Method and device for identifying positions of carbon-carbon double bonds based on electrospray gaseous derivative ionization

Abstract

The invention provides a method and a device for identifying positions of carbon-carbon double bonds based on electrospray gaseous derivative ionization. Specifically, the invention provides an electrospray ionization device based on ozone gaseous derivative ionization, the device comprises a device for supplying ozone to a mass spectrum ion source and the mass spectrum ion source, and the device for supplying ozone to the mass spectrum ion source supplies generated ozone to the mass spectrum ion source. The invention further provides a method for identifying the number and the positions of the carbon-carbon double bonds in the unsaturated organic matter by using the device and application of the device in identifying the number and the positions of the carbon-carbon double bonds in the unsaturated organic matter.

Description

Technical Field

[0001] The present invention belongs to the field of mass spectrometry analysis, and more specifically, the present invention relates to a method and a device for identifying the position of a carbon-carbon double bond based on electrospray gaseous derivative ionization. Background Art

[0002] Mass spectrometry is an analytical tool that measures the mass-to-charge ratio of ions. Mass spectrometry has high sensitivity, good selectivity, and strong versatility. Combined with the efficient separation capability of liquid chromatography, liquid chromatography-mass spectrometry is currently a commonly used tool for qualitative and quantitative analysis of organic matter.

[0003] The mass spectrometry ion source is an important front end for ionizing organic matter into charged ions and thus entering mass spectrometry analysis. Electrospray ionization is the standard ionization method of the mass spectrometry ion source, which is suitable for more than 80% of organic matter analysis. However, there are still great challenges in using mass spectrometry to identify the position of carbon-carbon double bonds in organic matter. The carbon-carbon double bonds in organic matter have high bond energies and cannot be directly cleaved and produce characteristic fragment ions in the collision-induced dissociation fragmentation mode of conventional commercial mass spectrometers, making it impossible to determine the position of the double bond.

[0004] Literature research shows that combining ozone-induced dissociation with mass spectrometry and introducing ozone into the mass spectrometer ion transmission or collision region can effectively reduce the risk of ozone contamination. -4 Below mbar, under high vacuum conditions, the activity of ozone-induced dissociation reaction is extremely weak and the reaction rate is low. At the same time, the components required for ozone introduction and flow control need to be resistant to ozone. Even after ozone resistance modification, it is impossible to use it in conjunction with conventional analysis because the time of ions in the mass spectrometer ion transmission or ion collision area needs to be extended. It is only suitable for analyzing standard products or specific compounds. At the same time, there are many components inside the mass spectrometer, and it is difficult to completely avoid the damage of high-concentration ozone to the component seals, resulting in poor durability and practical application value, and the experiment cannot be reproduced.

[0005] In some other literature, ozone-induced dissociation and mass spectrometry are combined by using an open ion source at normal pressure, such as introducing an ozone reaction through a nanoliter electrospray open ion source, and the reaction product enters mass spectrometry analysis. Mass spectrometry-based organic analysis is mostly performed on conventional liquid chromatography-mass spectrometry, with a flow rate generally between 0.3ml / min-1.0ml / min, but the flow rate adapted to the nanoliter ion source is generally 100-300nl / min, which is not suitable for conventional liquid chromatography-mass spectrometry for organic identification.

[0006] Therefore, there is an urgent need in the art to develop a method and device that is suitable for conventional liquid chromatography-mass spectrometry, which can solve the problems of increasing the ozone concentration under high vacuum and instrument damage caused by high concentration ozone, and realize the identification of the position of carbon-carbon double bonds in organic matter. Summary of the invention

[0007] The object of the present invention is to provide a method and a device for identifying the position of a carbon-carbon double bond based on electrospray gaseous derivative ionization.

[0008] In a first aspect of the present invention, an electrospray ionization device based on ozone gas phase derivative ionization is provided, the device comprising: a device for supplying ozone to a mass spectrometry ion source and a mass spectrometry ion source, wherein the device for supplying ozone to the mass spectrometry ion source supplies the generated ozone to the mass spectrometry ion source.

[0009] In one or more embodiments, the mass spectrometry ion source includes: an ion source and an ion inlet device.

[0010] In one or more embodiments, the ion source includes an electrospray needle and a high-voltage power supply, the outlet end of the electrospray needle is located in front of the ion inlet device of the mass spectrometry ion source, and the high-voltage power supply is connected to the electrospray needle and can trigger the sample solution in the electrospray needle to form ions, and the formed ions enter the mass spectrometry ion source through the ion inlet device.

[0011] In one or more embodiments, the distance between the outlet end of the electrospray needle and the ion inlet device of the mass spectrometry ion source is 10 to 100 mm, preferably 20 to 50 mm.

[0012] In one or more embodiments, the material of the electrospray needle includes, but is not limited to: metal, graphite, carbon fiber, and conductive polymer material.

[0013] In one or more embodiments, the ion source further includes an auxiliary gas line and / or a sheath gas line, wherein the auxiliary gas line and / or the sheath gas line are used to supply ozone to the mass spectrometry ion source.

[0014] In one or more embodiments, the device for supplying ozone to the mass spectrometer ion source is connected to the auxiliary gas line, or to the sheath gas line, or to both the auxiliary gas line and the sheath gas line.

[0015] In one or more embodiments, the axis of the electrospray needle is parallel to the axis of the auxiliary gas line and / or the axis of the sheath gas line.

[0016] In one or more embodiments, the device for supplying ozone to the mass spectrometry ion source comprises: an oxygen supplier and an ozone generator, oxygen is supplied to the ozone generator through the oxygen supplier to generate ozone, and the device for supplying ozone to the mass spectrometry ion source supplies the generated ozone to the ion source.

[0017] In one or more embodiments, the device for supplying ozone to the mass spectrometry ion source further comprises one, more or all components selected from the following: a gas pressure display, an ozone analyzer, and an exhaust destructor.

[0018] In one or more embodiments, the gas pressure display is connected to the ozone generator to detect the flow rate of ozone generated in the ozone generator.

[0019] In one or more embodiments, the ozone analyzer is used to analyze residual ozone in a device that supplies ozone to a mass spectrometry ion source.

[0020] In one or more embodiments, the tail gas destroyer removes excess ozone from the apparatus that supplies ozone to the mass spectrometer ion source.

[0021] In one or more embodiments, the device for supplying ozone to the mass spectrometry ion source also includes: an inert gas supplier, an ozone inlet channel converter and a nitrogen inlet channel converter, and the ozone inlet channel converter and the inert gas inlet channel converter are used to adjust the gas in the device for supplying ozone to the mass spectrometry ion source to ozone or an inert gas.

[0022] In one or more embodiments, the inert gas includes: one of nitrogen, argon, helium and air, or a mixture of any two, three or all of them, or a mixture of any one, two, three or all of them and ozone.

