Electrospray ionization device based on ozone gaseous derivative ionization

Abstract

The utility model provides an electrospray ionization device based on ozone gaseous derivative ionization, which comprises a device for supplying ozone to a mass spectrum ion source and the mass spectrum ion source, and the mass spectrum ion source comprises an ion source and an ion inlet device, the apparatus for supplying ozone to a mass spectrum ion source includes an oxygen supply device and an ozone generator, oxygen is supplied into the ozone generator through the oxygen supply device, ozone is generated, and the apparatus for supplying ozone to a mass spectrum ion source supplies the generated ozone to an ion source.

Description

TECHNICAL FIELD

[0001] The utility model belongs to the field of mass spectrum analysis, more particularly, the utility model relates to a kind of based on ozone gaseous derivatization ionization electric spray ionization device. BACKGROUND

[0002] Mass spectrum is an analytical tool for measuring ion mass-to-charge ratio. Mass spectrum has high sensitivity, good selectivity and strong universality. Combined with the high-efficiency 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 spectrum ion source is an important front end for ionization of organic matter into charged ions, thereby entering mass spectrum analysis. Electric spray ionization is a standard ionization method for mass spectrum ion source, which is suitable for analysis of more than 80% of organic matter. However, there are still great challenges in identifying the position of carbon-carbon double bond of organic matter by using mass spectrum. The carbon-carbon double bond in organic matter has high bond energy, which cannot be directly cleaved and produce characteristic fragment ions in the collision-induced dissociation fragmentation mode of conventional commercial mass spectrometers, so that the position of the double bond cannot be determined.

[0004] Literature research shows that ozone-induced dissociation is combined with mass spectrum, and ozone is introduced into the ion transmission or collision area of mass spectrum. Since the ion transmission and collision fragmentation area are in vacuum, especially the collision area has a vacuum as high as 10 -4 mbar, under high vacuum conditions, the activity of ozone-induced dissociation reaction is very weak, and the reaction rate is low. At the same time, the components required for ozone introduction and flow control need to be ozone-resistant. Even after ozone-resistant modification, since the time of ions in the ion transmission or ion collision area of mass spectrum needs to be extended, it cannot be used in combination with conventional analysis, and is only suitable for analysis of standard substances or specific compounds. At the same time, it is difficult to completely avoid the damage of high-concentration ozone to the sealing ring of the internal components of mass spectrum, resulting in poor durability and actual application value, and the experiment cannot be reproduced.

[0005] In addition, in some other literatures, ozone-induced dissociation is combined with mass spectrum by using atmospheric pressure open ion source, such as by using nanoliter electric spray open ion source to introduce ozone reaction, and the products after reaction enter mass spectrum analysis. Organic matter analysis based on mass spectrum is usually carried out on conventional liquid chromatography-mass spectrometry, and the flow rate is generally between 0.3 ml / min and 1.0 ml / min. However, the flow rate suitable for nanoliter ion source is generally between 100 nl / min and 300 nl / min, which is not suitable for conventional liquid chromatography-mass spectrometry for organic matter identification.

[0006] Therefore, there is an urgent need in the field to develop a device suitable for conventional liquid chromatography-mass spectrometry, which can solve the problems of increasing ozone concentration in high vacuum, instrument damage caused by high-concentration ozone, etc., and realize the identification of the position of carbon-carbon double bond of organic matter. UTILITY MODEL CONTENTS

[0007] The utility model discloses a kind of ozone gaseous derivative ionization-based electrospray ionization device.

[0008] In the first aspect of the utility model, provide a kind of ozone gaseous derivative ionization-based electrospray ionization device, the device includes: the device that ozone is supplied to mass spectrometry ion source and mass spectrometry ion source, the mass spectrometry ion source includes: ion source and ion inlet device, the device that ozone is supplied to mass spectrometry ion source includes: oxygen supply and ozone generator, oxygen is supplied to ozone generator by oxygen supply, and ozone is generated, the device that ozone is supplied to mass spectrometry ion source supplies generated ozone to ion source.

[0009] 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 to initiate the formation of ions from the sample solution in the electrospray needle, and the formed ions enter the mass spectrometry ion source through the ion inlet device.

[0010] In one or more embodiments, the ion source further includes an auxiliary gas pipeline and / or a sheath gas pipeline, and the auxiliary gas pipeline and / or the sheath gas pipeline are used to supply ozone to the mass spectrometry ion source.

[0011] In one or more embodiments, the device for supplying ozone to the mass spectrometry ion source can be connected to the auxiliary gas pipeline, or connected to the sheath gas pipeline, or connected to both the auxiliary gas pipeline and the sheath gas pipeline.

[0012] In one or more embodiments, the device for supplying ozone to the mass spectrometry ion source further includes a gas pressure display connected to the ozone generator for detecting the flow rate of the generated ozone in the ozone generator.

