Unsaturated lipid analysis method, unsaturated lipid analysis system and online supercritical fluid derivative extraction-phase change focusing device thereof

Abstract

The invention relates to an unsaturated lipid analysis method, an unsaturated lipid analysis system and an on-line supercritical fluid derivative extraction-phase change focusing device thereof, the unsaturated lipid analysis method comprises the following steps: S1, under the action of a selective adsorbent, unsaturated lipid and a derivatization reagent are subjected to an epoxidation reaction in carbon dioxide in a supercritical state to obtain an extract liquid; the selective adsorbent is TiO2, and the derivatization reagent is m-CPBA; s2, changing the carbon dioxide in the supercritical state into a gas state by controlling the temperature and / or pressure, so as to enrich an epoxidation product of unsaturated lipid; and S3, analyzing the epoxidation product by adopting chromatography-mass spectrometry to obtain diagnosis ions of the epoxidation product, and determining the positions of double bonds in the unsaturated lipid according to the diagnosis ions. According to the method, derivation and extraction can be carried out in the same container, the extract directly analyzed by the supercritical fluid chromatography-mass spectrometry is provided, and the sample pretreatment step is simplified, so that sample degradation and artificial errors are reduced.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry chromatographic analysis methods, and particularly relates to an analysis method for unsaturated lipids, a system for an unsaturated lipid analysis method, and an on-line supercritical fluid derivatization extraction-phase change focusing device thereof. Background Art

[0002] Lipids are hydrophobic or amphiphilic small metabolites that play a variety of key roles in cell, tissue, and organ physiology, including energy storage, intercellular signaling, and cell membrane formation. The biochemical and biophysical functional properties of lipids highly depend on their unique chemical structure. Once the structure changes, their properties also change. Most lipid compounds often contain varying numbers of unsaturated carbon-carbon double bonds (C═C) on their carbon chains, and the positions of these carbon-carbon double bonds (C═C) have important effects on their biological functions. In order to more deeply understand the metabolic pathways and biological effects of lipids, it is particularly important to perform structural analysis on unsaturated lipids, which is the basis for realizing qualitative and quantitative analysis of unsaturated lipids.

[0003] Before performing qualitative and quantitative analysis on unsaturated lipids, pretreatment steps such as chemical derivatization, extraction, and enrichment of unsaturated lipids are required. Please refer to Figure 1 , this pretreatment method first adds unsaturated lipids and a derivatization reagent into a reaction vessel, stands for a period of time to complete the derivatization reaction; then adds the reaction solution and an extraction agent into an extraction container (such as a syringe, separatory funnel) for extraction and separation to obtain an extraction solution; then takes a part of the extraction solution and dries it with nitrogen, and then redissolves it with 50% ACN (containing 0.1% formic acid) to obtain a redissolved solution; the redissolved solution is sent to a chromatograph-mass spectrometer for analysis of unsaturated lipids. The operation process of this pretreatment method is numerous and time-consuming, and it is easy to cause oxidation and decomposition of unsaturated lipids or their derivatization products, thereby affecting the accuracy of qualitative and quantitative analysis of unsaturated lipids; in addition, there are multiple steps in this pretreatment method that require manual operation. For example, during extraction and separation, it is necessary to manually judge the boundary line of the two-phase solution, which is prone to human error, affecting the purity and content of the derivatization products of unsaturated lipids, thereby affecting the accuracy and repeatability of qualitative and quantitative analysis of unsaturated lipids. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide an analysis method for unsaturated lipids, a system for an unsaturated lipid analysis method, and an on-line supercritical fluid derivatization extraction-phase change focusing device thereof.

[0005] The above object of the present invention is achieved by the following technical solutions:

[0006] An analysis method for unsaturated lipids, comprising the following steps:

[0007] S1. Under the action of a selective adsorbent, an epoxidation reaction occurs between unsaturated lipids and a derivatizing reagent in supercritical carbon dioxide to obtain an extract; the selective adsorbent is TiO2, and the derivatizing reagent is m-CPBA;

[0008] S2. By controlling the temperature and / or pressure, the supercritical carbon dioxide in the extract is transformed into a gas state, enabling the enrichment of the epoxidation product;

[0009] S3. Analyze the epoxidation product by chromatography-mass spectrometry to obtain the diagnostic ions of the epoxidation product, and determine the double bond position in the unsaturated lipid based on the diagnostic ions.