[0023] In one or more embodiments, the electrospray ionization device based on ozone gas phase derivative ionization further comprises: a mass spectrometer, and the electrospray ionization device based on ozone gas phase derivative ionization is connected to the mass spectrometer.

[0024] In one or more embodiments, the inlet of the mass spectrometer is connected to the outlet of a mass spectrometer ion source in an electrospray ionization device based on ozone gaseous derivative ionization, so as to perform mass spectrometry analysis on ions in the mass spectrometer ion source.

[0025] In one or more embodiments, the mass spectrometer includes a triple quadrupole mass spectrometer, an electrostatic field orbital trap mass spectrometer, a quadrupole tandem orbital trap mass spectrometer, a time-of-flight mass spectrometer, a quadrupole time-of-flight mass spectrometer, an ion trap mass spectrometer, a quadrupole linear ion trap mass spectrometer, and a Fourier transform ion cyclotron resonance mass spectrometer.

[0026] In one or more embodiments, the mass spectrometer includes a triple quadrupole mass spectrometer (e.g., QQQ, Triple Quad, TSQ, TSQ Plus, TQ, Stellar), an electrostatic field orbital trap mass spectrometer (e.g., Exactive, QExactive, Exactive Plus, Exactive Plus Orbitrap, Exactive HF, Orbitrap Exploris120, Orbitrap Exploris 240, Orbitrap Exploris 480), a quadrupole tandem orbital trap mass spectrometer (e.g., QExactive, Exactive Plus, Exactive HF, Orbitrap Exploris 120, Orbitrap Exploris240, Orbitrap Exploris 480, Orbitrap Astral, Orbitrap Eclipse Tribrid, Orbitrap Fusion Tribrid, Orbitrap Fusion Lumos Tribrid, Orbitrap ID-X Tribrid), a time-of-flight mass spectrometer (e.g., TOF), a quadrupole time-of-flight mass spectrometer (e.g., QTOF, timsTOF Pro, timsTOF Pro2, timsTOF SCP, timsTOF Ultra, timsTOF Ultra2), ion trap mass spectrometer (e.g. Ion Trap, LTQ, LCQ), quadrupole linear ion trap mass spectrometer (e.g. Qtrap), Fourier transform ion cyclotron resonance mass spectrometer (e.g. FT-ICR).

[0027] In one or more embodiments, the electrospray ionization device based on ozone gas phase derivative ionization further comprises: a liquid chromatography analysis device, and the electrospray ionization device based on ozone gas phase derivative ionization is connected to the liquid chromatography instrument.

[0028] In one or more embodiments, the outlet of the liquid chromatography instrument is connected to the inlet of the electrospray needle of a mass spectrometer ion source in an electrospray ionization device based on ozone gas phase derivative ionization.

[0029] In one or more embodiments, the liquid chromatography includes: high performance liquid chromatography, ultra high performance liquid chromatography, liquid chromatography, microliter liquid chromatography, and ion chromatography.

[0030] In one or more embodiments, the liquid chromatography includes: high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC), liquid chromatography (LC), microliter liquid chromatography (Micro LC), ion chromatography (IC)

[0031] In one or more embodiments, in the electrospray ionization device based on ozone gas phase derivative ionization, the liquid chromatography analysis device, the electrospray ionization device based on ozone gas phase derivative ionization, and the mass spectrometer are connected in sequence.

[0032] In one or more embodiments, the outlet end of the liquid chromatography instrument is connected to the inlet end of the electrospray needle of a mass spectrometry ion source in an electrospray ionization device based on ozone gas phase derivative ionization, and the inlet end of the mass spectrometry instrument is connected to the outlet end of the mass spectrometry ion source in an electrospray ionization device based on ozone gas phase derivative ionization, for performing mass spectrometry analysis on ions in the mass spectrometry ion source.

[0033] The second aspect of the present invention provides a method for identifying the number and position of carbon-carbon double bonds in unsaturated organic matter using the electrospray ionization device based on ozone gaseous derivative ionization described in any embodiment of the present invention, the method comprising:

[0034] (1) using the electrospray ionization device based on ozone gaseous derivative ionization, supplying ozone to a mass spectrometry ion source, so that ozone and unsaturated organic matter undergo an epoxide addition reaction in the mass spectrometry ion source to form charged characteristic ions;

[0035] (2) Using a mass spectrometer to detect the characteristic ions formed in (1), the number and position of carbon-carbon double bonds in the unsaturated organic matter are identified.

[0036] In one or more embodiments, the unsaturated organic compound contains at least one carbon-carbon double bond.

[0037] In one or more embodiments, the unsaturated organic matter includes a single organic matter, or a mixture or combination of two or more organic matters.

[0038] In one or more embodiments, the unsaturated organic substance is an unsaturated lipid.

[0039] In one or more embodiments, the unsaturated lipids include: one, more or all of fatty acids, glycerolipids, phospholipids, sphingolipids, glycolipids, glycerophospholipids, sterol esters, prenol lipids, and polyketides.

[0040] In one or more embodiments, the unsaturated lipids include fatty acids (FA), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylserine (LPS), sphingomyelin (SM), ceramide (Cer), cholesterol ester (CE), cardiolipin (CL), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), thioiso-rhamnosyldiacylglycerol (SQDG), monoglyceride (MG), diglyceride (DG), triglyceride (TG).

[0041] In one or more embodiments, the method further comprises one, more or all of the following conditions:

[0042] (A) In step (1), the method of supplying ozone to the mass spectrometer ion source by using the electrospray ionization device based on ozone gas phase derivative ionization comprises: turning on the ozone generator, and after the ozone analyzer shows that ozone has been generated, adjusting the inert gas inlet channel converter and the ozone inlet channel converter, turning off the inert gas and allowing the ozone to enter the mass spectrometer ion source;

[0043] (B) Step (1) further comprises the step of detecting and calibrating ozone to zero. Preferably, the oxygen supply device and the ozone analyzer are turned on, and the ozone analyzer is used to detect and calibrate ozone to zero.

[0044] (C) the concentration of the unsaturated organic matter in step (1) is 0.1 pmol / L-1 mol / L, preferably 1 μmol / L-10 mmol / L;

[0045] (D) in step (1), adjusting the concentration and flow rate of ozone by controlling the ozone generator;

[0046] (E) in step (1), the concentration of ozone is less than 100 mg / L, preferably less than 80 mg / L, less than 70 mg / L, less than 60 mg / L or less than 50 mg / L, more preferably, the concentration of ozone is 10 to 30 mg / L;

[0047] (F) In step (1), the flow rate of ozone is 0.1 L / min or more, preferably 0.2 L / min or more, 0.3 L / min or more, 0.4 L / min or more, or 0.5 L / min or more; more preferably, the flow rate of ozone is 1 to 10 L / min;

[0048] (G) In step (2), the method for obtaining the number and position information of carbon-carbon double bonds of unsaturated organic matter based on the characteristic ions formed in step (1) comprises: the characteristic ions formed in step (1) appear in pairs in the mass spectrum, and the number of characteristic peaks of the characteristic ions appearing in pairs is the number of carbon-carbon double bonds; and the position of the carbon-carbon double bonds is determined based on the difference between the mass information of the characteristic peaks of the characteristic ions appearing in pairs and the mass information of the parent ion.

