Method for identifying unsaturated organic compound, 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 organics. [Background technology]

[0002] Lipids are important nutrients and important components of living cells, and are closely related to some important immune functions and metabolic defects. 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-position), number and position of double bonds, and cis-trans isomeric orientation of double bonds. A variety of new mass spectrometry methods are being developed one after another to identify lipid isomers, especially the double bond positions of unsaturated lipids. Currently, the mainstream methods can be classified 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 the peak positions with those of standards. However, such methods require a large amount of standards, and the scope of application is limited for lipid analysis due to 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), remote charge fragmentation (Non-Patent Document 7), ultraviolet light dissociation (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 excessively fragment unsaturated lipids, producing high-complexity spectrograms with low abundances of relevant diagnostic ions at double bond positions and low sensitivity. Analysis of complex samples usually requires relatively complex pretreatment and separation schemes. Due to the use of precise mass spectrometers and limited sensitivity, such methods have not yet been applied to large-scale lipid analysis.

[0005] Currently, the most commonly used methods for identifying low-abundance lipid isomers in complex biological samples are the third type of chemical derivatization-mass spectrometry methods, including 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] Xia et al., in Non-Patent Document 27, developed a deep phospholipid analysis system with automatic data analysis capabilities by organically integrating hydrophilic interaction liquid chromatography (HILIC), trapped ion mobility spectrometry (TIMS), and isomer analysis tandem mass spectrometry (MS / MS), and realized high-speed, high-sensitivity, and high-comprehensive quantitative analysis of phospholipids in multiple biological samples. In this document, the offline Paterno-Buchi (PB) derivatization reaction is 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 a method to identify the location of unsaturated lipid-carbon double bonds using epoxidation reactions, respectively. Based on the Prilezhaev mechanism, the synergistic reaction of mCPBA with double bonds converts the double bonds to oxychloropropane, and the three-membered ring structure is susceptible to cleavage in the subsequent CID, generating diagnostic ions that can indicate the location of the double bonds.

[0008] However, epoxidation does not introduce any easily ionizable functional groups, and the reaction yield is poor, so the detection sensitivity is low, and the analysis of low-content lipids remains a major obstacle. For lipids containing multiple double bonds, the reaction produces multiple types of over-oxidized by-products, which not only significantly reduces the sensitivity but also greatly increases the difficulty of the analysis.

[0009] Nitrogen-containing heterocyclic propanation is a preferred method for introducing sites that are easily ionized. Yan et al. disclosed in Patent Document 1 a new mass labeling method that uses a combination of HOSA reagent, pyridine, and ethyl trifluoropyruvate to aziridine carbon-carbon double bonds and label the carbon-carbon double bonds. This reaction utilizes an electron-deficient ketone as a catalyst, which first reacts with the nitrogen source HOSA to generate the key intermediate oxyaziridine. This intermediate then reacts with a double-bonded compound to generate aziridine.

[0010] Guo et al. disclose a method of applying a chloroamine derivatization reagent to the identification of double bond positions 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 suffer from certain deficiencies. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] Patent WO2022 / 216767A1 [Patent Document 2] Patent CN114166921A [Patent Document 3] Patent CN113495112A [Non-Patent Document]

[0013] [Non-Patent Document 1] Holcapek, M. et al. J. Chromatogr. A 2011, 1218, 5146 [Non-Patent Document 2] Mclean, J. A. et al. Anal. Chem. 2014, 86, 2107 [Non-Patent Document 3] Groessl, M. et al. Analyst 2015, 140, 6904 [Non-Patent Document 4] Fernandez-Lima, F. et al. Anal. Chem. 2019, 91, 5021 [Non-Patent Document 5] Ouyang, Z. et al. Nat. Commun. 2023, 14, 1535 [Non-Patent Document 6] Gross, M. L. et al. J. Am. Chem. Soc. 1983, 105, 5487 [Non-Patent Document 7] McLuckey, S. A. et al. Anal. Chem. 2019, 91, 9032 [Non-Patent Document 8] Brodbelt, J. S. et al. Anal. Chem. 2017, 89, 1516 [Non-Patent Document 9] Brodbelt, JS et al. J. Am. Chem. Soc. 2017, 139, 15681