[0010] Compared with the prior art, the method for analyzing unsaturated lipids of the present invention simultaneously completes chemical derivatization and supercritical fluid extraction in the same container. The derivatization product is extracted by supercritical carbon dioxide, while impurities and excess derivatizing reagents are adsorbed by the selective adsorbent; then, by transforming the supercritical carbon dioxide, an enriched derivatization product can be obtained without transferring the reaction solution or extract, reducing the processing steps, processing time, and manual operations in the pretreatment method of unsaturated lipids, thereby reducing sample degradation and human error during the pretreatment process.

[0011] The present invention also provides an on-line supercritical fluid derivatization extraction-phase change focusing device, comprising:

[0012] a carbon dioxide pump, a modifier pump, a mixer, an extraction unit, a three-way connector, a separation unit, a first back pressure regulator, and a second back pressure regulator;

[0013] The mixer has a first inlet, a second inlet, and an outlet, which are respectively connected to the outlet of the carbon dioxide pump, the outlet of the modifier pump, and the inlet of the extraction unit; the three interfaces of the three-way connector are respectively connected to the outlet of the extraction unit, the inlet of the separation unit, and the inlet of the second back pressure regulator; the outlet of the separation unit is connected to the inlet of the first back pressure regulator;

[0014] The extraction unit includes an extraction tank for loading samples and derivatizing reagents; the extraction tank is filled with a selective adsorbent, and the extraction tank has a top inlet and a bottom inlet; the extraction unit includes a static mode and a dynamic mode. The static mode is that the outlet of the mixer is respectively connected to the top inlet of the extraction tank and the three-way connector; the dynamic mode is that the outlet of the mixer is connected to the bottom inlet, and the top inlet is connected to the three-way connector.

[0015] Among them, the supercritical fluid-derived extraction is to carry out a derivatization reaction and selective extraction in an extraction unit; the phase change focusing is to change the properties of the supercritical fluid inside the device by changing the pressure of the back pressure regulator.

[0016] Compared with the prior art, the present invention sets a static mode in the on-line supercritical fluid-derived extraction-phase change focusing device, enabling supercritical carbon dioxide to enter the extraction tank from the top inlet, promoting the full reaction of unsaturated lipids with the derivatization reagent under static high-pressure conditions, avoiding flow interference, and ensuring complete and uniform reaction; and a dynamic mode, enabling the supercritical fluid to enter the extraction tank from the bottom inlet and discharging the reacted derivatization product from the top inlet to the separation unit. The different flow directions of the fluid in the static mode and the dynamic mode can make the substances in the extraction tank fully contact with the selective adsorbent, reducing the influence of unreacted substances and impurities on the analysis of the derivatization product, thereby increasing the rate and yield of the derivatization reaction, ensuring the high purity of the derivatization product, providing more accurate and reliable detection results for subsequent sample analysis, and enhancing the controllability and reproducibility of the experimental operation.

[0017] Further, the extraction unit further includes an extraction valve. The extraction valve is provided with a liquid inlet, a first liquid outlet, and at least one second liquid outlet. The liquid inlet is communicated with the outlet of the mixer, and each second liquid outlet is respectively communicated with the bottom inlet of an extraction tank; the extraction valve can switch the valve position to communicate the liquid inlet with the first liquid outlet or with any second liquid outlet.

[0018] Further, the extraction unit further includes a drain valve, a T-shaped tube, and a sampling needle. The drain valve is provided with a first interface, a second interface, a third interface, and a fourth interface. The fourth interface is communicated with the inlet of the tee connector, and the third interface is communicated with the first liquid outlet; the T-shaped tube is respectively communicated with the first interface, the second interface, and the sampling needle, and the sampling needle can be communicated with the top inlet of any extraction tank; the drain valve can switch the valve position to the extraction position and the drain position. The extraction position is that the fourth interface is communicated with the first interface and the second interface is communicated with the third interface; the drain position is that the fourth interface is communicated with the third interface. With this setting, when the drain valve is switched to the extraction position, the first liquid inlet can be communicated with the top inlet of the extraction tank and the inlet of the tee connector through the drain valve, the T-shaped tube, and the sampling needle; when the drain valve is switched to the drain position, the first liquid inlet can be communicated with the inlet of the tee connector through the drain valve.

[0019] Further, in the static mode, the drain valve is in the extraction position, and the liquid inlet is communicated with the first liquid outlet; in the dynamic mode, the drain valve is in the extraction position, the liquid inlet is communicated with any one of the second liquid outlets, and any one of the second liquid outlets is communicated with the T-shaped tube through the extraction tank; the extraction unit further includes an analysis mode, in which the drain valve is in the drainage position and the liquid inlet is communicated with the first liquid outlet; wherein, in the dynamic mode and the analysis mode, the pressure of the second back pressure regulator is greater than that of the first back pressure regulator.

