Method for identifying unsaturated organic substances and mass spectrometry system

Abstract

To provide a method for identifying an unsaturated organic compound and a mass spectrometry system, which are particularly suitable for implementing position identification of a carbon-carbon double bond of an unsaturated organic compound.SOLUTION: An identification method includes: a derivatization reaction step of aziridinating a carbon-carbon double bond in the unsaturated organic compound by an aza-Prilezhaev reaction so as to obtain a derivatization product; and then specifically dissociating the derivatization product, and performing mass analysis. Compared with conventional methods, the identification method has at least one of advantages such as a mild reaction condition, an appropriate derivatization degree, a reduced side reaction, and a high conversion rate.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present invention relates to the field of mass spectrometry, and in particular to a method and a mass spectrometry system for identifying unsaturated organic compounds. [Background technology]

[0002] Lipids are important nutrients and components of living cells, and are closely related to some important immune functions and metabolic disorders. Currently, the Lipid Metabolites and Pathways Strategy (LIPID MAPS) has been launched to promote lipid omics research by establishing a classification database.

[0003] Complete lipid labeling and identification information includes classification, elemental composition, size and position of R-groups (sn-positions), number and position of double bonds, and cis-trans isomeric orientation of double bonds. A variety of new mass spectrometry methods have been developed to identify lipid isomers, particularly the double bond positions of unsaturated lipids. Currently, the mainstream methods can be divided into three types. The first type uses separation techniques such as liquid chromatography (Non-Patent Document 1) and ion mobility spectrometry (Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5) to identify lipids by comparing peak positions with those of standards. However, these methods require large amounts of standards, and their applicability to lipid analysis is limited by the large number of unknown samples. The second type is based on gas-phase ion excitation methods that can realize selective fragmentation of carbon-carbon double bonds in unknown lipids, including high-energy collision-induced dissociation (Non-Patent Document 6), long-distance charge fragmentation (Non-Patent Document 7), ultraviolet photodissociation (Non-Patent Document 8; Non-Patent Document 9), ozone-induced dissociation (Non-Patent Document 10; Non-Patent Document 11; Non-Patent Document 12; Non-Patent Document 13), electron impact excitation of organic ions (Non-Patent Document 14; Non-Patent Document 15), oxygen adsorption dissociation (Non-Patent Document 16), and radical-induced dissociation (Non-Patent Document 17).

[0004] However, most gas-phase ion excitation dissociation methods result in excessive fragmentation of unsaturated lipids, producing highly complex spectrograms with low abundances of diagnostic ions at double bond positions and low sensitivity. Analysis of complex samples usually requires relatively complex pretreatment and separation methods. Due to the need for sophisticated mass spectrometers and limited sensitivity, these methods have not yet been applied to large-scale lipid analysis.

[0005] Currently, the most commonly used third-class chemical derivatization-mass spectrometry methods for identifying low-abundance lipid isomers in complex biological samples include the Paterno-Buchi (PB) reaction (Non-Patent Document 18; Non-Patent Document 19), epoxidation reaction (Non-Patent Document 20; Non-Patent Document 21), singlet oxidation reaction (Non-Patent Document 22), aziridination reaction (Non-Patent Document 23; Non-Patent Document 24; Non-Patent Document 25), and several other reactions (Non-Patent Document 26).

[0006] In Non-Patent Document 27, Xia et al. developed a deep phospholipid analysis system with automated data analysis capabilities that organically integrated hydrophilic interaction liquid chromatography (HILIC), trapped ion mobility spectrometry (TIMS), and isomer analysis tandem mass spectrometry (MS / MS), enabling high-speed, high-sensitivity, and high-comprehensive quantitative analysis of phospholipids in multiple biological samples. In this paper, offline Paterno-Buchi (PB) derivatization was used to label the carbon-carbon double bonds of phospholipids.

[0007] Li's team (Non-Patent Document 20) and Hsu's team (Non-Patent Document 21) have previously proposed methods to identify the location of unsaturated lipid-carbon double bonds using an epoxidation reaction. Based on the Prilezhaev mechanism, the synergistic reaction of mCPBA with the double bond converts the double bond to oxychloropropane, and the three-membered ring structure is easily cleaved in the subsequent CID, generating diagnostic ions that can indicate the location of the double bond.

[0008] However, epoxidation does not introduce easily ionizable functional groups, and the reaction yield is poor, resulting in low detection sensitivity and significant obstacles to the analysis of low-content lipids. For lipids containing multiple double bonds, the reaction generates multiple excessively oxidized by-products, significantly reducing sensitivity and increasing the difficulty of analysis.

[0009] Nitrogen-containing heterocyclic propanation is a preferred method for introducing easily ionizable sites. Yan et al. disclosed a new mass labeling method using a combination of HOSA reagent, pyridine, and ethyl trifluoropyruvate to label carbon-carbon double bonds through aziridination. This reaction utilizes an electron-deficient ketone as a catalyst, first reacting with the nitrogen source HOSA to generate the key intermediate oxaziridine. This intermediate then reacts with a double-bonded compound to generate the aziridine.

[0010] Guo et al. disclose a method for applying a chloroamine derivatization reagent to identify the position of a double bond in Patent Document 2. In this patent, N-chloro-4-methyl-benzenesulfonamide sodium salt is mainly used as a reaction reagent for labeling carbon-carbon double bonds.