[0049] In one or more specific embodiments, the method for determining the number of carbon-carbon double bonds is as follows: if the mass difference of the characteristic ions appearing in pairs in step (1) is 16±0.1Da, they are considered to be characteristic ion pairs, and the number of characteristic ion pairs is the number of carbon-carbon double bonds.

[0050] In one or more specific embodiments, the position of the carbon-carbon double bond is determined based on the difference between the mass information of the characteristic peaks of the characteristic ions that appear in pairs and the mass information of the parent ion in combination with Table 1.

[0051] The third aspect of the present invention provides the use of the electrospray ionization device based on ozone gas phase derivative ionization described in any embodiment of the present invention for identifying the number and position of carbon-carbon double bonds in unsaturated organic matter.

[0052] In one or more embodiments, the unsaturated organic compound contains at least one carbon-carbon double bond.

[0053] In one or more embodiments, the unsaturated organic matter includes a single organic matter, or a mixture or combination of two or more organic matters.

[0054] In one or more embodiments, the unsaturated organic substance is an unsaturated lipid.

[0055] In one or more embodiments, the unsaturated lipids include: one, more or all of fatty acids, glycerolipids, phospholipids, sphingolipids, glycolipids, glycerophospholipids, sterol esters, prenol lipids, and polyketides.

[0056] In one or more embodiments, the unsaturated lipids include fatty acids (FA), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylserine (LPS), sphingomyelin (SM), ceramide (Cer), cholesterol ester (CE), cardiolipin (CL), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), thioiso-rhamnosyldiacylglycerol (SQDG), monoglyceride (MG), diglyceride (DG), triglyceride (TG).

[0057] In one or more embodiments, the method for identifying the number and position of carbon-carbon double bonds in an unsaturated organic compound is as described in any embodiment of the present invention.

[0058] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 , a block diagram of the connection system between the ozone generator and the mass spectrometer provided by the present invention.

[0060] Figure 2 , diagram of the internal structure of the electrospray ion source used in the present invention.

[0061] Figure 3. Ozone gas-derivative ionization PC 16:0 / 18:1(n-9)( Figure 3A )、PC 16:0 / 18:2(n-6,9)( Figure 3B ), PC 16:0 / 20:4(n-6,9,12,15)( Figure 3C )、PE 16:0 / 18:1(n-9)( Figure 3D )、PE 16:0 / 18:2(n-6,9)( Figure 3E )First-level high-resolution mass spectrum.

[0062] Figure 4, PC 16:0 / 18:1(n-9)( Figure 4A )、PC 16:0 / 18:2(n-6,9)( Figure 4B ), PC 16:0 / 20:4(n-6,9,12,15)( Figure 4C )、PE 16:0 / 18:1(n-9)( Figure 4D )、PE 16:0 / 18:2(n-6,9)( Figure 4E) Structural derivation of characteristic ion fragments of double bonds of carbon-carbon ions ionized by electrospray gaseous derivative ionization using ozone as atomizing gas.

[0063] Figure 5 , electrospray gaseous derivative ionization (ozone conditions, with ozone as the nebulizer gas) and electrospray ionization (nitrogen conditions, with nitrogen as the nebulizer gas) analysis of PC 16:0 / 18:1(n-9) comparison chart.

[0064] The reference numerals are as follows:

[0065] 1. Ozone generator;

[0066] 2. Ozone inlet channel converter;

[0067] 3. Inert gas inlet channel converter;

[0068] 4. Mass spectrometry ion source;

[0069] 5. Gas pressure display;

[0070] 6. Ozone analyzer;

[0071] 7. Exhaust destroyer;

[0072] 8. ion source; 801. electrospray needle; 802. auxiliary gas pipeline; 803. sheath gas pipeline; 804. high voltage power supply;

[0073] 9. Ion inlet device;

[0074] 10. Oxygen supply device;

[0075] 11. Inert gas supplier. DETAILED DESCRIPTION

[0076] After in-depth research, the inventors have provided an electrospray ionization device based on ozone gaseous derivative ionization, which realizes electrospray gaseous derivative ionization in the mass spectrometer ion source area, thereby avoiding the damage to the internal parts of the instrument such as the sealing ring caused by the introduction of ozone into the collision chamber or the ion transmission area. The device of the present invention can effectively identify the carbon-carbon double bonds in unsaturated lipid samples. The device of the present invention can also be used in conjunction with conventional liquid chromatography and mass spectrometry systems to expand the depth of mass spectrometry identification of organic matter.

[0077] Electrospray ionization device based on ozone gaseous derivative ionization

[0078] The present invention provides an electrospray ionization device based on ozone gaseous phase derivative ionization, which comprises: a device for supplying ozone to a mass spectrometry ion source and the mass spectrometry ion source.

[0079] The electrospray ionization device based on ozone gaseous derivative ionization of the present invention can be connected to a mass spectrometer. For example, the mass spectrometer can be connected to the mass spectrometer ion source. When ozone is supplied to the mass spectrometer ion source, sample ions are ionized at the mass spectrometer ion source to form sample ions. The mass of the sample ions is detected by the mass spectrometer connected to the mass spectrometer ion source, and the mass spectrum information of the sample ions can be obtained.

[0080] In one or more embodiments, the mass spectrometer ion source includes: an ion source and an ion inlet device. The ion source is a device used to ionize a sample in mass spectrometry analysis, which can ionize the ionized sample into a gaseous state so as to be detected by a mass spectrometer detector. In one or more specific embodiments, when ozone is supplied to the mass spectrometer ion source, the sample is ionized at the ion source, and after forming sample ions, it enters the mass spectrometer from the ion inlet device.

[0081] The mass spectrometer used in the present invention can be a triple quadrupole mass spectrometer, a time-of-flight mass spectrometer, an ion trap mass spectrometer, a Fourier transform ion cyclotron resonance mass spectrometer, or the like.

[0082] In one or more embodiments, Figure 2 As shown, the ion source includes: an electrospray needle, an auxiliary gas pipeline, a sheath gas pipeline and a high-voltage power supply. The high-voltage power supply is connected to the electrospray needle. By turning on the high-voltage power supply connected to the electrospray needle, the sample solution generates spray under the action of high voltage electricity and is ionized into sample ions.

[0083] In one or more embodiments, the device for supplying ozone to the mass spectrometer ion source can be connected to the auxiliary gas line, or to the sheath gas line, or to both the auxiliary gas line and the sheath gas line. Therefore, the ozone can be supplied to the mass spectrometer ion source through the auxiliary gas line and / or the sheath gas line.