[0020] Further, the static mode includes two working modes, namely, the derivatization extraction mode: the pressure of the first back pressure regulator is greater than that of the second back pressure regulator; and the phase change mode: the pressure of the first back pressure regulator is less than that of the second back pressure regulator, and the pressure inside the device is less than the supercritical point pressure of carbon dioxide.

[0021] Further, the separation unit is a chromatographic column.

[0022] Further, in the dynamic mode and the analysis mode, the pressure of the first back pressure regulator is set to a fixed value between 10 - 30 MPa, and the pressure of the second back pressure regulator is set to 40 MPa.

[0023] Further, the selective adsorbent is titanium dioxide, the derivatization reagent is m-chloroperoxybenzoic acid; the sample is a sample containing unsaturated lipids, and the unsaturated lipids include unsaturated fatty acids.

[0024] Further, the temperature of the extraction tank is set to a fixed value between 31 - 60 °C.

[0025] Further, the volume of the extraction tank is 200 μL.

[0026] The present invention also provides an unsaturated lipid analysis system, which includes a mass spectrometry detector and the above-mentioned on-line supercritical fluid derivatization extraction-phase change focusing device, and the inlet of the mass spectrometry detector is communicated with the outlet of the first back pressure regulator.

[0027] Further, when analyzing the sample in any extraction tank for unsaturated lipids, the time of the derivatization extraction mode is preferably 10 min; the time of the phase change mode is preferably 2 min. Description of the Drawings

[0028] Figure 1 It is a schematic flow chart of the prior art method for qualitative and quantitative analysis of unsaturated lipids.

[0029] Figure 2 It is a schematic flow chart of the unsaturated analysis method of the present invention.

[0030] Figure 3Schematic structural diagram of a supercritical fluid extraction device of the prior art.

[0031] Figure 4 Schematic structural diagram of the unsaturated lipid analysis system of the present invention.

[0032] Figure 5 is Figure 4 Flow path diagram of the static mode of supercritical fluid derivatization extraction of the system shown.

[0033] Figure 6 is Figure 4 Flow path diagram of the dynamic mode of supercritical fluid derivatization extraction of the system shown.

[0034] Figure 7 is Figure 4 Flow path diagram of the analysis mode of supercritical fluid derivatization extraction of the system shown.

[0035] Figure 8 Analysis flow chart corresponding to the system of the present invention.

[0036] Figure 9 Schematic diagram of the principle of supercritical fluid derivatization extraction of the system of the present invention.

[0037] Figure 10 Mechanism diagram for the system of the present invention to identify the positions of carbon-carbon double bonds of unsaturated lipids.

[0038] Figure 11 MRM chromatogram obtained by the system of the present invention for on-line derivatization of a 9-unsaturated fatty acid mixed standard solution.

[0039] Figure 12 Effect of different derivatizing reagents on the response of epoxidation products in the system of the present invention.

[0040] Figure 13 Effect of different dosages of m-CPBA derivatizing reagent on the response of epoxidation products in the system of the present invention.

[0041] Figure 14 Response effect diagram of different adsorbents adsorbing the derivatizing reagent m-CPBA in the system of the present invention.

[0042] Figure 15 Effect of different dosages of TiO2 adsorbent on the response of epoxidation products in the system of the present invention.

[0043] Figure 16a Effect of the temperature during derivatization on the response of epoxidation products in the system of the present invention.

[0044] Figure 16b Effect of the pressure during derivatization on the response of epoxidation products in the system of the present invention.

[0045] Figure 16c The influence of the derivatization time on the response of the epoxidation product in the system of the present invention.

[0046] Figure 16d The influence of the modifier ratio during derivatization on the response of the epoxidation product in the system of the present invention.

[0047] Corresponding relationship of reference numerals: carbon dioxide pump - 1, modifier pump - 2, mixer - 3, extraction unit - 4, extraction tank - 5, column oven - 6, chromatographic column - 7, makeup pump - 8, tee connector - 8, second back pressure regulator - 9, first back pressure regulator - 10, mass spectrometry detector - 11, sample rack - 12, extraction valve - 13, drain valve - 14, stainless steel injection needle - 15, T - tube - 16; CO2 storage tank - 101, cooling system - 102, high - pressure pump - 103, extraction kettle - 104, separation kettle - 105, circulating cooling system - 106. Detailed implementation manners