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

Non-licensed Document 18

Non-licensed Document 19

Non-licensed Document 20

Non-licensed Document 21

Non-licensed Document 22

Non-licensed Document 23

Non-licensed Document 24

Non-licensed Document 25

Non-licensed Document 26

Non-licensed Document 27

Non-licensed Document 28

Non-licensed Document 29

[0014] Through continued research into the prior art, the present inventor has found that the labeling methods for carbon-carbon double bonds in the prior art all 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 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, 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 at sites that correspondingly have original carbon-carbon double bonds. A mass spectrometry step in which the mass numbers of the multiple subions are measured to identify the location of the carbon-carbon double bonds in the unsaturated organics.

[0016] R 2 The mass number of 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 matter may be an unsaturated lipid.

[0022] The unsaturated lipid is a fatty acyls, 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, in which 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 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, 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 products. The dissociator dissociates the derivatization products ionized in the ion source to obtain a plurality of subions by cleaving the derivatization products at sites that naturally have carbon-carbon double bonds. The mass spectrometer measures the mass numbers of the subions to identify the positions of the carbon-carbon double bonds in the unsaturated organic compounds.

[0024] R 2 The mass number of 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, where 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. A first inlet is provided in the communication device and communicates with a sample inlet line. A second inlet is provided in the communication device and communicates with a derivatization reagent inlet line. A product outlet is provided in the communication device and transports the derivatization product to the ion source. The acceleration control accelerates collisions of molecules in 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. Effect of the Invention

[0036] Compared with conventional identification methods, the method for identifying unsaturated organic substances according to the present application has at least one of the following advantages. A rich 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. b. Good substrate versatility: Applicable to a wide variety 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, and no strong oxidizing or reducing agents are required. The reaction can be completed quickly at room temperature or at a slightly elevated temperature, and the stability of the reagents and products is good. 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, usually only a single carbon-carbon double bond is aziridinated, or it can be adjusted so that only a single carbon-carbon double bond in most molecules is aziridinated by optimizing the reaction conditions (e.g., temperature and time). In addition, the spectrogram is simple and easy to analyze. g.Can be done online. [Brief description 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 isomeric orientation in unsaturated lipids according to a first embodiment of the present invention. [Diagram 2]Yield results from aza-Prilezhaev reaction at different temperatures for 10 minutes in the first embodiment of the present invention. [Diagram 3] 1 is a relationship of the conversion rate over time reflected by the peak intensity of 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. 2 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. [Diagram 5] FIG. 2 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 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. 2 is a diagram illustrating the principle of the reaction flow of the identification method according to the 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 is 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 is 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. 2 is a schematic diagram of 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 of 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 is 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. 1 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 PREFERRED EMBODIMENTS

[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 some 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 a person skilled in the art can obtain without creative labor all belong to 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 acyls, glycerides, glycerophospholipids, sphingolipids, sterol esters, pregnenolone lipids, glycolipids or polyketides, and in particular applies to fatty acyls / fatty acids, glycerophospholipids, sterol esters, sphingolipids and glycerides.

[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 bonds, and Z indicates the cis geometric configuration.

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

[0042] In addition, the following English abbreviations refer to the following lipid types: CE: Cholesterol ester 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 a flow diagram 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 the present embodiment.

[0044] Referring to FIG. 1, the identification method includes the following steps. In the derivatization step S1, a carbon-carbon double bond in an unsaturated organic compound is aziridinated by the aza-Prilezhaev reaction to obtain a derivative. An ionization step S2 in which the derivatization product is ionized. A mobility selection step S3 selects derivatized compounds whose carbon-carbon double bonds have already been aziridinated based on their ion mobility. 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 a plurality of subions. A mass analysis step S5 is performed to measure the mass numbers of the multiple subions to 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, techniques 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 the present 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 thereto.