[0084] In one or more embodiments, the device for supplying ozone to the mass spectrometry ion source comprises: an oxygen supplier and an ozone generator. Oxygen is supplied to the ozone generator through the oxygen supplier to generate ozone. The generated ozone is supplied to the mass spectrometry ion source through an auxiliary gas pipeline and / or a sheath gas pipeline.

[0085] In one or more embodiments, the device for supplying ozone to the mass spectrometer ion source further comprises one, more or all of the following components: a gas pressure display, an ozone analyzer and an exhaust gas destroyer. The gas pressure display can be connected to the ozone generator to detect the flow rate of ozone generated in the ozone generator. The ozone analyzer is used to analyze the residual ozone in the device for supplying ozone to the mass spectrometer ion source. The exhaust gas destroyer can remove excess ozone in the device for supplying ozone to the mass spectrometer ion source.

[0086] In one or more embodiments, the device for supplying ozone to the mass spectrometer ion source further comprises: an inert gas supplier, an ozone inlet channel converter and an inert gas inlet channel converter. The ozone inlet channel converter and the inert gas inlet channel converter can be used to adjust the gas supplied to the ion source to be an inert gas (such as nitrogen, argon, etc.) or ozone.

[0087] In one or more specific embodiments, the device for supplying ozone to a mass spectrometer ion source is, for example, Figure 1 As shown, it includes: an inert gas supplier, an oxygen supplier, an ozone generator, an ozone inlet channel converter, a nitrogen inlet channel converter, a gas pressure display, an ozone analyzer and an exhaust gas destroyer.

[0088] In one or more embodiments, the device for supplying ozone to the mass spectrometer ion source includes two inlet channels, and an inert gas supplier and an inert gas inlet channel converter are connected together in sequence to form an inert gas supply channel. An oxygen supplier, an ozone generator, an ozone inlet channel converter, a gas pressure display, an ozone analyzer and an exhaust gas destroyer are connected together in sequence to form an ozone supply channel. The inert gas inlet channel converter is also connected to the ozone inlet channel converter. By adjusting the inert gas inlet channel converter and the ozone inlet channel converter, ozone or an inert gas can be supplied to the mass spectrometer ion source through an auxiliary gas pipeline and / or a sheath gas pipeline.

[0089] When ozone is not needed for the epoxidation addition reaction, the inert gas inlet channel converter is turned on, and the inert gas in the inert gas supplier enters the mass spectrometer ion source. When ozone is needed for the epoxidation addition reaction, the ozone generator is turned on, and the oxygen in the oxygen supplier generates ozone in the ozone generator, and the flow of ozone can be monitored by a gas pressure display. By adjusting the inert gas inlet channel converter and the ozone inlet channel converter, the inert gas is turned off and the ozone is allowed to enter the mass spectrometer ion source. The ozone analyzer is used to analyze the ozone remaining in the ozone supply channel, and the tail gas destroyer is used to remove the excess ozone.

[0090] During the reaction, the sample solution is introduced through the electrospray needle, and a high voltage is added to the electrospray needle by a high voltage power supply, so that the sample solution is atomized into very small droplets with surface charge through the electrospray needle. The device for supplying ozone to the mass spectrometer ion source introduces ozone to the vicinity of the ion inlet device through an auxiliary gas pipeline and / or a sheath gas pipeline, and the introduced ozone reacts with the sample solution in a vaporized state to form sample ions. Under the action of vacuum difference and potential traction, the sample ions in the vaporized state enter the mass spectrometer from the ion inlet device for detection.

[0091] The electrospray ionization device based on ozone gas phase derivative ionization of the present invention can also be connected to a liquid chromatography instrument. In one or more embodiments, the liquid chromatography instrument separates the unsaturated organic matter, and then the number and position of carbon-carbon double bonds in the separated unsaturated organic matter are detected by using the electrospray ionization device based on ozone gas phase derivative ionization and a mass spectrometer connected thereto.

[0092] Liquid chromatography instruments that can be applied to the present invention include, but are not limited to, high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC), liquid chromatograph (LC), microliter liquid chromatograph (Micro LC), and ion chromatograph (IC).

[0093] A method for identifying the position of carbon-carbon double bonds based on electrospray gaseous derivative ionization

[0094] The present invention also provides a method for identifying the position of the carbon-carbon double bond of an unsaturated organic compound using the electrospray ionization device and mass spectrometer based on ozone gaseous derivative ionization of the present invention, comprising:

[0095] (1) using the electrospray ionization device based on ozone gaseous derivative ionization, supplying ozone to a mass spectrometry ion source, so that ozone and unsaturated organic matter undergo an epoxide addition reaction in the mass spectrometry ion source to form charged characteristic ions;

[0096] (2) Using a mass spectrometer to detect the characteristic ions formed in (1), the number and position information of the carbon-carbon double bonds of the unsaturated organic matter are obtained.

[0097] In the present invention, the "unsaturated organic matter" refers to an organic matter containing at least one (for example, more than two, more than three or more) carbon-carbon double bond. Herein, "at least" and "more than" both include the number itself. The unsaturated organic matter described in the present invention may be a single organic matter, or a mixture or composition of two, three, four or more organic matters. The unsaturated organic matter of the present invention may be a chemically synthesized unsaturated organic matter, or an unsaturated organic matter separated and extracted from animal, plant or microbial tissues (for example, brain tissue), body fluids (for example, blood, plasma, urine, sweat, etc.), cells (for example, cell membranes, cell contents), etc.

[0098] In some embodiments, the unsaturated organic matter may be an unsaturated lipid, such as but not limited to: fatty acids, glycerolipids, phospholipids, sphingolipids, glycolipids, glycerophospholipids, sterol esters, prenol lipids, and polyketides. In some embodiments, the unsaturated organic matter includes but is not limited to: fatty acid (FA), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), lysophosphatidylserine (LPS), sphingomyelin (SM), ceramide (Cer), cholesterol ester (CE), cardiolipin (CL), monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), thioiso-rhamnosyl diacylglycerol (SQDG), monoglyceride (MG), diglyceride (DG), triglyceride (TG). In some specific embodiments, the unsaturated organic matter is unsaturated phosphatidylcholine (PC) or unsaturated phosphatidylethanolamine (PE).

[0099] In step (1), the method of supplying ozone to the mass spectrometry ion source using the electrospray ionization device based on ozone gas phase derivative ionization includes: turning on the ozone generator, and after the ozone analyzer shows that ozone has been generated, adjusting the inert gas inlet channel converter and the ozone inlet channel converter, turning off the inert gas and allowing ozone to enter the mass spectrometry ion source.

[0100] Step (1) may also include the step of detecting and calibrating the ozone, for example, turning on the oxygen supplier and the ozone analyzer, and detecting and calibrating the ozone using the ozone analyzer.