[0048] In order to simplify the sample pretreatment steps, reduce the influence of long - time treatment on sample degradation, and reduce the artificial errors caused by a large number of manual operation steps, the present invention proposes an unsaturated lipid analysis method, which combines chemical derivatization and supercritical fluid extraction and completes chemical derivatization and supercritical fluid extraction in the same container simultaneously. Please refer to Figure 2 and the method includes the following steps:

[0049] S1. Supercritical fluid derivatization extraction: Under the action of a selective adsorbent, an unsaturated lipid undergoes an epoxidation reaction with a derivatization reagent in supercritical carbon dioxide to obtain an extraction solution; the selective adsorbent is any one of Florisil, C18, and TiO2; the derivatization reagent is any one of MMPP and m - CPBA;

[0050] S2. Phase change: By controlling the temperature and / or pressure, the supercritical carbon dioxide in the extraction solution is changed into a gaseous state, so that the epoxidation products of the unsaturated lipid are enriched;

[0051] S3. Mass spectrometry analysis: The epoxidation product is analyzed by chromatography - mass spectrometry to obtain the diagnostic ions of the epoxidation product, and the double - bond position in the unsaturated lipid is determined according to the diagnostic ions.

[0052] Among them, steps S1 and S2 need to be carried out in a supercritical extraction device, such as Figure 3As shown, the existing supercritical extraction device includes a CO2 storage tank 101, a cooling system 102, a high-pressure pump 103, an extraction kettle 104, a separation kettle 105, and a circulating cooling system 106. The outlet of the CO2 storage tank 101 is connected to the cooling system 102 through a pipeline, and a one-way valve is provided in the pipeline. The outlet of the cooling system 102 is connected to the high-pressure pump 103. The outlet of the high-pressure pump 103 is connected to the extraction kettle 104. The outlet of the extraction kettle 104 is connected to the separation kettle 105. The outlet of the separation kettle 105 is connected to the circulating cooling system 106. When using this supercritical extraction device, the substance to be extracted is loaded into the extraction kettle. Then, the CO2 coming out of the CO2 storage tank is cooled into a liquid state, compressed by the high-pressure pump, and then enters the extraction kettle to contact and transfer mass with the substance to be extracted. The supercritical CO2 dissolved with the solute enters the separation kettle after heating and pressure reduction, and the substance to be extracted is enriched at the bottom of the separation kettle. However, when this supercritical extraction device is used for supercritical fluid derivatization extraction, the efficiency of the derivatization reaction and the purity of the product are limited, which affects the accuracy and repeatability of the qualitative and quantitative analysis results of unsaturated lipids.

[0053] Therefore, the present invention explores the reasons why the existing supercritical fluid extraction device affects the accuracy and repeatability of the qualitative and quantitative analysis results of unsaturated lipids, and finds that in this supercritical fluid extraction device, the supercritical fluid flows continuously, resulting in insufficient contact time between the derivatization reagent and unsaturated lipids, incomplete derivatization reaction, and thus reduced yield of the derivatization product and reduced repeatability of the analysis results. In addition, incomplete reaction may also cause unreacted unsaturated lipids to enter the separation kettle together with the derivatization product for separation, which will not only reduce the purity of the derivatization product, but also may cause signal interference to the subsequent mass spectrometry analysis, making the interpretation of the mass spectrometry difficult, and even affecting the accuracy of the qualitative and quantitative analysis of unsaturated lipids. Especially for low-abundance unsaturated lipids, incomplete reaction may cause them to be undetectable, affecting the application scenario of this analysis method.

[0054] To solve the above problems, the present invention improves the supercritical fluid extraction device so that the supercritical fluid extraction device has at least two states. In the first state, the derivatization reagent and unsaturated lipids can be in sufficient contact. In the second state, the supercritical fluid extracts the derivatization product of unsaturated lipids and transports it to the separation kettle for enrichment.

[0055] Based on the above concept, the present invention provides an unsaturated lipid analysis system, which includes an on-line supercritical fluid derivatization extraction-phase change focusing device, a chromatographic detection unit 6, a mass spectrometry detector 11, and connecting pipes for connecting each component. Please refer to Figure 4, the on-line supercritical fluid derivatization extraction-phase focusing device includes a carbon dioxide pump 1, a modifier pump 2, a mixer 3, an extraction unit 4, a three-way connector 8, a separation unit 7, a first back pressure regulator 10 and a second back pressure regulator 9.