[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 only undergo a one-step synergistic reaction to remove one molecule of arylsulfonic acid to obtain aziridine.

[0050] The derivatization reaction step S1 can be completed offline, i.e., an experimenter completes steps such as mixing raw materials and controlling temperature by himself. On the other hand, it can also be completed online, i.e., the analytical device completes it according to a preset program. In this embodiment, the derivatization reaction step S1 is 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 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, 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] R 1 By using an aromatic group as R, the stability of the reagent can be improved. 2 is usually a group having a mass number of more than 15 Da. 2 is a group having a mass number of more than 50 Da. More preferably, R 2 is a group with a mass number greater than 80 Da, or R 2 is a group with a mass number greater than 100 Da. 2By reasonably increasing the mass number of R, the overlapping of mass peaks in the spectrogram can be effectively prevented. 2 may contain a relatively sterically hindered group such as a methyl group, 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] Other alternative and readily available aza-Prilezhaev reagent mass labels and their CAS numbers are listed in 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 Take a 10-minute reaction as an example. With tert-butyl N-tosyloxycarbamate as the mass label and hexafluoroisopropanol as the solvent, Figure 2 shows the results of the reaction yield at different temperatures for 10 minutes. Experiments have found that the yield of aza-Prilezhaev can be significantly improved with increasing temperature at about 20°C, and tends to be almost stable at about 50°C-55°C. The reaction temperature is more preferably set in the range of 20°C-55°C, more preferably in the range of 30°C-55°C, for example 50°C. In this temperature range, a higher yield can be obtained 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 of the mass spectrum of the target substance at a reaction temperature of 50°C. Referring to Figure 3, research has shown that the reaction rate of aza-Prilezhaev is quite high when the reaction temperature is 50°C. It only takes 30s for the reaction to reach a detectable peak intensity. When the reaction lasts for more than 10 minutes, the conversion rate approaches 100%.

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

[0060] <ims-ms ms> a) System configuration Referring to FIG. 4, a system used for off-line analysis of derivatization 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 source 1 includes electrospray ionization source (ESI), atmospheric pressure photoionization source (APPI); atmospheric pressure chemical ionization source (APCI), matrix assisted laser desorption ionization source (MALDI), laser desorption ionization source (LDI), atmospheric pressure ionization source (API), desorption ionization source on silicon (DIOS), electron impact ionization source (EI), chemical ionization source (CI), field ionization source (FI), field desorption ionization source (FD), inductively coupled plasma ionization source (ICP), fast atom bombardment ionization source (FAB), liquid secondary ion mass spectrometry ionization source (LSIMS), direct electrospray ionization (D The ion source includes one selected from the group consisting of an electrospray ionization source, a nickel-63 radioactive ion source, an atmospheric pressure matrix-assisted laser desorption ionization source, a thermal spray ionization source, an air 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 inlet ionization source (MAII), a solvent-assisted inlet ionization source (SAII), a Penning ionization source, a laser ablation electrospray ionization source (LAESI), and a He plasma ionization source (HePl). 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. Between the ion mobility spectrometer 2 and the ion source one or more ion guide devices 6 may be provided.

[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 device structure of the U-type ion mobility spectrometer and the description of the filtering mode applied to this identification method can be referred to Patent Document 3, and the description is omitted here. Similarly, the identification method according to the embodiment of the present invention is not limited to the type of the applied ion mobility spectrometer.

[0065] The ion mobility spectrometer 2 can provide a second dimension of data for tandem mass spectrometry. Different ion mobilities can differentiate isomers. In particular, differences in molecular structure are enhanced after aziridination of carbon-carbon double bonds by aza-Prilezhaev reaction. In some embodiments, ion mobility spectrograms can be utilized 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 spectrometer 5, which are connected in series. Between the ion mobility spectrometer 2 and the mass filter 3, one or more ion guide devices 6 may be provided.