[0011] However, all of the prior art methods for labeling carbon-carbon double bonds in lipids have some deficiencies. [Prior art documents] [Patent documents]

[0012] [License 1] License WO2022 / 216767A1 [License 2] Patent CN114166921A [License 3] Patent CN113495112A [Non-licensed literature]

[0013] [Non-licensed Document 1] Holcapek, M. et al. J. Chromatogr. A 2011, 1218, 5146 [Non-licensed Document 2] Mclean, JA et al. Anal. Chem. 2014, 86, 2107 [Non-licensed Document 3] Groessl, M. et al. Analyst 2015, 140, 6904 [Non-licensed Document 4] Fernandez-Lima, F. et al. Anal. Chem. 2019, 91, 5021

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Non-licensed literature 10

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[0014] Through continuous research into the prior art, the present inventors have found that all of the prior art methods for labeling carbon-carbon double bonds have at least one problem selected from the group consisting of harsh reaction conditions, serious over-derivatization, a large number of side reactions, and low conversion rates. [Means for solving the problem]

[0015] A first aspect of the present application provides a method for identifying unsaturated organic matter, comprising the steps of: A derivatization reaction step in which the carbon-carbon double bond in the unsaturated organic compound is aziridinated by the aza-Prilezhaev reaction to obtain a derivatized product. The derivatization reagent used in the aza-Prilezhaev reaction includes a compound represented by the following general formula (1): [ka] In general formula (1), R1 is selected from substituted or unsubstituted aromatic groups having 6 to 18 ring carbon atoms. R2 is selected from H, a substituted or unsubstituted linear alkyl, alkoxy or thioalkyl group having 1 to 20 C atoms, a substituted or unsubstituted branched or cyclic alkyl, alkoxy or thioalkyl group having 3 to 20 C atoms, a substituted or unsubstituted alkenyl or alkynyl group having 2 to 20 C atoms, or a substituted or unsubstituted aromatic or heteroaromatic ring system having 5 to 30 aromatic ring atoms. A dissociation step in which the ionized derivatization product is dissociated to yield multiple subions by cleavage at sites corresponding to the original carbon-carbon double bond. A mass spectrometry step for determining the mass numbers of the multiple subions to identify the position of the carbon-carbon double bond in the unsaturated organic compound.

[0016] The mass number of R2 may be greater than 80 Da.

[0017] The compound represented by general formula (1) may be as shown below. [ka]

[0018] The solvent for the derivatization reagent may be an acidic solvent.

[0019] The acidic solvent may be one or a combination of trifluoroethanol, hexafluoroisopropanol, and perfluoro-t-butanol.

[0020] The reaction temperature for the aza-Prilezhaev reaction may be 20-100°C.

[0021] The unsaturated organic material may be an unsaturated lipid.

[0022] The unsaturated lipid is a fatty acyl, glyceride, glycerophospholipid, sphingolipid, sterol ester, pregnenolone lipid, glycolipid or polyketide.

[0023] A second aspect of the present application is a mass spectrometry system including a derivatization reactor, an ion source, a dissociation device, and a mass spectrometer, wherein the derivatization reactor mixes and reacts a sample with an aza-Prilezhaev derivatization reagent to aziridinate a carbon-carbon double bond of an unsaturated organic compound in the sample using the aza-Prilezhaev reaction to obtain a derivatized product. The derivatization reagent includes a compound represented by the following general formula (1): [ka] In general formula (1), R1 is selected from substituted or unsubstituted aromatic groups having 6 to 18 ring carbon atoms. R2 is selected from H, a substituted or unsubstituted linear alkyl, alkoxy or thioalkyl group having 1 to 20 C atoms, a substituted or unsubstituted branched or cyclic alkyl, alkoxy or thioalkyl group having 3 to 20 C atoms, a substituted or unsubstituted alkenyl or alkynyl group having 2 to 20 C atoms, or a substituted or unsubstituted aromatic or heteroaromatic ring system having 5 to 30 aromatic ring atoms. The ion source receives and ionizes the derivatization product. The dissociator dissociates the derivatization product ionized in the ion source, cleaving the product at sites corresponding to the carbon-carbon double bond to obtain multiple subions. The mass spectrometer measures the mass numbers of the multiple subions to identify the positions of the carbon-carbon double bonds in the unsaturated organic compound.

[0024] The mass number of R2 may be greater than 80 Da.

[0025] The compound represented by general formula (1) may be as shown below. [ka]

[0026] The ion source may be an electrospray ionization source, a nanospray ionization source, a desorption electrospray ionization source, an atmospheric pressure chemical ionization source, an atmospheric pressure photoionization source, or a matrix-assisted laser desorption ionization source.

[0027] The dissociation device may be one or more of a high energy collision dissociation device, a collision induced dissociation device, an oxygen attachment dissociation device, a hydrogen attachment dissociation device, an electron capture dissociation device, a radical directed dissociation device, an ultraviolet light induced dissociation device, and a charge remote fragmentation device.

[0028] The dissociation device may be a collision-induced dissociation device, and the dissociation energy of the collision-induced dissociation device may be 30-40 eV.

[0029] The derivatization reactor includes a reaction vessel and an acceleration control section, in which the sample and the derivatization reagent are mixed in the reaction vessel, and the acceleration control section may be an offline reaction device that accelerates collisions of molecules in the reaction vessel.

[0030] The derivatization reactor may be an online reactor including a communication device and an acceleration control unit. The first inlet is provided in the communication device and communicates with a sample introduction line. The second inlet is provided in the communication device and communicates with a derivatization reagent introduction line. The product outlet is provided in the communication device and transports the derivatization product to the ion source. The acceleration control unit accelerates collisions of molecules within the communication device.

[0031] The acceleration control unit may be one or more of a temperature control unit, an ultrasonic device, a microwave device, an infrared device, and an oscillator device.

[0032] The reaction temperature of the aza-Prilezhaev reaction may be controlled to 20-100°C by a temperature control unit.

[0033] The mass spectrometry system may further include a liquid chromatograph device installed in the sample introduction line.

