claims 7 to 9, wherein the compound of formula II, IIa or IIb is in - R is labeled with a deuterium, tritium or carbon-13 atom and is selected from the group consisting of: methyl, ethyl, n-propyl and n-butyl; and –G 2 Selected from the group consisting of: –F, –Cl, –Br and –I.

11. A compound according to any one of claims 7 to 9, wherein for Z is C═O, and wherein n is 1-3.

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