[0013] In one or more embodiments, the device for supplying ozone to the mass spectrometry ion source further includes an ozone analyzer for analyzing the residual ozone in the device for supplying ozone to the mass spectrometry ion source.

[0014] In one or more embodiments, the device for supplying ozone to the mass spectrometry ion source further includes an exhaust gas destroyer for removing excess ozone in the device for supplying ozone to the mass spectrometry ion source.

[0015] In one or more embodiments, the device for supplying ozone to the mass spectrometry ion source further includes an inert gas supply, an ozone gas inlet channel converter, and a nitrogen gas inlet channel converter, and the ozone gas inlet channel converter and the inert gas gas inlet channel converter are used to adjust the gas in the device for supplying ozone to the mass spectrometry ion source to be ozone or inert gas.

[0016] In one or more embodiments, the electrospray ionization device further comprises a mass spectrometry instrument, the inlet end of the mass spectrometry instrument being connected to the outlet end of the mass spectrometry ion source for mass spectrometry analysis of ions in the mass spectrometry ion source.

[0017] In one or more embodiments, the electrospray ionization device further comprises a liquid chromatography analysis device, the liquid chromatography analysis device being connected to the mass spectrometry ion source, the outlet end of the liquid chromatography analysis device being connected to the inlet end of the electrospray needle of the mass spectrometry ion source.

[0018] Other aspects of the present application will be apparent to those skilled in the art from consideration of the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The ozone generation device and the connection system block diagram of the mass spectrometry instrument are provided.

[0020] Figure 2 The internal structure diagram of the electrospray ion source used in the present application.

[0021] Figure 3, ozone gaseous 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 ) primary high-resolution mass spectrum.

[0022] 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 diagram of carbon-carbon ion double bond characteristic ion fragments using ozone as atomizing gas electrospray gaseous derivative ionization.

[0023] Figure 5Figure 1 is a comparison chart of the first mass spectrum of PC 16:0 / 18:1 (n-9) analyzed by electrospray gaseous derivatization ionization (ozone condition, ozone as atomizing gas) and electrospray ionization (nitrogen condition, nitrogen as atomizing gas).

[0024] Reference signs are as follows:

[0025] 1. Ozone generator

[0026] 2. Ozone gas inlet channel converter

[0027] 3. Inert gas inlet channel converter

[0028] 4. Mass spectrometry ion source

[0029] 5. Gas pressure display

[0030] 6. Ozone analyzer

[0031] 7. Tail gas destroyer

[0032] 8. Ion source; 801, electrospray needle; 802, auxiliary gas pipeline; 803, sheath gas pipeline; 804, high-voltage power supply

[0033] 9. Ion inlet device

[0034] 10. Oxygen supply

[0035] 11. Inert gas supply DETAILED DESCRIPTION

[0036] The utility model discloses a kind of electrospray ionization devices based on ozone gaseous derivatization ionization, which realizes electrospray gaseous derivatization ionization in mass spectrometry ion source area, so as to avoid the damage to internal parts of instrument such as sealing ring caused by introducing ozone into collision chamber or ion transmission area. The device of the utility model can effectively identify carbon-carbon double bond in unsaturated lipid sample. The device of the utility model can also be used with conventional liquid chromatography mass spectrometry system, and expand the identification depth of mass spectrometry on organic matter.

[0037] Electrospray ionization device based on ozone gaseous derivatization ionization

[0038] In the first aspect of the utility model, an electrospray ionization device based on ozone gaseous derivatization ionization is provided, which comprises: a device for supplying ozone to a mass spectrometry ion source and the mass spectrometry ion source, wherein the mass spectrometry ion source comprises: an ion source and an ion inlet device, and the device for supplying ozone to the mass spectrometry ion source comprises: an oxygen supply and an ozone generator, oxygen is supplied to the ozone generator through the oxygen supply to generate ozone, and the device for supplying ozone to the mass spectrometry ion source supplies the generated ozone to the ion source.

[0039] An ion source is a device for ionizing a sample for mass spectrometric analysis, which can ionize the sample into gas state after ionization, so as to be detected and analyzed in a mass spectrometer.

[0040] In one or more embodiments, when ozone is supplied to the mass spectrometric ion source, the sample is ionized at the ion source to form sample ions, and then enters the mass spectrometric ion source from the ion inlet device.

[0041] In one or more embodiments, the ion source comprises 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 spectrometric ion source, and the high-voltage power supply is connected to the electrospray needle to initiate ionization of the sample solution in the electrospray needle, and the formed ions enter the mass spectrometric ion source through the ion inlet device.

[0042] In one or more embodiments, the distance between the outlet end of the electrospray needle and the ion inlet device of the mass spectrometric ion source is 10-100 mm, preferably 20-50 mm.

[0043] 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.

[0044] In one or more embodiments, the sample is a sample containing unsaturated organic matter.

[0045] In one or more embodiments, the unsaturated organic matter contains at least one carbon-carbon double bond.