[0034] The mass spectrometry system may further include a mass filter located between the ion source and the dissociator.

[0035] The mass spectrometry system may further include an ion mobility spectrometer located between the ion source and the dissociator. [Effects of the Invention]

[0036] Compared with conventional identification methods, the method for identifying unsaturated organic substances according to the present invention has at least one of the following advantages. A wide variety of identifiable information, such as the position of the carbon-carbon double bond in the aliphatic chain, the position of sn in the aliphatic chain, and the cis-trans isomeric orientation of the carbon-carbon double bond, can be identified. b. Good substrate versatility: Can be applied to a wide range of substrates, including FA, GP, ST, SP, and GL. c. Mild reaction conditions. No metal catalyst or additional additives are required, and no ultraviolet light, electrolysis, high temperature, inert atmosphere, or dry environment is required. No strong redox agents are used. The reaction can be completed quickly at room temperature or slightly elevated temperatures, and the reagents and products are stable. d. High conversion rate: The conversion rate can reach 90% or more. e. Reduction of side reactions and improvement of ionization efficiency. f. Appropriate degree of derivatization. In each unsaturated organic molecule, typically only a single carbon-carbon double bond is aziridinated, or by optimizing the reaction conditions (e.g., temperature and time), it is possible to adjust the degree of derivatization so that only a single carbon-carbon double bond in most molecules is aziridinated. In addition, the spectrogram is simple and easy to analyze. g.Can be done online. [Brief explanation of the drawings]

[0037] [Figure 1] FIG. 1 is a schematic diagram showing the flow of a method for identifying the carbon-carbon double bond position and cis-trans isomer orientation in unsaturated lipids according to a first embodiment of the present invention. [Figure 2]1 shows the yield results of the aza-Prilezhaev reaction at different temperatures for 10 minutes in the first embodiment of the present invention. [Figure 3] 1 shows the relationship between the time course of the conversion rate reflected by the peak intensity in the mass spectrum of the target substance at a reaction temperature of 50° C. in the first embodiment of the present invention. [Figure 4] FIG. 1 is a system diagram of a mass spectrometry system used for offline analysis of derivatization products according to a first embodiment of the present invention. [Figure 5] FIG. 2 is a graph showing the change in the detection peak intensity of the aziridinated lipid according to the first embodiment of the present invention after different standing times. [Figure 6] FIG. 2 is a graph showing the fold change in peak intensity of the mass spectrum before and after the derivatization reaction according to the first embodiment of the present invention. [Figure 7] FIG. 1 is a diagram illustrating the principle of a reaction flow of an identification method according to a first embodiment of the present invention. [Figure 8] 1 is a mass spectrum obtained by aziridinating a C18:1(9Z) standard sample according to the first embodiment of the present invention. [Figure 9] 1 is a mass spectrum obtained by aziridinating a C18:1(6Z) standard sample according to the first embodiment of the present invention. [Figure 10] 1 shows a mass spectrum obtained by aziridinating a standard sample of PC16:0 / 18:1(9Z) according to the first embodiment of the present invention. [Figure 11] 1 shows a mass spectrum obtained by aziridinating a PC18:1(9Z) / 16:0 standard sample according to the first embodiment of the present invention. [Figure 12] FIG. 1 is a schematic diagram showing the molecular disruption process after aziridination of PC16:0 / 18:1(9Z) according to the first embodiment of the present invention. [Figure 13] FIG. 2 is a schematic diagram showing the molecular disruption process after aziridination of PC18:1(9Z) / 16:0 according to the first embodiment of the present invention. [Figure 14]1 shows spectrograms of ion mobility spectra of C18:1(9E)[M+2Na-H]+, C18:1(9Z)[M+2Na-H]+, and a mixture of both, in the aziridination according to the first embodiment of the present invention. [Figure 15] 1 is a mass spectrum of C20:4 (5Z, 8Z, 11Z, 14Z) obtained by aziridination according to the first embodiment of the present invention. [Figure 16] 1 shows a tandem mass spectrometry spectrum of aziridination C20:4 (5Z, 8Z, 11Z, 14Z) according to the first embodiment of the present invention. [Figure 17] FIG. 10 is a system diagram of a mass spectrometry system used for online analysis of derivatization products according to a second embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0038] The technical solutions in the embodiments of the present invention will be described below clearly and completely in accordance with the drawings in the embodiments of the present invention, but it is clear that the described embodiments are only a part of the embodiments of the present invention, and are not all of the embodiments. Based on the embodiments of the present invention, all other embodiments that can be obtained by those skilled in the art without creative efforts fall within the protection scope of the present invention.

[0039] <Lipids> The unsaturated organic matter according to an embodiment of the present invention includes at least unsaturated lipids, such as unsaturated fatty acyl, glyceride, glycerophospholipid, sphingolipid, sterol ester, pregnenolone lipid, glycolipid or polyketide, and is particularly applicable to fatty acyl / fatty acid, glycerophospholipid, sterol ester, sphingolipid and glyceride.

[0040] Lipid naming rules: The following lipids use the Δ-nomenclature, e.g., FA18:1(9Z), where FA is the fatty acid, 18 is the number of carbon atoms, 1 is the number of carbon-carbon double bonds, 9 is the position of the carbon-carbon double bond, and Z indicates the cis geometry.

[0041] In TG 18:1(9Z) / 18:1(9Z) / 18:1(9Z), the three fragments connected by " / " represent lipid structures corresponding to the three fatty chains in the triglyceride, respectively.