[0101] In step (1), the concentration of the unsaturated organic matter can be any concentration that allows it to undergo an epoxide addition reaction with ozone. The "epoxide addition reaction" generally refers to a ring-forming reaction of the carbon-carbon double bond in the substrate under ozone conditions to generate an intermediate product containing a five-membered ring structure:

[0102]

[0103] The intermediate product is unstable and will break quickly to generate an ion Aldehyde with one oxygen atom added and an ion Criegee with two oxygen atoms added. The mass difference between the two ions is 16 (deviation 0.1Da). The ions Aldehyde and Criegee are the characteristic ions in step (1):

[0104]

[0105] It should be understood that the epoxy addition reaction can only be completed under the condition of ozone, and the epoxy addition reaction will not occur under the condition of inert gas (such as nitrogen). Therefore, under the condition of inert gas, when mass spectrometry is used for detection, the characteristic peak of the characteristic ion will not be observed, and only the mass spectrum peak of the unsaturated organic parent ion can be observed.

[0106] In a specific embodiment, the concentration of the unsaturated organic matter may be 0.1 pmol / L-1 mol / L, preferably 1 μmol / L-10 mmol / L, such as 10 mmol / L.

[0107] In some embodiments, by controlling the ozone generator, the concentration and flow rate of ozone can be adjusted.

[0108] In a specific embodiment, in step (1), the concentration of ozone is less than 100 mg / L, such as less than 80 mg / L, less than 70 mg / L, less than 60 mg / L or less than 50 mg / L. More specifically, the concentration of ozone may be 10 to 30 mg / L.

[0109] In a specific embodiment, in step (1), the flow rate of ozone is 0.1 L / min or more, such as 0.2 L / min or more, 0.3 L / min or more, 0.4 L / min or more, or 0.5 L / min or more. More specifically, the flow rate of ozone can be 1 to 10 L / min.

[0110] In step (2), the method for obtaining the number and position information of carbon-carbon double bonds of unsaturated organic matter according to the characteristic ions formed in step (1) includes: the characteristic ions formed in step (1) appear in pairs in the mass spectrum, and the number of characteristic peaks of the characteristic ions appearing in pairs is the number of carbon-carbon double bonds. The position of the carbon-carbon double bond is determined according to the difference between the mass information of the characteristic peaks of the characteristic ions appearing in pairs and the mass information of the parent ion.

[0111] More specifically, the method for determining the number of carbon-carbon double bonds is as follows: if the mass difference of the characteristic ions appearing in pairs in step (1) is 16±0.1 Da, they are considered to be characteristic ion pairs, and the number of characteristic ion pairs is the number of carbon-carbon double bonds.

[0112] More specifically, the method for determining the position of the carbon-carbon double bond is to determine the position of the carbon-carbon double bond based on the difference between the mass information of the characteristic peak of the characteristic ion that appears in pairs and the mass information of the parent ion in combination with Table 1.

[0113] When the electrospray ionization device based on ozone gaseous derivative ionization of the present invention is connected to a liquid chromatograph, the liquid chromatograph can be used to separate unsaturated organic matter, thereby obtaining a single unsaturated organic matter. At this time, the method for identifying the position of carbon-carbon double bonds based on electrospray gaseous derivative ionization of the present invention can be used to identify the number and position of carbon-carbon double bonds in each unsaturated organic matter obtained by separation, thereby being able to analyze the number and position of carbon-carbon double bonds for a sample containing multiple unsaturated organic matter.

[0114] Compared with the prior art, the present invention has the following beneficial effects:

[0115] (1) Ozone is introduced into the electrospray ion source as an online reaction gas to react with the sample ions in the vapor state and obtain characteristic information related to the position of the carbon-carbon double bond through scanning by a high-resolution mass analyzer. This avoids the damage to the internal components of the instrument, such as the sealing ring, that may be caused by the introduction of ozone into the collision chamber or the ion transmission area.

[0116] (2) The present invention allows the compound to react and fragment in the ion source to form characteristic ions related to the position of the carbon-carbon double bond, thereby effectively locating the double bond position in the compound. It has strong versatility and can be used in conjunction with a conventional liquid chromatography-mass spectrometry system to expand the depth of mass spectrometry identification of organic matter.

[0117] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0118] Example 1: An electrospray ionization device based on ozone gas-derived ionization

[0119] This embodiment provides an electrospray ionization device based on ozone gaseous derivative ionization, which includes: a device for supplying ozone to a mass spectrometer ion source and a mass spectrometer ion source 4. The mass spectrometer ion source 4 includes: an ion source 8 and an ion inlet device 9. Figure 2 As shown, the ion source 8 includes: an electrospray needle 801, an auxiliary gas pipeline 802, a sheath gas pipeline 803 and a high voltage power supply 804. The device for supplying ozone to the mass spectrometer ion source can be connected to the auxiliary gas pipeline 802, or connected to the sheath gas pipeline 803, or connected to the auxiliary gas pipeline 802 and the sheath gas pipeline 803 at the same time.

[0120] The device for supplying ozone to the mass spectrometer ion source is as follows: Figure 1As shown, it includes: an inert gas supplier 11, an oxygen supplier 10, an ozone generator 1, an ozone inlet channel converter 2, an inert gas inlet channel converter 3, a gas pressure display 5, an ozone analyzer 6 and an exhaust gas destroyer 7. The device includes two inlet channels, and the inert gas supplier 11 and the inert gas inlet channel converter 3 are connected together in sequence to form an inert gas supply channel. The oxygen supplier 10, the ozone generator 1, the ozone inlet channel converter 2, the gas pressure display 5, the ozone analyzer 6 and the exhaust gas destroyer 7 are connected together in sequence to form an ozone supply channel. The inert gas inlet channel converter 3 is also connected to the ozone inlet channel converter 2. By adjusting the inert gas inlet channel converter 3 and the ozone inlet channel converter 2, ozone can be supplied to the mass spectrometer ion source 4 through the auxiliary gas pipeline 802 and / or the sheath gas pipeline 803.

[0121] When ozone is not needed for the epoxidation addition reaction, the inert gas inlet channel converter 3 is opened, and the inert gas in the inert gas supplier 11 enters the mass spectrometry ion source 4 .

[0122] When ozone is needed for the epoxidation addition reaction, the ozone generator 1 is turned on, and the oxygen in the oxygen supply device 10 generates ozone in the ozone generator 1, and the flow rate of ozone can be monitored by the gas pressure display 5. By adjusting the inert gas inlet channel converter 3 and the ozone inlet channel converter 2, the inert gas is turned off and the ozone is allowed to enter the mass spectrometer ion source 4. The ozone analyzer 6 is used to analyze the ozone remaining in the ozone supply channel, and the tail gas destroyer 7 is used to remove the excess ozone.