[0056] The carbon dioxide pump 1 is used to transport carbon dioxide as the mobile phase; the modifier pump 2 is used to transport the modifier to adjust the polarity of the mobile phase. The mixer 3 has a first inlet a, a second inlet b and an outlet c, which are respectively communicated with the outlet of the carbon dioxide pump 1, the outlet of the modifier pump 2 and the inlet of the extraction unit 4. The three-way connector 8 includes a first interface d, a second interface e and a third interface f, which are respectively communicated with the outlet of the extraction unit 4, the inlet of the separation unit 7 and the inlet of the second back pressure regulator 9. The outlet of the second back pressure regulator 9 is communicated with a waste liquid pipe for draining liquid. The outlet of the separation unit 7 is communicated with the inlet of the mass spectrometry detector 11 through the first back pressure regulator 10. In this embodiment, the separation unit 7 is a chromatographic column, and the chromatographic detection unit 6 includes an oven, and the chromatographic column is arranged in the oven. In other embodiments, the separation unit 7 can be a separation kettle.

[0057] Please refer to Figures 5 - 7 , the extraction unit 4 includes an extraction tank 5, and a selective adsorbent is placed in the extraction tank 5. The extraction tank 5 has a top inlet and a bottom inlet. The extraction unit 4 can be switched between a static mode, a dynamic mode and an analysis mode. Among them, the static mode is that the outlet of the mixer 3 is respectively communicated with the top inlet of the extraction tank 5 and the three-way connector 8; the dynamic mode is that the outlet of the mixer 3 is communicated with the bottom inlet of the extraction tank 5, and the top inlet of the extraction tank 5 is communicated with the first interface d of the three-way connector 8. In this embodiment, the extraction tank 5 is composed of two metal filter sheets, a tank body, a tank jacket and a tank cap. The two metal filter sheets are relatively clamped at both ends of the tank body; a PTFP membrane can be arranged between the metal filter sheet and the tank body of the extraction tank 5 to prevent the extracted substances from blocking the metal filter sheet. Preferably, the specification of the PTFP membrane is 47mm, 0.45μm.

[0058] Specifically, the extraction unit 4 further includes a sample rack 12, an extraction valve 13 and a drain valve 14 for switching the flow path, a stainless steel sampling needle, and a T-shaped tube 16. The sample rack 12 is provided with a sample position for placing the extraction tank 5. The extraction valve 13 is provided with a liquid inlet, a first liquid outlet, and a second liquid outlet. The liquid inlet is communicated with the outlet of the mixer 3. The first liquid outlet is communicated with the drain valve 14. The second liquid outlet is communicated with the bottom inlet of the extraction tank 5. The extraction valve 13 can switch the valve position to communicate the liquid inlet with the first liquid outlet or the second liquid outlet. The drain valve 14 is sequentially provided with six interfaces in the counterclockwise order: a first interface m, a fifth interface n, a sixth interface o, a second interface p, a third interface q, and a fourth interface r. The fourth interface r is communicated with the inlet of the three-way connector 8. The first interface m and the second interface p are respectively communicated with the T-shaped tube 16. The third interface q is communicated with the first liquid outlet. The drain valve 14 can switch the valve position to an extraction position and a drain position. The extraction position is that the fourth interface r is communicated with the first interface m, and the second interface p is communicated with the third interface q. The drain position is that the fourth interface r is communicated with the third interface q. The T-shaped tube 16 is respectively communicated with the first interface m, the second interface p, and the stainless steel sampling needle 15. The stainless steel sampling needle 15 is detachably connected to the top inlet of the extraction tank 5.

[0059] In this embodiment, the sample positions are set to 4, and 4 extraction tanks 5 are respectively placed. The extraction valve 13 is sequentially provided with six interfaces in the counterclockwise order, namely: a first interface g, a second interface h, a third interface i, a fourth interface j, that is, 4 second liquid outlets, which are respectively communicated with the bottom inlets of 4 extraction tanks 5; and a fifth interface k, which is connected to an origin port for placing the stainless steel sampling needle 15; and a sixth interface l, that is, the first liquid outlet, which is communicated with the drain valve 14. The stainless steel sampling needle 15 can be moved above any one of the extraction tanks 5 and connected to the top inlet of the extraction tank 5 to fill the extraction tank 5 with the mobile phase. Or, the mobile phase in the extraction tank 5 is discharged to the drain valve 14 through the stainless steel sampling needle 15. By connecting the stainless steel sampling needle 15 to the extraction tanks 5 at different sample positions, batch processing of the samples in different extraction tanks 5 is realized, and the efficiency of derivatizing and extracting the samples is improved.