[0042] In addition, the following English abbreviations respectively represent the following lipid types: CE: cholesterol esters PC: phosphatidylcholine FA: fatty acyl / fatty acid GP: Glycerol phosphate ST: sterol ester SP: sphingolipid GL: Glycerides TG: Triglyceride

[0043] [First embodiment] <Overall flow> FIG. 1 is a schematic diagram of the flow of a method for identifying the positions of carbon-carbon double bonds (including positions in the fatty chain and sn positions) and cis-trans isomeric orientation in unsaturated lipids according to this embodiment.

[0044] Referring to FIG. 1, the identification method includes the following steps. Aza-Prilezhaev reaction allows aziridination and derivatization of carbon-carbon double bonds in unsaturated organic compounds Generate Step S1: Derivatization reaction to obtain the product. an ionization step S2 in which the derivatization product is ionized; Based on ion mobility, derivatization where the carbon-carbon double bond has already been aziridinated Generate A mobility selection step S3 for selecting an object. A dissociation step S4 in which the ionized derivatization product is dissociated to break the original carbon-carbon double bond at the corresponding site to yield multiple subions. A mass analysis step S5 is performed to measure the mass numbers of the multiple subions and identify the positions of the carbon-carbon double bonds in the unsaturated organic compounds.

[0045] In the above steps, before the derivatization reaction step S1, technologies such as liquid chromatography or gas chromatography may be used to separate unsaturated organic substances, and further separate components having carbon-carbon double bonds, thereby reducing the complexity of the spectrogram and improving the analysis rate.

[0046] In the mass spectrometry step S5, the measured sub-ions include at least diagnostic ions, that is, sub-ions cleaved at the site of the carbon-carbon double bond or sub-ions cleaved at the sn position.

[0047] In this embodiment, the ionization step S2 is performed after the derivatization reaction step S1, the mobility selection step S3 is performed after the ionization step S2, the dissociation step S4 is performed after the mobility selection step S3, and the mass spectrometry step S5 is performed after the dissociation step S4. In other embodiments of the present invention, the order between some steps may be interchanged, and this is not limited.

[0048] <aza-Prilezhaev reaction> In the derivatization reaction step S1, the carbon-carbon double bond is labeled by the aza-Prilezhaev reaction. The reaction mechanism of the aza-Prilezhaev reaction is shown below.

Chemical formula

[0049] Different from the two-step catalytic reaction of HOSA and pyridyl with the double bond, the aza-Prilezhaev reaction does not require a catalyst. The aromatic hydroxyamine and the double bond obtain aziridine by removing one molecule of arylsulfonic acid through only a one-step synergistic reaction.

[0050] The derivatization reaction step S1 can be completed offline, i.e., the experimenter completes the steps of mixing raw materials, controlling the temperature, etc., by themselves. Alternatively, it can be completed online, i.e., the analytical device completes the step according to a preset program. In this embodiment, the derivatization reaction step S1 is described as being completed offline. In a second embodiment, a mass spectrometry system capable of completing the derivatization reaction step S1 online is also provided.

[0051] a) aza-Prilezhaev reagent a.1 Mass label The derivatization reagent comprises a mass label dissolved in an acidic solvent, the mass label being a compound represented by the following general formula (1): [ka]

[0052] In general formula (1), R1 is selected from substituted or unsubstituted aromatic groups having 6 to 18 ring carbon atoms. R2 is selected from H, a substituted or unsubstituted linear alkyl, alkoxy or thioalkyl group having 1 to 20 C atoms, a substituted or unsubstituted branched or cyclic alkyl, alkoxy or thioalkyl group having 3 to 20 C atoms, a substituted or unsubstituted alkenyl or alkynyl group having 2 to 20 C atoms, or a substituted or unsubstituted aromatic or heteroaromatic ring system having 5 to 30 aromatic ring atoms.

[0053] The use of an aromatic group as R1 can improve the stability of the reagent. R2 is typically a group having a mass number greater than 15 Da. Preferably, R2 is a group having a mass number greater than 50 Da. More preferably, R2 is a group having a mass number greater than 80 Da, or R2 is a group having a mass number greater than 100 Da. By rationally increasing the mass number of R2, overlapping of mass peaks in the spectrogram can be effectively prevented. In some embodiments, R2 may include a relatively sterically hindered group such as a methyl group or a benzene ring, for example, a tert-butoxycarbonyl group or a benzoyl group.

[0054] In this embodiment, the mass label in the aza-Prilezhaev reagent is tert-butyl N-tosyloxycarbamate (N-Boc-O-tosylhydroxylamine, CAS: 105838-14-0).

[0055] For other alternative and readily available aza-Prilezhaev reagent mass labels and their CAS numbers, see the table below. [Table 1]

[0056] a.2 Solvent The acidic solvent used in the derivatization reagent may be an organic acid such as trifluoroethanol, hexafluoroisopropanol, or perfluoro-t-butanol, or an inorganic acid, preferably hexafluoroisopropanol.

[0057] b) Reaction temperature Taking a 10-minute reaction as an example, using tert-butyl N-tosyloxycarbamate as the mass marker and hexafluoroisopropanol as the solvent, Figure 2 shows the reaction yields for 10 minutes at different temperatures. Experiments have shown that the yield of aza-Prilezhaev increases significantly with increasing temperature at around 20°C, but tends to stabilize at around 50-55°C. The reaction temperature is preferably set between 20°C and 55°C, and even more preferably between 30°C and 55°C, e.g., 50°C. This temperature range allows for higher yields while reducing the occurrence of side reactions.

[0058] c) Reaction time Figure 3 shows the time course of conversion as reflected by the peak intensity in the mass spectrum of the target substance at a reaction temperature of 50°C. Research has shown that the reaction rate of aza-Prilezhaev is quite high at a reaction temperature of 50°C. It only takes 30 seconds for the reaction to reach a detectable peak intensity. After 10 minutes of reaction, the conversion rate approaches 100%.