[0046] In one or more embodiments, the unsaturated organic matter includes a single organic matter, or a mixture or composition of two or more organic matters.

[0047] In one or more embodiments, the unsaturated organic matter is an unsaturated lipid.

[0048] In one or more embodiments, the unsaturated lipid includes one, more or all of fatty acid, glycerolipid, phospholipid, sphingolipid, glycolipid, glycerophospholipid, sterol ester, prenol lipid, and polyketide.

[0049] 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), cholesteryl ester (CE), cardiolipin (CL), monogalactosyldiacylglyceride (MGDG), digalactosyldiacylglyceride (DGDG), sulphoquinovosyl diacylglyceride (SQDG), monoglyceride (MG), diglyceride (DG), triglyceride (TG).

[0050] In one or more embodiments, as shown in Figure 2 The ion source includes an electrospray needle, an auxiliary gas line, a sheath gas line, 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 is sprayed and ionized into sample ions under the action of high voltage electricity.

[0051] In one or more embodiments, the ion source further includes an auxiliary gas line and / or a sheath gas line for supplying ozone to the mass spectrometry ion source.

[0052] In one or more embodiments, the device for supplying ozone to the mass spectrometry ion source can be connected to the auxiliary gas line, or connected to the sheath gas line, or connected to both the auxiliary gas line and the sheath gas line. Therefore, the ozone can be supplied to the mass spectrometry ion source through the auxiliary gas line and / or the sheath gas line.

[0053] 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.

[0054] In one or more embodiments, the device for supplying ozone to the mass spectrometry ion source further includes a gas pressure display connected to the ozone generator for detecting the flow rate of the ozone generated in the ozone generator.

[0055] In one or more embodiments, the device for supplying ozone to the mass spectrometry ion source further includes an ozone analyzer for analyzing the residual ozone in the device for supplying ozone to the mass spectrometry ion source.

[0056] In one or more embodiments, the device for supplying ozone to a mass spectrometry ion source further comprises an exhaust gas destroyer that eliminates excess ozone in the device for supplying ozone to a mass spectrometry ion source.

[0057] In one or more embodiments, the device for supplying ozone to a mass spectrometry ion source further comprises: an inert gas supplier, an ozone inlet channel switcher, and a nitrogen inlet channel switcher, the ozone inlet channel switcher and the inert gas inlet channel switcher being used to adjust the gas in the device for supplying ozone to a mass spectrometry ion source to be ozone or an inert gas.

[0058] In one or more embodiments, the inert gas comprises: one of nitrogen, argon, helium, and air, or a mixture of any two, three, or all of the gases, or a mixture of any one, two, three, or all of the gases with ozone.

[0059] In one or more specific embodiments, the device for supplying ozone to a mass spectrometry ion source, as shown in Figure 1 comprises: an inert gas supplier, an oxygen supplier, an ozone generator, an ozone inlet channel switcher, a nitrogen inlet channel switcher, a gas pressure display, an ozone analyzer, and an exhaust gas destroyer.

[0060] In one or more embodiments, the device for supplying ozone to a mass spectrometry ion source comprises two inlet channels, the inert gas supplier and the inert gas inlet channel switcher being connected in sequence to form an inert gas supply channel. The oxygen supplier, the ozone generator, the ozone inlet channel switcher, the gas pressure display, the ozone analyzer, and the exhaust gas destroyer are connected in sequence to form an ozone supply channel. The inert gas inlet channel switcher is also connected to the ozone inlet channel switcher, and by adjusting the inert gas inlet channel switcher and the ozone inlet channel switcher, ozone or inert gas can be supplied to the mass spectrometry ion source through the auxiliary gas pipeline and / or the sheath gas pipeline.

[0061] When the addition reaction of epoxidation does not require ozone, the inert gas inlet channel switcher is turned on, and the inert gas in the inert gas supplier enters the mass spectrometry ion source. When the addition reaction of epoxidation requires ozone, the ozone generator is turned on, and the oxygen in the oxygen supplier generates ozone in the ozone generator, the flow rate of the ozone can be monitored using the gas pressure display. By adjusting the inert gas inlet channel switcher and the ozone inlet channel switcher, the inert gas is turned off and the ozone enters the mass spectrometry ion source. The ozone remaining in the ozone supply channel is analyzed using the ozone analyzer, and the excess ozone is eliminated using the exhaust gas destroyer.

[0062] In the reaction, the sample solution is introduced through an electrospray needle, and a high voltage is applied to the ion source by a high-voltage power supply, and the sample solution is atomized into small surface-charged droplets through the needle. The device for supplying ozone to the mass spectrometry ion source introduces ozone into 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 gaseous sample solution to form sample ions. The gaseous sample ions enter the mass spectrometer for detection under the action of vacuum difference and electric potential traction from the ion inlet device.