[0123] During the reaction, the sample solution is introduced through the electrospray needle 801, and a high voltage is added to the electrospray needle 801 by using the high voltage power supply 804, and the sample solution is atomized into very small droplets with surface charge through the electrospray needle 801. The device for supplying ozone to the mass spectrometer ion source introduces ozone to the vicinity of the ion inlet device 9 through the auxiliary gas pipeline 802 and / or the sheath gas pipeline 803, and the introduced ozone reacts with the sample solution in the vaporized state to form sample ions. The sample ions in the vaporized state enter the ion inlet device 9 under the action of the vacuum difference and the potential traction force, and the full scan and secondary fragmentation spectrum of the characteristic ions of the reaction between the sample and ozone are detected by mass spectrometry, and the carbon-carbon double bond position information can be obtained by mass number analysis.

[0124] The mass spectrometer used in the present invention can be a triple quadrupole mass spectrometer, a time-of-flight mass spectrometer, an ion trap mass spectrometer, a Fourier transform ion cyclotron resonance mass spectrometer or the like.

[0125] Example 2: Using the device of the present invention to identify the position of carbon-carbon double bonds in unsaturated lipid samples

[0126] In this embodiment, five unsaturated lipid samples, namely PC 16:0 / 18:1 (n-9), PC 16:0 / 18:2 (n-6,9), PE 16:0 / 18:1 (n-9), PE 16:0 / 18:2 (n-6,9), and PC 16:0 / 20:4 (n-6,9,12,15), are used as examples to identify the positions of carbon-carbon double bonds of unsaturated lipid samples using the device of Example 2. The identification method comprises the following steps:

[0127] a) turning on the oxygen supplier 10 and the ozone analyzer 6, and using the ozone analyzer 6 to detect and calibrate the ozone to zero;

[0128] b) Turn on the ozone generator 1, and after the ozone analyzer 6 indicates that ozone has been generated, adjust the inert gas inlet channel converter 3 and the ozone inlet channel converter 2, turn off the inert gas and allow the ozone to enter the mass spectrometer ion source 4. The flow rate of ozone entering the mass spectrometer ion source 4 is controlled by the gas pressure display 5. Excess ozone is analyzed and detected by the ozone analyzer 6, and removed by the tail gas destroyer 7;

[0129] c) using chloroform / methanol (1:1, v / v) solution, PC 16:0 / 18:1(n-9), PC 16:0 / 18:2(n-6,9), PE 16:0 / 18:1(n-9), PE 16:0 / 18:2(n-6,9), PC 16:0 / 20:4(n-6,9,12,15) were prepared into 10 mmol / L five kinds of unsaturated lipid sample solutions;

[0130] d) introducing the unsaturated lipid sample solutions prepared in c) into the ion source 8, respectively, and the sample solutions are sprayed out from the electrospray needle 801 to form charged droplets;

[0131] e) The device for supplying ozone to the mass spectrometer ion source introduces ozone through the auxiliary gas pipeline 802 and / or the sheath gas pipeline 803, wherein the ozone flow rate is 1 to 10 liters / minute and the ozone concentration is 10-120 mg / L. The introduced ozone reacts with the sample solution in the vaporized state to form sample ions, and the sample ions enter the mass spectrometer ion source 4. Through mass spectrometry scanning and detection, a primary mass spectrum of the unsaturated lipid sample under ozone conditions is obtained;

[0132] f) by analyzing the primary mass spectrum of the unsaturated lipid sample under ozone conditions obtained in e), identifying the position and number of carbon-carbon double bonds of the unsaturated lipid sample, the identification method is as follows:

[0133] Because ozone as atomizing gas reacts with carbon-carbon double bonds to form a ring, an intermediate product containing a five-membered ring structure is generated. The intermediate product is unstable and will break quickly to generate the ion Aldehyde with one oxygen atom added and the ion Criegee with two oxygen atoms added. The mass spectrometer detects the charged Aldehyde and Criegee ions, and the mass difference between the two ions is 16 (deviation 0.1Da).

[0134]

[0135] Therefore, the number of carbon-carbon double bonds can be determined by using the characteristic peaks of charged Aldehyde and Criegee ions with a difference of 16Da. The number of characteristic peaks with a difference of 16Da that appear in pairs in the mass spectrum is the number of carbon-carbon double bonds. Further, the mass-to-charge ratio information of the characteristic peaks with a difference of 16Da that appear in pairs in the mass spectrum is compared with the mass-to-charge ratio information of the parent ion, and the position of the carbon-carbon double bond can be determined according to Table 1.

[0136] Table 1

[0137]

[0138]

[0139] The mass spectra of PC 16:0 / 18:1(n-9), PC 16:0 / 18:2(n-6,9), PC 16:0 / 20:4(n-6,9,12,15), PE16:0 / 18:1(n-9) and PE 16:0 / 18:2(n-6,9) for carbon-carbon double bond position identification are shown in the figure below. Figures 3A-3E The structures of PC 16:0 / 18:1(n-9), PC 16:0 / 18:2(n-6,9), PC 16:0 / 20:4(n-6,9,12,15), PE 16:0 / 18:1(n-9) and PE 16:0 / 18:2(n-6,9) compounds and the structural deduction of the characteristic ion fragments of carbon-carbon ion double bonds after ozone reaction are shown in Figures 4A-4E shown.

[0140] The results show that the device of the present invention can accurately identify the position of carbon-carbon double bonds in unsaturated lipid samples. In addition, the device of the present invention does not require changing the mass spectrometry collision time, and can also avoid damage to internal instrument components such as sealing rings caused by introducing ozone into the collision chamber or ion transmission area, and reduces the requirement for ozone concentration during the reaction process.

[0141] Example 3: Application of the device of the present invention under inert gas conditions

[0142] The device of the present invention can also perform mass spectrometry scanning and detection under inert gas (e.g., nitrogen) conditions. In this embodiment, nitrogen is used as the inert gas, and a method for performing mass spectrometry scanning and detection under nitrogen conditions by the device of the present invention is provided, the method being as follows: opening the inert gas supplier 11, adjusting the inert gas inlet channel converter 3 and the ozone inlet channel converter 2, turning off ozone, introducing nitrogen through the auxiliary gas pipeline 802 and / or the sheath gas pipeline 803, and the sample solution in the vaporized state forms sample ions, which enter the mass spectrometry ion source 4, and obtaining the mass analysis results of the sample under nitrogen conditions through mass spectrometry scanning and detection.

[0143] Since unsaturated lipid samples will not undergo cyclization reaction under inert gas conditions, the characteristic peaks of Aldehyde ions with one oxygen atom added and Criegee ions with two oxygen atoms added cannot be generated under inert gas conditions.