[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 heat or temperature source dissociation device, an electric field-induced dissociation device, a magnetic field-induced dissociation device, an ion-ion reaction dissociation device, an ion-molecule reaction dissociation device, an ion-atom reaction dissociation device, an ion metastable ion reaction dissociation device, an ion metastable molecule reaction 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, that is, the site where the aziridine ring is present can be cleaved with high selectivity, and the occurrence of side reactions can be avoided.

[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 a part of the derivatization product can be cleaved, thereby detecting the sn-position of the carbon-carbon double bond. 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 jointly by the mass filter 3 and the 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, ie 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 In addition, the aziridinyl lipid obtained by the reaction can be stable. Figure 5 is a graph showing the change in detection peak intensity of the aziridinyl lipid according to the first embodiment of the present invention with different standing times. Referring to Figure 5, the aziridinyl lipid has almost no effect on the detection peak intensity even after standing for more than 48 hours, so that it can meet the requirements of different types of tests.

[0073] b) Sensitivity Figure 6 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. Referring to Figure 6, since an easily ionizable group is introduced, the aza-Prilezhaev reaction can improve the sensitivity of mass spectrum detection by aziridinating the carbon-carbon double bond. Specifically, the signal intensity of the mass spectrum of the parent ion can be improved by 1-3 orders of magnitude.

[0074] c) Isomers that distinguish between different positions of carbon-carbon double bonds 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, -NH 2 + Two scenarios can occur: either the - is assigned to the aliphatic chain or to the head group. 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 different positions of the carbon-carbon double bonds in the fatty chain, specifically, the carbon-carbon double bond of C18:1(9Z) is located between the 9th and 10th carbon atoms of the main chain, while the carbon-carbon double bond of C18:1(6Z) is located between the 6th and 7th carbon atoms of the main chain. The following describes a method for determining the position of the carbon-carbon double bond based on mass spectrum.

[0076] FIG. 8 shows a mass spectrum obtained by aziridinating a standard sample of C18:1(9Z). Referring to the aziridinated molecular structure shown in FIG. 8, when the aziridine ring is specifically cleaved by the dissociation device 4, the fatty chain side is cleaved to give C 8 H 17 -CH=NH 2 + 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 C 8 H 13 O-CH=NH 2 + Subions are formed, namely a mass peak at m / z=154 appears.

[0077] FIG. 9 shows a mass spectrum obtained by aziridinating a standard sample of C18:1(6Z). Referring to the aziridinated molecular structure shown in FIG. 9, when the aziridine ring is specifically cleaved by the dissociation device 4, the fatty chain side is cleaved to give C 11 H 23 -CH=NH 2 + 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 C 5 H 7 O-CH=NH 2 + 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 it has the above characteristic mass peaks and characteristic mass peaks corresponding to each of the other different positions, or based on the intensity of the mass peaks. When dissociation energy is applied to the aziridine ring site, it is easily broken specifically, so the peak intensity of the characteristic mass peak is strong, and the detection sensitivity and accuracy are high.

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

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

[0081] Referring to Non-Patent Document 29, the characteristic peaks 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. Accordingly, for the aza-Prilezhaev aziridination reaction, the characteristic peaks m / z=274, 290, and 360 can be used to distinguish the sn positions.

[0082] Specifically, PC16:0 / 18:1(9Z), in which the carbon-carbon double bond is 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, in which the carbon-carbon double bond is at the sn1 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 by the above two characteristic peaks.

[0083] c.3. Identification of 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 expanding the difference in ion mobility between different molecules and further separating the ion mobility peaks.