[0063] In one or more embodiments, the electrospray ionization device further comprises a mass spectrometer instrument connected to the outlet end of the mass spectrometry ion source for mass spectrometry analysis of the ions in the mass spectrometry ion source. When ozone is supplied to the mass spectrometry ion source, sample ions are ionized at the mass spectrometry ion source to form sample ions, and the mass of the sample ions is detected by the mass spectrometer instrument connected to the mass spectrometry ion source, so that the mass spectrometry information of the sample ions can be obtained.

[0064] In one or more embodiments, the mass spectrometer instrument 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, etc.

[0065] In one or more embodiments, the mass spectrometry instrument comprises a triple quadrupole mass spectrometer (e.g., QQQ, Triple Quad, TSQ, TSQ Plus, TQ, Stellar), an electrostatic field orbitrap mass spectrometer (e.g., Exactive, QExactive, Exactive Plus, Exactive Plus Orbitrap, Exactive HF, Orbitrap Exploris 120, Orbitrap Exploris 240, Orbitrap Exploris 480), a quadrupole rod tandem orbitrap mass spectrometer (e.g., QExactive, Exactive Plus, Exactive HF, Orbitrap Exploris 120, Orbitrap Exploris 240, 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), an ion trap mass spectrometer (e.g., Ion Trap, LTQ, LCQ), a quadrupole linear ion trap mass spectrometer (e.g., Qtrap), a Fourier transform ion cyclotron resonance mass spectrometer (e.g., FT-ICR).

[0066] In one or more embodiments, the electrospray ionization device further comprises a liquid chromatography analysis device, the liquid chromatography analysis device is connected with the mass spectrometry ion source, and an outlet end of the liquid chromatography analysis device is connected with an inlet end of an electrospray needle of the mass spectrometry ion source.

[0067] In one or more embodiments, the liquid chromatography instrument separates unsaturated organic substances, and then detects the number and position of carbon-carbon double bonds in the separated unsaturated organic substances by using an electrospray ionization device based on ozone gaseous derivative ionization and a mass spectrometry instrument connected therewith.

[0068] In one or more embodiments, the liquid chromatography comprises a high-performance liquid chromatograph, an ultra-high-performance liquid chromatograph, a liquid chromatograph, a microliter liquid chromatograph, and an ion chromatograph.

[0069] In one or more embodiments, the liquid chromatography comprises: a high performance liquid chromatograph (HPLC), an ultra-high performance liquid chromatograph (UHPLC), a liquid chromatograph (LC), a micro-liter liquid chromatograph (Micro LC), an ion chromatograph (IC).

[0070] The method for identifying a sample containing unsaturated organic matter by using the electrospray ionization device based on ozone gaseous derivative ionization has the advantages of simple structure, convenient operation, high efficiency, high accuracy, and the like.

[0071] (1) The ozone is supplied to the mass spectrometry ion source by using the electrospray ionization device based on ozone gaseous derivative ionization, so that the ozone and the unsaturated organic matter are subjected to an epoxide addition reaction in the mass spectrometry ion source to form charged characteristic ions.

[0072] (2) The characteristic ions formed in (1) are detected by using a mass spectrometer, and the number and position information of carbon-carbon double bonds of the unsaturated organic matter are obtained.

[0073] In the utility model, the "unsaturated organic matter" refers to an organic matter containing at least one (for example, two or more, three or more or more) carbon-carbon double bond. In this paper, "at least", "above" includes the number. The unsaturated organic matter described in the utility model can be a single organic matter, or a mixture or composition of two, three, four or more organic matters. The unsaturated organic matter of the utility model can be a chemically synthesized unsaturated organic matter, or an unsaturated organic matter isolated and extracted from tissues (for example, brain tissue), body fluids (for example, blood, plasma, urine, sweat and the like), cells (for example, cell membrane, cell content) and the like of animals, plants or microorganisms.

[0074] In some embodiments, the unsaturated organic compound can be an unsaturated lipid, such as, but not limited to, a fatty acid, a glycerolipid, a phospholipid, a sphingolipid, a glycolipid, a glycerophospholipid, a sterol ester, a prenol lipid, a polyketide. In some embodiments, the unsaturated organic compound includes, but is not limited to, a fatty acid (FA), a phosphatidic acid (PA), a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylglycerol (PG), a phosphatidylinositol (PI), a phosphatidylserine (PS), a lysophosphatidic acid (LPA), a lysophosphatidylcholine (LPC), a lysophosphatidylethanolamine (LPE), a lysophosphatidylglycerol (LPG), a lysophosphatidylinositol (LPI), a lysophosphatidylserine (LPS), a sphingomyelin (SM), a ceramide (Cer), a cholesteryl ester (CE), a cardiolipin (CL), a monogalactosyldiacylglyceride (MGDG), a digalactosyldiacylglyceride (DGDG), a sulfogalactosyldiacylglyceride (SQDG), a monoglyceride (MG), a diglyceride (DG), a triglyceride (TG). In some specific embodiments, the unsaturated organic compound is an unsaturated phosphatidylcholine (PC) or an unsaturated phosphatidylethanolamine (PE).