[0060] In the static mode, the extraction valve 13 is switched to the position of the sixth interface l, and the drain valve 14 is switched to the extraction position, that is, the liquid inlet is communicated with the sixth interface l (the first liquid outlet) of the extraction valve 13, the third interface q is communicated with the second interface p, and the first interface m is communicated with the fourth interface r. The mobile phase passes through the extraction valve 13, the drain valve 14, the T-shaped tube 16, and the stainless steel sampling needle 15, and enters the extraction tank 5 from the top inlet of the extraction tank 5 to apply pressure to the extraction tank 5 for derivatizing extraction, such as Figure 5As shown. The excess mobile phase passes through the extraction valve 13, the drain valve 14, the T-shaped tube 16, and the drain valve 14, and flows to the tee connector 8. In this embodiment, the static mode includes two working modes, namely, the derivatization extraction mode: the pressure of the first back pressure regulator 10 is greater than the pressure of the second back pressure regulator 9, and at this time, the excess mobile phase can be discharged from the second back pressure regulator 9; and the phase change mode: the pressure of the first back pressure regulator 10 is less than the pressure of the second back pressure regulator 9, and the pressure inside the system is less than the supercritical point pressure of carbon dioxide.

[0061] In the dynamic mode, the drain valve 14 is still in the extraction position. The liquid inlet of the extraction valve 13 is connected to the bottom inlet of the extraction tank 5 through any one of the second liquid outlets (the first interface g, or the second interface h, or the third interface i, or the fourth interface j), and the pressure of the second back pressure regulator 9 is greater than the pressure of the first back pressure regulator 10. That is, the mobile phase passes through the extraction valve 13, enters from the bottom inlet of the extraction tank 5, and exits from the top inlet of the extraction tank 5, and then passes through the stainless steel injection needle 15, the T-shaped tube 16, the first interface m and the fourth interface r of the drain valve 14, and the tee connector 8 to flow to the separation unit 7, as Figure 6 shown.

[0062] In the analysis mode, the extraction valve 13 is switched to the position of the sixth interface l, the drain valve 14 is switched to the drain position, and the pressure of the second back pressure regulator 9 is greater than the pressure of the first back pressure regulator 10. That is, the third interface q is connected to the fourth interface r. As Figure 7 shown, the mobile phase passes through the sixth interface l of the extraction valve 13, the third interface q and the fourth interface r of the drain valve 14, the tee connector 8, the separation unit 7, and the first back pressure regulator 10 and enters the mass spectrometer detector 11.

[0063] The on-line supercritical fluid derivatization extraction-phase change focusing device of the present invention has a static mode and a dynamic mode. The sample and the derivatization reagent are pre-added into the extraction tank. Then, in the static mode, the supercritical fluid enters the extraction tank 5 from the top inlet 11, so that the sample and the derivatization reagent move from above the selective adsorbent to the middle or bottom of the adsorbent. The sample, the derivatization reagent and the selective adsorbent are fully mixed to realize the full reaction of the sample and the derivatization reagent and the full adsorption of impurities by the selective adsorbent. In the dynamic mode, the supercritical fluid enters the extraction tank 5 from the bottom inlet, so that the extraction liquid in the extraction tank 5 moves to the top inlet. During this process, the extraction liquid is mixed and contacted with the selective adsorbent again to further improve the impurity removal effect of the selective adsorbent, so as to improve the purity of the derivatization product in the extraction liquid.

[0064] Referring to Figures 8 - 10 , based on the above unsaturated lipid analysis system, the unsaturated analysis method includes the following steps:

[0065] (1) Supercritical fluid derivatization extraction: The extraction unit 4 is maintained in the static mode, and the pressure of the first back pressure regulator 10 is greater than that of the second back pressure regulator 9, i.e., the derivatization extraction mode; the sample and the derivatization reagent are successively dropped onto the selective adsorbent in the extraction tank 5; the carbon dioxide pump 1 and the modifier pump 2 respectively transport supercritical carbon dioxide and the modifier into the mixer 3 for mixing to obtain the first mobile phase, and the first mobile phase enters the extraction tank 5 from the top inlet of the extraction tank 5, and pressure is applied to perform supercritical fluid derivatization extraction on the sample. Subsequently, the excess first mobile phase flows out of the system through the second back pressure regulator 9. That is, under the action of the selective adsorbent, the unsaturated lipids in the sample undergo an epoxidation reaction with the derivatization reagent in the first mobile phase to obtain the extract. The carbon dioxide in the first mobile phase is in the supercritical state.

[0066] (2) Phase change: The extraction unit 4 is maintained in the static mode, and the pressures of the second back pressure regulator 9 and the first back pressure regulator 10 are respectively adjusted so that the pressure of the first back pressure regulator is less than that of the second back pressure regulator, and the pressure inside the device is less than the supercritical point pressure of carbon dioxide, that is, the carbon dioxide in the system changes from the supercritical state to the gaseous state, and the phase change is completed.