[0144] Taking PC 16:0 / 18:1 (n-9) as an example, the mass spectrum under ozone conditions (i.e., electrospray gaseous derivative ionization) obtained using the apparatus of the present invention and the method of Example 3, and the mass spectrum under nitrogen conditions (i.e., electrospray ionization) obtained using the apparatus of the present invention and the method of this example, as shown in FIG. Figure 5 It can be seen that when the device of the present invention is used for mass spectrometry scanning and detection under nitrogen conditions, only the characteristic peak information of the sample parent ion can be obtained, while when the device of the present invention is used for mass spectrometry scanning and detection under ozone conditions, the sample parent ion and the Aldehyde and Criegee characteristic peak information can be obtained simultaneously.

[0145] Example 4: The device of the present invention is used in combination with liquid chromatography

[0146] The device of the present invention can also be used in conjunction with liquid chromatography to separate different lipid samples using liquid chromatography, and then the device of the present invention is used to identify the position of carbon-carbon double bonds in the separated lipid samples, thereby completing the identification of the position of carbon-carbon double bonds in complex lipid samples.

[0147] Exemplarily, the present invention provides a liquid chromatography that can be used in conjunction with the device of the present invention and a method for separating lipid samples using the liquid chromatography:

[0148] The liquid phase device was ACQUITY UPLC I-Class, manufactured by Waters, and the lipids were analyzed using Accucore TMC30 column (2.1mm×150mm, 3μm) was used for separation. The column temperature was 50℃ and the flow rate was 0.35mL / min. Mobile phase A was acetonitrile: water (60:40, v / v), 2mmol / L ammonium formate; mobile phase B was isopropanol: acetonitrile (90:10, v / v), 2mmol / L ammonium formate. Gradient (B%): 0-18min, 30%-80%; 18-23min, 80%-98%; 23-26min, 98%; 26-26.1min, 98%-30%; 26.1-30min, 30%.

[0149] It should be understood that although the present invention takes ACQUITY UPLC I-Class liquid chromatography as an example, the liquid chromatography that can be used in conjunction with the present invention is not limited to this device. As long as it is a liquid chromatography that can separate lipid samples, it can be used in conjunction with the device of the present invention to complete the identification of the position of the carbon-carbon double bond in the lipid sample.

[0150] Example 5: Comparison between the device of the present invention and other electrospray ionization devices

[0151] In this example, the device of the present invention is compared with other electrospray ionization devices, as shown in Table 2.

[0152] As can be seen from Table 2, the device of the present invention introduces ozone in a closed manner in the mass spectrometry ion source region, which reduces the requirement for ozone concentration during the reaction process. Only 10-30 mg / L is required to instantaneously complete the reaction. In addition, the device of the present invention introduces ozone in the mass spectrometry ion source region, and the vacuum degree requirement of the mass spectrometry ion source region is low. There is no need to modify the inside of the mass spectrometer, and it can also avoid the damage to the internal parts of the instrument such as the sealing ring caused by the introduction of ozone into the collision chamber or the ion transmission region. In addition, the device of the present invention can be used in conjunction with liquid chromatography, which expands its scope of application.

[0153] Table 2

[0154]

[0155]

[0156]

[0157] Notes:

[0158] Reference 1: Poad, B. L., et al. (2010). Ozone-induced dissociation on a modified tandem linear ion-trap: observations of different reactivity for isomeric lipids. Journal of the American Society for Mass Spectrometry, 21(12), 1989–1999.

[0159] Reference 2: Kozlowski, R. L., et al. (2015). Combining liquid chromatography with ozone-induced dissociation for the separation and identification of phosphatidylcholine double bond isomers. Analytical and bioanalytical chemistry, 407(17), 5053–5064.

[0160] Reference 3: Poad, B. L., et al. (2017). High-Pressure Ozone-Induced Dissociation for Lipid Structure Elucidation on Fast Chromatographic Timescales. Analytical chemistry, 89(7), 4223–4229.

[0161] Reference 4: Vu, N., et al. (2017). Ozone-induced dissociation on a traveling wave high-resolution mass spectrometer for determination of double-bond position in lipids. Rapid Communications in Mass Spectrometry, 31(17), 1415-1423.

[0162] Document 5: Batarseh, AM, et al. (2018). Discrimination of isobaric and isomeric lipids in complex mixtures by combining ultra-high pressure liquidchromatography with collision and ozone-induced dissociation. International Journal of Mass Spectrometry, 431, 27-36.

[0163] Document 6: Marshall, DL, et al. (2019). Mapping Unsaturation in Human PlasmaLipids by Data-Independent Ozone-Induced Dissociation. Journal of The American Society for Mass Spectrometry, 30(9), 1621-1630.

[0164] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. An electrospray ionization device based on ozone gas-derived ionization, characterized in that: The device comprises: a device for supplying ozone to a mass spectrometry ion source and the mass spectrometry ion source, wherein the device for supplying ozone to the mass spectrometry ion source supplies the generated ozone to the mass spectrometry ion source.

2. The electrospray ionization device based on ozone gaseous derivative ionization according to claim 1, characterized in that: The mass spectrometer ion source comprises: an ion source and an ion inlet device.

3. The electrospray ionization device based on ozone gaseous derivative ionization according to claim 2, characterized in that: The ion source includes an electrospray needle and a high-voltage power supply. The outlet end of the electrospray needle is located in front of the ion inlet device of the mass spectrometer ion source. The high-voltage power supply is connected to the electrospray needle and can trigger the sample solution in the electrospray needle to form ions. The formed ions enter the mass spectrometer ion source through the ion inlet device.

4. The electrospray ionization device based on ozone gaseous derivative ionization according to claim 3, characterized in that: The ion source further comprises an auxiliary gas pipeline and / or a sheath gas pipeline, wherein the auxiliary gas pipeline and / or the sheath gas pipeline are used to supply ozone to the mass spectrometry ion source; Preferably, the device for supplying ozone to the mass spectrometer ion source is connected to the auxiliary gas pipeline, or is connected to the sheath gas pipeline, or is connected to both the auxiliary gas pipeline and the sheath gas pipeline.

5. The electrospray ionization device based on ozone gaseous derivatization ionization according to any one of claims 1 to 4, characterized in that: The device for supplying ozone to the mass spectrometer ion source comprises: an oxygen supplier and an ozone generator, oxygen is supplied to the ozone generator through the oxygen supplier to generate ozone, and the device for supplying ozone to the mass spectrometer ion source supplies the generated ozone to the ion source.

6. The electrospray ionization device based on ozone gaseous derivative ionization according to claim 5, characterized in that: The device for supplying ozone to the mass spectrometer ion source further comprises one, more or all components selected from the following: a gas pressure display, an ozone analyzer and an exhaust gas destroyer; Preferably, the gas pressure display is connected to the ozone generator to detect the flow rate of ozone generated in the ozone generator; The ozone analyzer is used to analyze the residual ozone in the device for supplying ozone to the mass spectrometer ion source; the tail gas destroyer removes the excess ozone in the device for supplying ozone to the mass spectrometer ion source.