[0075] In step (1), the method of supplying ozone to the ion source of the mass spectrometer using the ozone-based gaseous derivatization ionization device includes: turning on the ozone generator, adjusting the inert gas inlet channel switcher and the ozone inlet channel switcher after the ozone analyzer shows that ozone has been generated, and closing the inert gas and allowing ozone to enter the ion source of the mass spectrometer.

[0076] Step (1) can further include a step of detecting and zeroing the ozone, for example: turning on the oxygen supply and the ozone analyzer, and detecting and zeroing the ozone using the ozone analyzer.

[0077] In step (1), the concentration of the unsaturated organic compound is only required to be able to react with ozone to form an epoxide addition reaction. The "epoxide addition reaction" generally refers to the formation of a five-membered ring structure intermediate product under ozone conditions:

[0078]

[0079] The intermediate product is unstable and quickly breaks down to form an ion Aldehyde with one oxygen atom added and an ion Criegee with two oxygen atoms added, and the mass difference between the two ions is 16 (deviation 0.1 Da). The ion Aldehyde and the ion Criegee are the characteristic ions in step (1):

[0080]

[0081] It should be understood that the epoxide addition reaction needs to be completed under the condition of ozone, and the epoxide addition reaction will not occur under the condition of inert gas (for example, nitrogen). Therefore, under the condition of inert gas, the characteristic peak of the characteristic ion will not be observed when detected by mass spectrometry, and only the mass spectrometry peak of the parent ion of the unsaturated organic matter can be observed.

[0082] In specific embodiments, the concentration of the unsaturated organic matter can be 0.1 pmol / L-1 mol / L, preferably 1 μmol / L-10 mmol / L, for example, 10 mmol / L.

[0083] In some embodiments, by controlling the ozone generator, the concentration and flow rate of ozone can be adjusted.

[0084] In specific embodiments, in step (1), the concentration of ozone is less than 100 mg / L, for example, 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 can be 10-30 mg / L.

[0085] In specific embodiments, in step (1), the flow rate of ozone is 0.1 liters / minute or more, for example, 0.2 liters / minute or more, 0.3 liters / minute or more, 0.4 liters / minute or more, or 0.5 liters / minute or more. More specifically, the flow rate of ozone can be 1-10 liters / minute.

[0086] In step (2), according to the characteristic ions formed in step (1), the method for obtaining the number and position information of the carbon-carbon double bond of the unsaturated organic matter 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. 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, the position of the carbon-carbon double bond is determined.

[0087] More specifically, the method for determining the number of carbon-carbon double bonds is: if the mass difference of the characteristic ions appearing in pairs in step (1) is 16±0.1 Da, it is considered that it is a pair of characteristic ions, and the number of the pair of characteristic ions is the number of carbon-carbon double bonds.

[0088] More specifically, the method for determining the position of the carbon-carbon double bond is: 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, and combining Table 1, the position of the carbon-carbon double bond is determined.

[0089] When the ozone gaseous derivative ionization-based electrospray ionization device is connected with a liquid chromatograph, the liquid chromatograph can be used to separate unsaturated organic matters, so that a single unsaturated organic matter is obtained.

[0090] Compared with the prior art, the ozone gaseous derivative ionization-based electrospray ionization device has the following beneficial effects:

[0091] (1) The ozone is introduced into the electrospray ion source as an online reaction gas, reacts with sample ions in a gaseous state, and is scanned by a high-resolution mass analyzer to obtain characteristic information related to the position of the carbon-carbon double bond.

[0092] (2) The ozone gaseous derivative ionization-based electrospray ionization device 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, so that the position of the double bond in the compound is effectively located, and the device has strong universality.

[0093] The ozone gaseous derivative ionization-based electrospray ionization device will be further described in combination with specific embodiments. It should be understood that these embodiments are only used to illustrate the ozone gaseous derivative ionization-based electrospray ionization device and are not used to limit the scope of the ozone gaseous derivative ionization-based electrospray ionization device. The experimental methods not specified in the following embodiments are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

[0094] Example 1, an ozone gaseous derivative ionization-based electrospray ionization device

[0095] The ozone gaseous derivative ionization-based electrospray ionization device provided in the embodiment includes a device for supplying ozone to a mass spectrometry ion source and the mass spectrometry ion source 4. The mass spectrometry ion source 4 includes an ion source 8 and an ion inlet device 9. Figure 2 As shown in the figure, 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 spectrometry ion source can be connected with the auxiliary gas pipeline 802, or connected with the sheath gas pipeline 803, or connected with both the auxiliary gas pipeline 802 and the sheath gas pipeline 803.