[0067] (3) Sample loading and column head focusing: The extraction unit 4 is switched to the dynamic mode, supercritical carbon dioxide and the modifier are mixed to obtain the second mobile phase, and the second mobile phase enters the extraction tank 5 from the bottom inlet of the extraction tank 5, and the extract in the extraction tank 5 is discharged to the chromatographic column, and the epoxidation products in the extract are focused at the column head of the chromatographic column.

[0068] (4) Chromatographic separation and mass spectrometry analysis: The extraction unit 4 is switched to the analysis mode, the carbon dioxide pump 1 and the modifier pump 2 respectively transport supercritical carbon dioxide and the modifier according to the set analysis gradient, and are mixed in the mixer 3 to obtain the third mobile phase. The third mobile phase enters the chromatographic column to elute the epoxidation products on the chromatographic column, and the obtained eluate enters the mass spectrometry detector 11 for analysis.

[0069] Based on this, in the following embodiments of the present invention, the above unsaturated lipid analysis system is used to verify the effect of the above unsaturated lipid analysis method. First, the present invention explores the compatibility of different chemical derivatization methods with the above unsaturated lipid analysis system, hoping to find the most suitable derivatization reagent and selective adsorbent to improve the response and sensitivity of the mass spectrometry to the derivatization products and reduce the interference of the derivatization reagent and impurities on the mass spectrometry. Then the present invention explores how to minimize the sample preparation time, improve the accuracy and repeatability of sample analysis through the system by adopting different key operating conditions, such as the temperature and pressure of the system. In addition, the present invention explores whether to use a supplementary pump for the analysis system and finds that using a supplementary pump results in a decrease in the response of the epoxidation products.

[0070] In the specific examples, the reagents used, unless otherwise specified, are commercially available; the methods used in the specific examples, unless otherwise specified, can be realized by conventional methods.

[0071] Example 1

[0072] The unsaturated fatty acid mixed standard solution composed of 9 unsaturated fatty acids was subjected to on-line supercritical epoxidation derivatization extraction and analysis detection using the unsaturated lipid analysis system of the present invention. The 9 unsaturated fatty acids are FA 14:1(9Z)-1, FA16:1(9Z)-2, FA 17:1(10Z)-3, FA18:1(9Z)-4, FA18:1(11Z)-5, FA18:2(9Z,12Z)-6, FA 18:3(6Z,9Z,12Z)-7, FA18:3(9Z,12Z,15Z)-8, FA20:4(5Z,8Z,11Z,14Z)-9 in sequence.

[0073] In this example, the extraction tank 5 has a specification of 200 μL and is filled with 10 mg of the selective adsorbent TiO2; the chromatographic column is a Shim-pack UC-X-RP column (specification: 4.6 mm × 250 mm, 3.0 μm) of Shimadzu Corporation of Japan, and the temperature of the column oven is set at 35 °C; the modifier is methanol plus 1% water.

[0074] In step (1), 2.5 μL of the unsaturated fatty acid mixed standard solution (200 μM) and 2.5 μL of the meta-chloroperbenzoic acid (m-CPBA) solution (500 mM) were successively dropped onto the TiO2 in the extraction tank 5, and the extraction tank 5 was placed at the sample position of the sample rack 12. The stainless steel injection needle 15 was connected to the top inlet of the extraction tank 5 to perform supercritical fluid derivatization extraction on the unsaturated fatty acids. The temperature of the extraction tank 5 was set at 35 °C; the pressure of the first back pressure regulator 10 was set at 40 MPa, and the pressure of the second back pressure regulator 9 was set at 15 MPa; the total flow rate of the first mobile phase was 1.2 mL / min, and the delivery ratio of the modifier was 8%; the derivatization extraction time was 10 min.

[0075] In step (2), the pressure of the second back pressure regulator 9 was set at 40 MPa, and the first back pressure regulator 10 was closed. The carbon dioxide in the system changed from the supercritical fluid state to the gaseous state. Among them, the delivery ratio of the carbon dioxide pump 1 was set at 100%, and the flow rate was set at 0.4 mL / min; the phase change time was 2 min.

[0076] In step (3), the pressure of the second backpressure regulator 9 is set to 40 MPa, and the pressure of the first backpressure regulator 10 is set to 20 MPa. The total flow rate of the second mobile phase is set to 1.2 mL / min, wherein the delivery ratio of the carbon dioxide pump 1 is set to 92%, and the carbon dioxide is in a supercritical state; the focusing time is 2 min.