7. The electrospray ionization device based on ozone gaseous derivative ionization according to claim 5 or 6, characterized in that: The device for supplying ozone to the mass spectrometer ion source further comprises: an inert gas supplier, an ozone inlet channel converter and a nitrogen inlet channel converter, wherein the ozone inlet channel converter and the inert gas inlet channel converter are used to adjust the gas in the device for supplying ozone to the mass spectrometer ion source to ozone or inert gas; Preferably, the inert gas includes: one of nitrogen, argon, helium and air, or a mixture of any two, three or all of them, or a mixture of any one, two, three or all of them and ozone.

8. The electrospray ionization device based on ozone gaseous derivatization ionization according to any one of claims 1 to 7, characterized in that: The electrospray ionization device based on ozone gaseous phase derivative ionization further comprises a mass spectrometer, and the electrospray ionization device based on ozone gaseous phase derivative ionization is connected to the mass spectrometer; Preferably, the inlet end of the mass spectrometer is connected to the outlet end of a mass spectrometer ion source in an electrospray ionization device based on ozone gaseous derivative ionization, so as to perform mass spectrometry analysis on ions in the mass spectrometer ion source; More preferably, the mass spectrometer includes a triple quadrupole mass spectrometer, an electrostatic field orbital trap mass spectrometer, a quadrupole tandem orbital trap mass spectrometer, a time-of-flight mass spectrometer, a quadrupole time-of-flight mass spectrometer, an ion trap mass spectrometer, a quadrupole linear ion trap mass spectrometer, and a Fourier transform ion cyclotron resonance mass spectrometer.

9. The electrospray ionization device based on ozone gaseous derivatization according to any one of claims 1 to 8, characterized in that: The electrospray ionization device based on ozone gaseous phase derivative ionization further comprises: a liquid chromatography analysis device, wherein the electrospray ionization device based on ozone gaseous phase derivative ionization is connected to the liquid chromatography instrument; Preferably, the outlet end of the liquid chromatography instrument is connected to the inlet end of the electrospray needle of the mass spectrometry ion source in the electrospray ionization device based on ozone gas phase derivative ionization; More preferably, the liquid chromatography includes: high performance liquid chromatography, ultra high performance liquid chromatography, liquid chromatograph, microliter liquid chromatography, and ion chromatograph.

10. A method for identifying the number and position of carbon-carbon double bonds in unsaturated organic matter using the electrospray ionization device based on ozone gaseous derivative ionization according to any one of claims 1 to 9, characterized in that: The method comprises: (1) using the electrospray ionization device based on ozone gaseous derivative ionization, supplying ozone to a mass spectrometry ion source, so that ozone and unsaturated organic matter undergo an epoxide addition reaction in the mass spectrometry ion source to form charged characteristic ions; (2) using a mass spectrometer to detect the characteristic ions formed in (1) and identify the number and position of carbon-carbon double bonds in the unsaturated organic matter; Preferably, the unsaturated organic matter contains at least one carbon-carbon double bond; more preferably, the unsaturated organic matter includes a single organic matter, or a mixture or combination of two or more organic matters; more preferably, the unsaturated organic matter is an unsaturated lipid; more preferably, the unsaturated lipid includes: one, more or all of fatty acids, glycerolipids, phospholipids, sphingolipids, glycolipids, glycerophospholipids, sterol esters, isopentenol lipids, and polyketides; more preferably, the unsaturated lipid includes: fatty acids, glycerolipids, phospholipids, sphingolipids, glycolipids, glycerophospholipids, sterol esters, One, more than one or all of isopentanol lipids and polyketides; more preferably, the unsaturated lipids include: fatty acids, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, lysophosphatidylserine, sphingomyelin, ceramide, cholesterol ester, cardiolipin, monogalactosyl diacylglycerol, digalactosyl diacylglycerol, thioiso-rhamnosyl diglyceride, monoglyceride, diglyceride, triglyceride.

11. The method according to claim 10, characterized in that The method further comprises one, more or all of the following conditions: (A) In step (1), the method of supplying ozone to the mass spectrometer ion source by using the electrospray ionization device based on ozone gas phase derivative ionization comprises: turning on the ozone generator, and after the ozone analyzer shows that ozone has been generated, adjusting the inert gas inlet channel converter and the ozone inlet channel converter, turning off the inert gas and allowing the ozone to enter the mass spectrometer ion source; (B) Step (1) further comprises the step of detecting and calibrating ozone. Preferably, the oxygen supply device and the ozone analyzer are turned on, and the ozone analyzer is used to detect and calibrate ozone. (C) the concentration of the unsaturated organic matter in step (1) is 0.1 pmol / L-1 mol / L, preferably 1 μmol / L-10 mmol / L; (D) in step (1), adjusting the concentration and flow rate of ozone by controlling the ozone generator; (E) in step (1), the concentration of ozone is less than 100 mg / L, preferably less than 80 mg / L, less than 70 mg / L, less than 60 mg / L or less than 50 mg / L, more preferably, the concentration of ozone is 10 to 30 mg / L; (F) In step (1), the flow rate of ozone is 0.1 L / min or more, preferably 0.2 L / min or more, 0.3 L / min or more, 0.4 L / min or more, or 0.5 L / min or more; more preferably, the flow rate of ozone is 1 to 10 L / min; (G) In step (2), the method for obtaining the number and position information of carbon-carbon double bonds of unsaturated organic matter based on the characteristic ions formed in step (1) comprises: the characteristic ions formed in step (1) appear in pairs in the mass spectrum, and the number of characteristic peaks of the characteristic ions appearing in pairs is the number of carbon-carbon double bonds; and the position of the carbon-carbon double bonds is determined based on the difference between the mass information of the characteristic peaks of the characteristic ions appearing in pairs and the mass information of the parent ion.

12. Use of the electrospray ionization device based on ozone gaseous derivative ionization according to any one of claims 1 to 9 for identifying the number and position of carbon-carbon double bonds in unsaturated organic matter; Preferably, the unsaturated organic matter contains at least one carbon-carbon double bond; more preferably, the unsaturated organic matter includes a single organic matter, or a mixture or composition of two or more organic matters; more preferably, the unsaturated organic matter is an unsaturated lipid; more preferably, the unsaturated lipid includes: One, more than one or all of fatty acids, glycerolipids, phospholipids, sphingolipids, glycolipids, glycerophospholipids, sterol esters, isopentenol lipids, and polyketides; more preferably, the unsaturated lipids include: fatty acids, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, lysophosphatidylserine, sphingomyelin, ceramide, cholesterol ester, cardiolipin, monogalactosyl diacylglycerol, digalactosyl diacylglycerol, thioiso-rhamnosyl diglyceride, monoglyceride, diglyceride, triglyceride; Preferably, the method for identifying the number and position of carbon-carbon double bonds in unsaturated organic matter is as described in claim 10 or 11.

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