[0096] The device for supplying ozone to the mass spectrometry ion source, as shown in the figure, Figure 1As shown, the device comprises: an inert gas supply 11, an oxygen supply 10, an ozone generator 1, an ozone inlet channel switcher 2, an inert gas inlet channel switcher 3, a gas pressure display 5, an ozone analyzer 6 and a tail gas destroyer 7. The device comprises two inlet channels, the inert gas supply 11, the inert gas inlet channel switcher 3 are connected in sequence to form an inert gas supply channel. The oxygen supply 10, the ozone generator 1, the ozone inlet channel switcher 2, the gas pressure display 5, the ozone analyzer 6 and the tail gas destroyer 7 are connected in sequence to form an ozone supply channel. The inert gas inlet channel switcher 3 is also connected with the ozone inlet channel switcher 2, and by adjusting the inert gas inlet channel switcher 3 and the ozone inlet channel switcher 2, the ozone can be supplied to the mass spectrometry ion source 4 through the auxiliary gas pipeline 802 and / or the sheath gas pipeline 803.

[0097] When the epoxidation addition reaction does not need to use ozone, the inert gas inlet channel switcher 3 is opened, and the inert gas in the inert gas supply 11 enters the mass spectrometry ion source 4.

[0098] When the epoxidation addition reaction needs to use ozone, the ozone generator 1 is opened, and the oxygen in the oxygen supply 10 generates ozone in the ozone generator 1, and the flow of the ozone can be monitored by using the gas pressure display 5. By adjusting the inert gas inlet channel switcher 3 and the ozone inlet channel switcher 2, the inert gas is closed and the ozone enters the mass spectrometry ion source 4. The residual ozone in the ozone supply channel is analyzed by using the ozone analyzer 6, and the excess ozone is removed by using the tail gas destroyer 7.

[0099] During the reaction, the sample solution is introduced through the electrospray needle 801, and a high voltage is added at the ion source by using the high-voltage power supply 804, and the sample solution is atomized into small surface-charged droplets by the needle. The device for supplying ozone to the mass spectrometry ion source introduces ozone near 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 gaseous sample solution to form sample ions. The gaseous sample ions enter the ion inlet device 9 under the action of vacuum difference and electric potential traction, and the full scan and secondary fragment spectrum of the sample and ozone reaction characteristic ions are detected by mass spectrometry, and the carbon-carbon double bond position information can be obtained by mass number analysis.

[0100] The mass spectrometer used in the utility model 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 and the like.

[0101] Embodiment 2: The device is used for identifying the carbon-carbon double bond position of an unsaturated lipid sample.

[0102] In this embodiment, five unsaturated lipid samples, 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 used as examples, and the device of Example 2 was used to identify the position of carbon-carbon double bonds in the unsaturated lipid samples. The identification method comprises the following steps:

[0103] a) Turn on the oxygen supply 10 and the ozone analyzer 6, and use the ozone analyzer 6 to detect and calibrate the zero of the ozone;

[0104] b) Turn on the ozone generator 1, and after the ozone analyzer 6 shows that ozone has been generated, adjust the inert gas inlet channel switch 3 and the ozone inlet channel switch 2, close the inert gas and make the ozone enter the mass spectrometry ion source 4. The flow rate of ozone entering the mass spectrometry ion source 4 is controlled by the gas pressure display 5. Excess ozone is analyzed and detected by the ozone analyzer 6, and is removed by the tail gas destroyer 7;

[0105] c) Use a chloroform / methanol (1:1, v / v) solution to prepare five unsaturated lipid sample solutions of 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) at a concentration of 10 mmol / L, respectively;

[0106] d) Introduce the unsaturated lipid sample solutions prepared in c) into the ion source 8, respectively, and the sample solutions are sprayed from the electrospray needle 801 to form charged droplets;

[0107] e) The device for supplying ozone to the mass spectrometry ion source introduces ozone through the auxiliary gas pipeline 802 and / or the sheath gas pipeline 803, and the flow rate of the ozone is 1-10 liters per minute, and the ozone concentration is 10-120 mg / L. The introduced ozone reacts with the gaseous sample solution to form sample ions, which enter the mass spectrometry ion source 4, and the mass spectrum of the unsaturated lipid sample under ozone conditions is obtained by mass spectrometry scanning and detection;

[0108] f) The position and number of carbon-carbon double bonds in the unsaturated lipid sample are identified by analyzing the mass spectrum of the unsaturated lipid sample under ozone conditions obtained in e), and the identification method is as follows:

[0109] Since ozone as atomization gas reacts with carbon-carbon double bond to form a five-membered ring structure intermediate, the intermediate is unstable and quickly breaks to form ion Aldehyde and ion Criegee with one and two oxygen atoms, respectively, and the charged Aldehyde and Criegee ions are detected by mass spectrometry, and the mass difference between the two ions is 16 (deviation 0.1 Da).