[0059] Further details and mechanistic discussion of the aza-Prilezhaev reaction may be found in Non-Patent Document 28, which will not be repeated here.

[0060] <ims-ms ms> a) System configuration Referring to FIG. 4, a system used for off-line analysis of derivatized products comprises an ion source 1, an ion mobility spectrometer 2 and a tandem mass analyzer connected in series in sequence.

[0061] b) Ion Source 1 The ionization step S2 is performed by the ion source 1. Ion sources include electrospray ionization sources (ESI), atmospheric pressure photoionization sources (APPI); atmospheric pressure chemical ionization sources (APCI), matrix-assisted laser desorption ionization sources (MALDI), laser desorption ionization sources (LDI), atmospheric pressure ionization sources (API), desorption ionization sources on silicon (DIOS), electron impact ionization sources (EI), chemical ionization sources (CI), field ionization sources (FI), field desorption ionization sources (FD), inductively coupled plasma ion sources (ICP), fast atom bombardment ion sources (FAB), liquid secondary ion mass spectrometry ion sources (LSIMS), and direct electrospray ionization (D The ion source may be one selected from the group consisting of an ESI (electrospray ionization source), a nickel-63 radioactive ion source, an atmospheric pressure matrix-assisted laser desorption ionization source, a thermal spray ionization source, an atmospheric sampling glow discharge ionization source (ASGDI), a glow discharge ionization source (GD), an impactor ionization source, a real-time direct analysis ionization source (DART), a laser spray ionization source (LSI), an acoustic wave spray ionization source (SSI), a matrix-assisted entrance ionization source (MAII), a solvent-assisted entrance ionization source (SAII), a Penning ionization source, a laser ablation electrospray ionization source (LAESI), and a He plasma ionization source (HeP1). Preferably, the ion source 1 is an electrospray ionization source, a nanospray ionization source, a desorption electrospray ionization source, an atmospheric pressure chemical ionization source, an atmospheric pressure photoionization source, or a matrix-assisted laser desorption ionization source. In this embodiment, the ion source is preferably an electrospray ionization source.

[0062] c) Ion mobility spectrometer 2 The mobility selection step S3 is performed by an ion mobility spectrometer 2. One or more ion guide devices 6 may be provided between the ion mobility spectrometer 2 and the ion source.

[0063] The ion mobility spectrometer 2 includes one ion mobility analyzer selected from the group consisting of a drift tube ion mobility spectrometer (DTIMS), a differential mobility analysis (DMA) device, a field asymmetric-waveform ion-mobility spectrometry (FAIMS) device, a traveling wave ion mobility spectrometer (TW-IMS), a differential mobility spectrometry (DMS) device, a transverse modulation ion mobility spectrometer, a trapped ion mobility spectrometer (TIMS), and a U-shaped ion mobility analyzer (UMA).

[0064] In this embodiment, the ion mobility spectrometer 2 is preferably a U-type ion mobility spectrometer. The structure of the U-type ion mobility spectrometer and the filtering mode applicable to the present identification method can be described in Patent Document 3, and the description thereof will be omitted here. Similarly, the identification method according to the embodiment of the present invention is not limited to the type of ion mobility spectrometer used.

[0065] The ion mobility spectrometer 2 can provide a second dimension of data for tandem mass spectrometry. Different ion mobilities can distinguish isomers. In particular, differences in molecular structure are enhanced after aziridination of carbon-carbon double bonds by the aza-Prilezhaev reaction. In some embodiments, ion mobility spectrograms can be used to identify differences in the position or cis-trans isomeric orientation of carbon-carbon double bonds.

[0066] d) Tandem mass spectrometer The tandem mass spectrometer comprises a mass filter 3, a dissociator 4 and a mass analyzer 5 connected in series. One or more ion guide devices 6 may be provided between the ion mobility spectrometer 2 and the mass filter 3.

[0067] The tandem mass spectrometer may include one or more mass filters 3 selected from the group consisting of a quadrupole mass filter, a 2D or linear quadrupole ion trap, a Paul or 3D quadrupole ion trap, a Penning ion trap, an ion trap, a magnetic sector mass filter, a time-of-flight mass filter, and a Wien filter.

[0068] The dissociation step S4 is performed by a dissociation device 4. The tandem mass spectrometer may include one or more dissociation devices 4 selected from the group consisting of a collision-induced dissociation (CID) device, a surface-induced dissociation (SID) device, an electron transfer dissociation (ETD) device, an electron capture dissociation (ECD) device, an electron collision or collision dissociation device, a photoinduced dissociation (PID) device, a laser-induced dissociation device, an infrared radiation-induced dissociation device, an ultraviolet radiation-induced dissociation device, a nozzle-separator interface dissociation device, an in-source dissociation device, an in-source collision-induced dissociation device, a thermal or temperature source dissociation device, an electric field-induced dissociation device, a magnetic field-induced dissociation device, an ion-ion reactive dissociation device, an ion-molecular reactive dissociation device, an ion-atom reactive dissociation device, an ion metastable ion reactive dissociation device, an ion metastable molecular reactive dissociation device, and an electron ionization dissociation (EID) device. Preferably, the dissociation energy of the collision-induced dissociation device 4 is 30-40 eV. By rationally setting the dissociation energy, the dissociation process can be made site-specific, i.e., the site where the aziridine ring is present can be cleaved with high selectivity, thereby avoiding the occurrence of side reactions.

[0069] In this embodiment, in the dissociation step S4, not only can the carbon-carbon double bond site of the derivatization product be cleaved, but also the sn-position of the carbon-carbon double bond can be detected by cleaving the sn-position of some of the derivatization products. The dissociation step S4 may be completed by one dissociation or multiple dissociations, and the present application is not limited thereto.