[0084] Figure 14 shows the spectrograms (from top to bottom) of the UMA ion mobility spectrum of aziridinated C18:1 (9E), C18:1 (9Z) and a mixture of both. 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 Figure 15-Figure 16 are mass spectra of aziridination of C20:4 (5Z, 8Z, 11Z, 14Z). Referring to Figure 15, the aza-Prilezhaev reaction can effectively prevent the excessive derivatization of carbon-carbon double bonds, so that in most cases, only a single carbon-carbon double bond in each unsaturated organic molecule is aziridinated, making the spectrogram simpler and easier to analyze. Referring to Figure 16, the eight characteristic mass peaks corresponding to the four carbon-carbon double bonds are still clear 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 rich variety of identifiable information. It is possible to identify 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. b. Good substrate versatility: Applicable to a wide variety 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, and no strong oxidizing or reducing agents are required. The reaction can be completed quickly at room temperature or at a slightly elevated temperature, and the stability of the reagents and products is good. 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, usually only a single carbon-carbon double bond is aziridinated, or it can be adjusted so that only a single carbon-carbon double bond in most molecules is aziridinated by optimizing the reaction conditions (e.g., temperature and time). 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 a derivatization product according to a second embodiment of the present invention. The difference from the first embodiment is that this embodiment uses an online derivatization reactor 7 to carry out the aza-Prilezhaev reaction.

[0088] Specifically, refer to FIG. 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 a sample inlet pipe 81 and is used to take in the sample. The second inlet 712 communicates with a derivatization reagent inlet pipe 82 and is used to take in the derivatization reagent. The product outlet 713 is connected to the ion source 1 and transports the derivatization product generated 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. Among them, any suitable type of temperature control unit, such as a microwave heating device, a water bath, an oil bath, etc., may be used for heating, and the reaction temperature of the derivatization reaction may be controlled, particularly within the temperature range of 20-100°C, to accelerate the reaction and reduce 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. Specifically, the liquid chromatograph is connected to a first inlet 711, and a column 9 of the liquid chromatograph is installed in a sample introduction pipe 81. By using them in combination to form an LC-IMS-MS / MS, a rapid and highly sensitive analysis of multiple lipids can be easily and quickly completed by simply injecting the sample once.

[0091] The above are merely preferred embodiments of the present invention, and the present invention is not limited thereto. Modifications and equivalents thereof are also included within the technical scope of the present invention, provided 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 section 81-Sample introduction pipe 82 - Derivatization reagent introduction line 9-Column

Claims

1. A method for identifying unsaturated organic matter, comprising the steps of: a derivatization step of converting a carbon-carbon double bond in an unsaturated organic compound into an aziridinyl compound by an aza-Prilezhaev reaction to obtain a derivative; 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; and a mass spectrometry step for determining the positions of carbon-carbon double bonds in the unsaturated organic compound by measuring the mass numbers of the subions. The derivatization reagent used in the aza-Prilezhaev reaction includes a compound represented by the following general formula (1): 【Chemistry 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 an unsaturated organic substance according to claim 1, wherein the mass number of is greater than 80 Da.

3. The compound represented by the general formula (1) is 【Chemistry 8】 The method for identifying unsaturated organic matter according to claim 2, characterized in that

4. 2. The method for identifying an unsaturated organic substance 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. The method for identifying an unsaturated organic matter according to claim 1, characterized in that the unsaturated organic matter is an unsaturated lipid.

8. 8. The method for identifying an unsaturated organic substance 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 the sample with an aza-Prilezhaev derivatization reagent to aziridinate the carbon-carbon double bonds of unsaturated organic compounds in the sample using the aza-Prilezhaev reaction to obtain a derivatized product; an ion source which receives and ionizes the derivatized product; a dissociation device for dissociating the derivatization product ionized by 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 a plurality of the subions to identify the positions of the carbon-carbon double bonds in the unsaturated organic compound. 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,

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. 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 for accelerating 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 pipe; a second inlet provided in the communication device and communicating with an inlet line for 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 comprising: a product outlet provided in the communication device for transporting the derivatization 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. 20. The mass spectrometry system according to claim 18, further comprising a liquid chromatograph device disposed in the sample introduction line.

20. 10. The mass spectrometry system of claim 9, further comprising a mass filter disposed 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.

Images