[0110]

[0111] Therefore, by using the characteristic peak that the charged Aldehyde and Criegee ions differ by 16 Da, the number of carbon-carbon double bonds can be determined. The number of characteristic peaks with a mass difference of 16 Da appearing in pairs in the mass spectrum is the number of carbon-carbon double bonds. Further, by comparing the mass-to-charge ratio information of the characteristic peaks with a mass difference of 16 Da appearing in pairs in the mass spectrum with the mass-to-charge ratio information of the parent ion, the position of the carbon-carbon double bond can be determined according to Table 1.

[0112] Table 1

[0113]

[0114] The mass spectrum results 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) for carbon-carbon double bond position identification are shown in Figures 3A-3E The structure 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) and the structure derivation diagram of carbon-carbon double bond characteristic ion fragments after ozone reaction are shown in Figures 4A-4E

[0115] The results show that the device of the utility model can accurately identify the position of the carbon-carbon double bond in the unsaturated lipid sample, and the device of the utility model can avoid damage to the internal parts such as the sealing ring of the instrument caused by introducing ozone into the collision chamber or the ion transmission region without changing the mass spectrometry collision time, and reduce the requirement for ozone concentration during the reaction process.

[0116] Example 3, application of the device of the utility model under inert gas conditions

[0117] ​The device of the utility model can also carry out mass spectrum scanning and detection under the condition of inert gas (for example, nitrogen). In this embodiment, nitrogen is taken as the inert gas, and a method for carrying out mass spectrum scanning and detection under the condition of nitrogen by the device of the utility model is provided, and the method is as follows: opening the inert gas feeder 11, adjusting the inert gas inlet channel converter 3 and the ozone inlet channel converter 2, closing the ozone, introducing nitrogen through the auxiliary gas pipeline 802 and / or the sheath gas pipeline 803, forming sample ions in the gaseous state of the sample solution, entering the mass spectrum ion source 4, carrying out mass spectrum scanning and detection, and obtaining the mass analysis result of the sample under the condition of nitrogen.

[0118] Under the condition of inert gas, the unsaturated lipid sample cannot generate cyclization reaction, and thus the characteristic peaks of ion Aldehyde and ion Criegee of adding one oxygen atom and adding two oxygen atoms cannot be generated under the condition of inert gas.

[0119] Taking PC 16:0 / 18:1 (n-9) as an example, the mass spectrum diagram under the condition of ozone (namely, gaseous derivative ionization of electrospray) obtained by the device of the utility model and the method of embodiment 3, and the mass spectrum diagram under the condition of nitrogen (namely, ionization of electrospray) obtained by the device of the utility model and the method of this embodiment are as shown in Figure 5 It can be seen that when the device of the utility model is applied to carry out mass spectrum scanning and detection under the condition of nitrogen, only the characteristic peak information of the sample parent ion can be obtained, and when the device of the utility model is applied to carry out mass spectrum scanning and detection under the condition of ozone, the characteristic peak information of the sample parent ion and Aldehyde and Criegee can be obtained at the same time.

[0120] Embodiment 4, the device of the utility model is combined with liquid chromatography

[0121] The device of the utility model can also be combined with liquid chromatography, the separation of different lipid samples is carried out by using liquid chromatography, and then the carbon-carbon double bond position of the separated lipid sample is identified by using the device of the utility model, and the identification of the carbon-carbon double bond position in the complex lipid sample can be completed.

[0122] For example, the utility model provides a liquid chromatography which can be combined with the device of the utility model and a method for separating the lipid sample by using the liquid chromatography:

[0123] The liquid phase device is ACQUITY UPLC I-Class, the manufacturer is Waters, and the lipids are separated by using an AccucoreTMC30 column (2.1 mm x 150 mm, 3 μm). The column temperature is 50°C, and the flow rate is 0.35 mL / min. The mobile phase A is acetonitrile: water (60:40, v / v), 2 mmol / L ammonium formate; the mobile phase B is isopropanol: acetonitrile (90:10, v / v), 2 mmol / L ammonium formate. Gradient (B%): 0-18 min, 30%-80%; 18-23 min, 80%-98%; 23-26 min, 98%; 26-26.1 min, 98%-30%; 26.1-30 min, 30%.

[0124] It should be understood that, although the utility model takes ACQUITY UPLC I-Class liquid chromatography as an example, the liquid chromatography that can be used with the utility model is not limited to the device, as long as the liquid chromatography that can separate the lipid sample can be used with the device of the utility model, so as to complete the identification of the position of the carbon-carbon double bond in the lipid sample.

[0125] Example 5, comparison of the device of the utility model with other electrospray ionization devices

[0126] In this embodiment, the device of the utility model is compared with other electrospray ionization devices, as shown in Table 2.