[0070] The mass analysis step S5 is performed by a mass spectrometer 5, or is performed jointly by a mass filter 3 and a mass spectrometer 5. The tandem mass analyzer may comprise one or more mass spectrometers 5 selected from the group consisting of a 2D or linear quadrupole mass spectrometer, a Paul or 3D quadrupole mass spectrometer, a Penning trap mass spectrometer, an ion trap mass spectrometer, a magnetic sector mass spectrometer, an ion cyclotron resonance mass spectrometer (ICR) mass spectrometer, a Fourier transform ion cyclotron resonance (FTIR) mass spectrometer, an electrostatic mass spectrometer arranged to generate an electrostatic field having a quadrupole logarithmic potential distribution, a Fourier transform electrostatic mass spectrometer, a Fourier transform mass spectrometer, a time-of-flight mass spectrometer, an orthogonal acceleration time-of-flight mass spectrometer and a linear acceleration time-of-flight mass spectrometer.

[0071] In this embodiment, the tandem mass spectrometer is a Q-TOF tandem mass spectrometer, i.e. the mass filter 3 is a quadrupole mass filter and the mass spectrometer 5 is a time-of-flight mass spectrometer. The dissociator 4 is a collision induced dissociator.

[0072] <Experimental Results> a) Stability Furthermore, the aziridinated lipid obtained by the reaction can be stable. Figure 5 is a graph showing the change in detection peak intensity of the aziridinated lipid according to the first embodiment of the present invention with different incubation times. Referring to Figure 5, the aziridinated lipid has almost no effect on the detection peak intensity even after being incubated for 48 hours or more, which can meet the requirements of various types of tests.

[0073] b) Sensitivity Figure 6 shows the fold change in peak intensity of the mass spectrum before and after the derivatization reaction according to the first embodiment of the present invention. Referring to Figure 6, the introduction of an easily ionizable group allows the aza-Prilezhaev reaction to aziridinate the carbon-carbon double bond, thereby improving the sensitivity of mass spectrum detection. Specifically, the signal intensity of the parent ion mass spectrum can be improved by 1-3 orders of magnitude.

[0074] c) Isomers that distinguish between different positions of the carbon-carbon double bond c.1. Identifying the location of carbon-carbon double bonds in aliphatic chains 7 is a principle diagram of the reaction flow of the identification method according to the first embodiment of the present invention. Referring to FIG. 7, when the aziridine ring is specifically cleaved, -NH2 + Two cases can occur: - can be assigned to the aliphatic chain or to the head group, and accordingly, two different mass-to-charge ratio groups are formed, which allows the position of the carbon-carbon double bond to be determined based on these two mass peaks.

[0075] C18:1(9Z) and C18:1(6Z) are isomers, and the difference between them is the position of the carbon-carbon double bond in the fatty chain. Specifically, the carbon-carbon double bond in C18:1(9Z) is located between the 9th and 10th carbon atoms in the main chain, while the carbon-carbon double bond in C18:1(6Z) is located between the 6th and 7th carbon atoms in the main chain. The following describes a method for determining the position of the carbon-carbon double bond based on mass spectrometry.

[0076] Figure 8 shows a mass spectrum obtained by aziridinating a C18:1(9Z) standard sample. Referring to the aziridinated molecular structure shown in Figure 8, when the aziridine ring is specifically cleaved by the dissociation device 4, the aliphatic chain side is cleaved to produce C8H 17 -CH=NH2 + A subion is formed, i.e., a mass peak at m / z=142 appears. Also, one side of the carboxylic acid-containing group is cleaved to form CH 13 O-CH=NH2 + A subion is formed, namely a mass peak at m / z=154.

[0077] Figure 9 shows a mass spectrum obtained by aziridinating a C18:1(6Z) standard sample. Referring to the aziridinated molecular structure shown in Figure 9, when the aziridine ring is specifically cleaved by the dissociation device 4, the fatty acid chain is cleaved to give C 11 H 23 -CH=NH2 + A subion is formed, i.e., a mass peak at m / z=184 appears. Also, one side of the carboxylic acid-containing group is cleaved to form C5H7O-CH=NH2 + is formed, i.e., a mass peak at m / z=112 appears.

[0078] As described above, since the mass numbers of the subions on the aliphatic chain side and the lipid side obtained by breaking the carbon-carbon double bond at different positions are different, the specific position of the carbon-carbon double bond in the aliphatic chain can be determined based on whether or not there are characteristic mass peaks corresponding to the above and other different positions, or based on the intensity of those mass peaks. Because the aziridine ring is easily broken specifically when dissociative energy is applied, the peak intensity of the characteristic mass peak is strong, resulting in high detection sensitivity and accuracy.

[0079] c.2. Identification of the sn position of a carbon-carbon double bond FIG. 10 shows the mass spectrum obtained by aziridinating a standard sample of PC16:0 / 18:1(9Z).

[0080] FIG. 11 shows the mass spectrum obtained by aziridinating a PC18:1(9Z) / 16:0 standard sample.

[0081] According to Non-Patent Document 29, the characteristic peaks at m / z = 380, 396, and 466 can be used to distinguish the sn positions of PC16:0 / 18:1(9Z) and PC18:1(9Z) / 16:0. These characteristic peaks correspond to the PB reaction. Correspondingly, the characteristic peaks at m / z = 274, 290, and 360 can be used to distinguish the sn positions of the aza-Prilezhaev aziridination reaction.

[0082] Specifically, PC16:0 / 18:1(9Z) with a carbon-carbon double bond at the sn-2 position can give a subion at m / z = 290 based on the process shown in Figure 12. On the other hand, PC18:1(9Z) / 16:0 with a carbon-carbon double bond at the sn-1 position can give a subion at m / z = 360 based on the process shown in Figure 13. Furthermore, the sn position of the carbon-carbon double bond can be identified based on these two characteristic peaks.

[0083] c.3. Identifying the cis-trans isomeric orientation of carbon-carbon double bonds The cis-trans isomeric orientation of the carbon-carbon double bond can be identified by the ion mobility spectrometer 2. In particular, because the aziridine ring has rigidity, the aziridination reaction can further increase the structural difference between different cis-trans constituent molecules, thereby amplifying the difference in ion mobility between different molecules and further separating the ion mobility peaks.

[0084] Figure 14 shows the spectrograms (top to bottom) of the UMA ion mobility spectra of aziridinated C18:1(9E), C18:1(9Z), and a mixture of the two. As can be seen from Figure 14, the peaks of molecules with different cis-trans configurations in the ion mobility spectrograms are clearly shifted, indicating that the identification method has good resolution for molecules with different cis-trans configurations.

[0085] d) Excessive derivatization Figures 15 and 16 show mass spectra of aziridinated C20:4 (5Z, 8Z, 11Z, 14Z). Referring to Figure 15, the aza-Prilezhaev reaction effectively prevents excessive derivatization of carbon-carbon double bonds. Therefore, in most cases, only a single carbon-carbon double bond in each unsaturated organic molecule is aziridinated, resulting in a simpler spectrogram that is easier to analyze. Referring to Figure 16, the eight characteristic mass peaks corresponding to the four carbon-carbon double bonds remain clearly visible in the mass spectrum.

[0086] As described above, the method for identifying unsaturated organic substances according to this embodiment has at least one of the following advantages over conventional identification methods. A wide variety of identifiable information can be identified. The position of the carbon-carbon double bond in the aliphatic chain, the sn position in the aliphatic chain, and the cis-trans isomeric orientation of the carbon-carbon double bond can be identified. b. Good substrate versatility: Can be applied to a wide range of substrates, including FA, GP, ST, SP, and GL. c. Mild reaction conditions. No metal catalyst or additional additives are required, and no ultraviolet light, electrolysis, high temperature, inert atmosphere, or dry environment is required. No strong redox agents are used. The reaction can be completed quickly at room temperature or slightly elevated temperatures, and the reagents and products are stable. d. High conversion rate: The conversion rate can reach 90% or more. e. Reduction of side reactions and improvement of ionization efficiency. f. Appropriate degree of derivatization. In each unsaturated organic molecule, typically only a single carbon-carbon double bond is aziridinated, or by optimizing the reaction conditions (e.g., temperature and time), it is possible to adjust the degree of derivatization so that only a single carbon-carbon double bond in most molecules is aziridinated. In addition, the spectrogram is simple and easy to analyze.

[0087] [Second embodiment] 17 is a system diagram of a mass spectrometry system used for online analysis of derivatization products according to a second embodiment of the present invention. The difference from the first embodiment is that this embodiment performs the Aza-Prilezhaev reaction using an online derivatization reactor 7.

[0088] Specifically, see Figure 17. The derivatization reactor 7 includes a communication device 71 and an acceleration control unit 72. The communication device 71 has a first inlet 711, a second inlet 712, and a product outlet 713. The first inlet 711 communicates with the sample inlet conduit 81 and is used to introduce the sample. The second inlet 712 communicates with the derivatization reagent inlet conduit 82 and is used to introduce the derivatization reagent. The product outlet 713 is connected to the ion source 1 and transports the derivatization product produced by the derivatization reaction to the ion source 1.

[0089] The acceleration control unit 72 may be any suitable type of device for increasing molecular collisions, such as heating, microwave, ultrasound, oscillation, laser, etc. For heating, any suitable type of temperature control unit may be used, such as a microwave heating device, a water bath, or an oil bath, to control the reaction temperature of the derivatization reaction, particularly within the temperature range of 20-100°C, thereby accelerating the reaction and reducing the occurrence of side reactions.

[0090] In this embodiment, a liquid chromatograph and an IMS-MS / MS mass spectrometry system may be used in combination to form an LC-IMS-MS / MS system. Specifically, the liquid chromatograph is connected to a first inlet 711, and the column 9 of the liquid chromatograph is installed in a sample introduction conduit 81. By using these in combination to form an LC-IMS-MS / MS system, a rapid and highly sensitive analysis of multiple lipids can be easily and quickly completed with just a single sample injection.

[0091] The above are merely preferred embodiments of the present invention, and the present invention is not limited to these. Modifications and equivalents thereof are also included in the technical scope of the present invention, provided that they do not deviate from the gist of the present invention. [Explanation of symbols]

[0092] 1-Ion Source 2-Ion Mobility Spectrometer 3-Mass Filter 4-Dissociation device 5-Mass spectrometer 6-Ion guide device 7-Derivatization reactor 71-Communication device 711-First entrance 712-Second Entrance 713-Product outlet 72-Acceleration control unit 81-Sample introduction pipe 82-Derivatization reagent introduction line 9-column

Claims

1. A method for identifying unsaturated organic matter, comprising: a derivatization step of aziridinating the carbon-carbon double bond in the unsaturated organic compound by the aza-Prilezhaev reaction to obtain a derivatized product; a dissociation step of dissociating the ionized derivatization product to cleave the carbon-carbon double bond at a site corresponding to the original carbon-carbon double bond to obtain a plurality of subions; a mass analysis step of measuring the mass numbers of the plurality of subions to identify the positions of the carbon-carbon double bonds in the unsaturated organic compounds; The derivatization reagent used in the aza-Prilezhaev reaction includes a compound represented by the following general formula (1): 【Transformation 7】 In the general formula (1), R １ is selected from substituted or unsubstituted aromatic groups having 6 to 18 ring carbon atoms; R ２ is selected from H, a substituted or unsubstituted linear alkyl group, alkoxy group, or thioalkyl group having 1 to 20 C atoms, a substituted or unsubstituted branched or cyclic alkyl group, alkoxy group, or thioalkyl group having 3 to 20 C atoms, a substituted or unsubstituted alkenyl group or alkynyl group having 2 to 20 C atoms, or a substituted or unsubstituted aromatic or heteroaromatic ring system having 5 to 30 aromatic ring atoms.