[0127] As can be seen from Table 2, the device of the utility model introduces ozone in a closed manner in the mass spectrometry ion source area, reduces the requirement for ozone concentration in the reaction process, only needs 10-30 mg / L, and can complete the reaction instantaneously. Moreover, the device of the utility model introduces ozone in the mass spectrometry ion source area, the requirement for the vacuum degree of the mass spectrometry ion source area is low, the interior of the mass spectrometer does not need to be modified, and damage to the internal parts such as sealing rings caused by introducing ozone into the collision chamber or ion transmission area can be avoided. Moreover, the device of the utility model can be used with liquid chromatography, and the application range thereof is expanded.

[0128] Table 2

[0129]

[0130]

[0131] Notes:

[0132] Document 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.

[0133] Document 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.

[0134] Document 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.

[0135] Document 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.

[0136] Document 5: Batarseh, A.M., et al. (2018). Discrimination of isobaric and isomeric lipids in complex mixtures by combining ultra-high pressure liquid chromatography with collision and ozone-induced dissociation. International Journal of Mass Spectrometry, 431, 27-36.

[0137] Document 6: Marshall, D.L., et al. (2019). Mapping Unsaturation in Human Plasma Lipids by Data-Independent Ozone-Induced Dissociation. Journal of The American Society for Mass Spectrometry, 30(9), 1621-1630.

[0138] The above-described embodiments only express several embodiments of the present utility model, and the description is more specific and detailed, but it cannot be understood as the limitation of the scope of the present utility model patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present utility model, a number of modifications and improvements can be made, which belong to the protection scope of the present utility model. Therefore, the protection scope of the present utility model patent should be subject to the appended claims. At the same time, all the documents mentioned in the present utility model are cited as reference in the present application, just like each document is cited as reference individually.

Claims

1. An electrospray ionization device based on ozone gaseous derivatization ionization, characterized in that, The device comprises: a device for supplying ozone to a mass spectrometry ion source and the mass spectrometry ion source, the mass spectrometry ion source comprising: an ion source and an ion inlet device, the device for supplying ozone to the mass spectrometry ion source comprising: an oxygen supply and an ozone generator, oxygen is supplied to the ozone generator through the oxygen supply to generate ozone, and the device for supplying ozone to the mass spectrometry ion source supplies the generated ozone to the ion source.

2. The ozone gaseous derivative ionization based electrospray ionization device of claim 1, wherein, The ion source comprises 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 to initiate the formation of ions in the sample solution in the electrospray needle, and the formed ions enter the mass spectrometry ion source through the ion inlet device.

3. The ozone gaseous derivative ionization based electrospray ionization device of claim 2, wherein, The ion source further comprises an auxiliary gas pipeline and / or a sheath gas pipeline for supplying ozone to the mass spectrometry ion source.

4. The ozone gaseous derivative ionization based electrospray ionization device of claim 3, wherein, The device for supplying ozone to the mass spectrometry ion source is connected to the auxiliary gas pipeline, or connected to the sheath gas pipeline, or connected to both the auxiliary gas pipeline and the sheath gas pipeline.

5. The ozone gaseous derivative ionization based electrospray ionization device of claim 1, wherein, The device for supplying ozone to the mass spectrometry ion source further comprises a gas pressure display connected to the ozone generator for detecting the flow of ozone generated in the ozone generator.

6. The ozone gaseous derivative ionization based electrospray ionization device of claim 1, wherein, The device for supplying ozone to the mass spectrometry ion source further comprises an ozone analyzer for analyzing the residual ozone in the device for supplying ozone to the mass spectrometry ion source.

7. The ozone gaseous derivative ionization based electrospray ionization device of claim 1, wherein, The device for supplying ozone to the mass spectrometry ion source further comprises an exhaust gas destroyer for removing excess ozone in the device for supplying ozone to the mass spectrometry ion source.

8. The ozone gaseous derivative ionization based electrospray ionization device of claim 1, wherein, The device for supplying ozone to the mass spectrometry ion source further comprises: an inert gas supply, an ozone gas inlet channel switcher and a nitrogen gas inlet channel switcher, the ozone gas inlet channel switcher and the inert gas inlet channel switcher are used to adjust the gas in the device for supplying ozone to the mass spectrometry ion source to be ozone or inert gas.

9. The ozone gas-phase derivative ionization based electrospray ionization device of any one of claims 1-8, wherein, The electrospray ionization device based on ozone gaseous derivative ionization further comprises: a mass spectrometer instrument, the inlet end of the mass spectrometer instrument is connected to the outlet end of the mass spectrometry ion source, and the mass spectrometer instrument is used for mass spectrometry analysis of the ions in the mass spectrometry ion source.

10. The ozone gaseous derivative ionization based electrospray ionization device of claim 9, wherein, The electrospray ionization device based on ozone gaseous derivative ionization further comprises: a liquid chromatography analysis device, the liquid chromatography analysis device is connected to the mass spectrometry ion source, and the outlet end of the liquid chromatography analysis device is connected to the inlet end of the electrospray needle of the mass spectrometry ion source.

Images