2. R ２ 2. The method for identifying unsaturated organic substances according to claim 1, wherein the mass number of the unsaturated organic substance is greater than 80 Da.

3. The compound represented by the general formula (1) is 【Transformation 8】 3. The method for identifying unsaturated organic substances according to claim 2, wherein:

4. 2. The method for identifying unsaturated organic compounds according to claim 1, wherein the solvent for the derivatization reagent is an acidic solvent.

5. 5. The method for identifying unsaturated organic substances according to claim 4, wherein the acidic solvent is one or a combination of a plurality of trifluoroethanol, hexafluoroisopropanol, and perfluoro-t-butanol.

6. 2. The method for identifying unsaturated organic compounds according to claim 1, wherein the reaction temperature of the Aza-Prilezhaev reaction is 20-100°C.

7. 2. The method for identifying an unsaturated organic substance according to claim 1, wherein the unsaturated organic substance is an unsaturated lipid.

8. 8. The method for identifying unsaturated organic substances according to claim 7, wherein the unsaturated lipid is a fatty acyl, a glyceride, a glycerophospholipid, a sphingolipid, a sterol ester, a pregnenolone lipid, a glycolipid, or a polyketide.

9. 1. A mass spectrometry system comprising: a derivatization reactor for mixing and reacting a sample with an aza-Prilezhaev derivatization reagent to aziridinate a carbon-carbon double bond of an unsaturated organic compound in the sample using the aza-Prilezhaev reaction to obtain a derivatized product; an ion source that receives and ionizes the derivatized product; a dissociation device for dissociating the derivatization product ionized in the ion source, so that the carbon-carbon double bond is broken at a site corresponding to the carbon-carbon double bond to obtain a plurality of subions; a mass spectrometer for measuring the mass numbers of the plurality of subions to identify the positions of the carbon-carbon double bonds in the unsaturated organic compounds. The derivatization reagent contains a compound represented by the following general formula (1): 【Chemistry 9】 In the general formula (1), R １ is selected from substituted or unsubstituted aromatic groups having 6 to 18 ring carbon atoms; R ２ is selected from H, a substituted or unsubstituted linear alkyl group, alkoxy group or thioalkyl group having 1 to 20 C atoms, a substituted or unsubstituted branched or cyclic alkyl group, alkoxy group or thioalkyl group having 3 to 20 C atoms, a substituted or unsubstituted alkenyl group or alkynyl group having 2 to 20 C atoms, or a substituted or unsubstituted aromatic or heteroaromatic ring system having 5 to 30 aromatic ring atoms.

10. R ２ 10. The mass spectrometry system of claim 9, wherein the mass number of is greater than 80 Da.

11. The compound represented by the general formula (1) is 【Chemistry 10】 11. The mass spectrometry system according to claim 10, wherein:

12. 10. The mass spectrometry system of claim 9, wherein the ion source is an electrospray ionization source, a nanospray ionization source, a desorption electrospray ionization source, an atmospheric pressure chemical ionization source, an atmospheric pressure photoionization source, or a matrix-assisted laser desorption ionization source.

13. 10. The mass spectrometry system of claim 9, wherein the dissociation device is one or more of a high-energy collision dissociation device, a collision-induced dissociation device, an oxygen attachment dissociation device, a hydrogen attachment dissociation device, an electron capture dissociation device, a radical-directed dissociation device, an ultraviolet light-induced dissociation device, and a charge remote fragmentation device.

14. 14. The mass spectrometry system according to claim 13, wherein the dissociation device is a collision-induced dissociation device, and the dissociation energy of the collision-induced dissociation device is 30-40 eV.

15. The derivatization reactor comprises: a reaction vessel in which the sample and the derivatization reagent are mixed; 10. The mass spectrometry system according to claim 9, which is an offline reaction device comprising an acceleration control section that accelerates collisions of molecules in the reaction vessel.

16. The derivatization reactor A communication device; a first inlet provided in the communication device and communicating with the sample introduction conduit; a second inlet provided in the communication device and communicating with an inlet conduit for introducing the derivatization reagent; an acceleration control unit that accelerates collisions of molecules in the communication device; 10. The mass spectrometry system of claim 9, wherein the system is an online reactor, the system comprising: a product outlet provided in the communication device for transporting the derivatized product to the ion source.

17. 17. The mass spectrometry system according to claim 16, wherein the acceleration control unit is one or more of a temperature control unit, an ultrasonic device, a microwave device, an infrared device, and an oscillator device.

18. 18. The mass spectrometry system according to claim 17, wherein the temperature control unit controls the reaction temperature of the aza-Prilezhaev reaction to 20-100°C.

19. 19. The mass spectrometry system according to claim 18, further comprising a liquid chromatograph device installed in the sample introduction line.

20. 10. The mass spectrometry system of claim 9, further comprising a mass filter located between the ion source and the dissociator.

21. 10. The mass spectrometry system of claim 9, further comprising an ion mobility spectrometer located between the ion source and the dissociator.

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