[0077] In step (4), the pressure of the second backpressure regulator 9 is set to 40 MPa, and the pressure of the first backpressure regulator 10 is set to 20 MPa, so that the carbon dioxide in the mobile phase is in a supercritical state. Among them, the total flow rate of the third mobile phase is 1.2 mL / min, and the delivery ratio of carbon dioxide and modifier therein changes uniformly from 92:8 to 85:15; the elution time is 10 min.

[0078] In steps (1) to (4), the temperature of the column oven is a selected value in the range of 31 - 70 °C. Preferably, the temperature of the column oven is the same as the temperature of the extraction tank 5.

[0079] Please refer to Figure 10 , which is the mechanism diagram of the system of the present invention for identifying the position of carbon-carbon double bonds in unsaturated lipids: The epoxidizing reagent m-CPBA undergoes a derivatization reaction with the carbon-carbon double bonds in unsaturated lipids in a supercritical fluid to generate an epoxidation product. After the epoxidation product of unsaturated lipids undergoes CID fragmentation by mass spectrometry, each epoxy group generates two characteristic diagnostic ions, and the mass-to-charge ratios of the two detectable characteristic fragment ions differ by 16 Da, so as to accurately determine the position of C=C. Due to the mass difference between the structures, the two detectable fragment ions are always spaced 16 Da apart, which can be used as a characteristic to accurately determine the position of C=C.

[0080] Refer to Figure 11 , the obtained chromatographic peaks are sharp and have the highest sensitivity, and can accurately identify the position of carbon-carbon double bonds in unsaturated lipids.

[0081] Example 2

[0082] The analytical method provided in this example is similar to that in Example 1, except that in this example, magnesium monoperphthalate hexahydrate (MMPP) is used as the derivatization reagent to carry out an epoxidation reaction on unsaturated lipids.

[0083] Refer to Figure 12, Online supercritical fluid derivatization extraction with m-CPBA gave a higher response for the epoxidation products, indicating that m-CPBA is more suitable for the online supercritical fluid derivatization extraction system. It may be that the polarity of m-CPBA is lower than that of MMPP, and the MMPP reagent is prepared with water while m-CPBA is prepared with an organic solvent (acetonitrile / isopropanol / water = 65:30:5). Therefore, m-CPBA is more compatible with supercritical carbon dioxide and has a better reaction effect. The derivatization reagent is preferably meta-chloroperoxybenzoic acid (m-CPBA).

[0084] Examples 3 - 6

[0085] The analytical methods provided in Examples 3 - 6 are similar to those in Example 1, except that the concentrations of the derivatization reagent m-CPBA are 50 mM, 100 mM, 200 mM, and 400 mM respectively.

[0086] Please refer to Figure 13 , the response of the lipid epoxidation products increased with the increase in the concentration of m-CPBA. When the concentration of m-CPBA was 500 mM, the responses of all epoxidation products reached the maximum. However, there were slight suspensions in solutions with a higher concentration (such as 600 mM), that is, m-CPBA could not be completely dissolved. Therefore, the concentration of the m-CPBA solution is preferably 500 mM.

[0087] Examples 7 - 9

[0088] The analytical methods provided in Examples 7 - 9 are similar to those in Example 1, except that in Example 7, no adsorbent is used, and the adsorbents in Examples 8 and 9 are Florisil and C18 respectively.

[0089] Refer to Figure 14 , without an adsorbent, the response of excessive m-CPBA was high, which contaminated the mass spectrometry and thus affected the response of the epoxidation products. After introducing the selective adsorbents Florisil, C18, and TiO2, the responses of m-CPBA decreased with all three adsorbents, indicating that the adsorbents have a good adsorption effect on m-CPBA. Thus, the contamination of the mass spectrometry and the influence on the response by the excessive derivatization reagent can be reduced. Among them, TiO2 has the best effect on adsorbing excessive m-CPBA. Therefore, the selective adsorbent is preferably TiO2.

[0090] Examples 10 - 12

[0091] The analytical methods provided in Examples 10 - 12 are similar to those in Example 1, except that the dosages of the adsorbent TiO2 are 20 mg, 30 mg, and 40 mg respectively.

[0092] Please refer to Figure 15, when the dosage of the adsorbent is 10 mg, the response intensity of the epoxidation product is the highest. With the increase in the TiO2 content, the responses of most target substances slightly decrease. This may be because too large a dosage of the adsorbent will occupy a relatively large volume of the extraction tank, which is not conducive to the full contact between the extraction solvent and the target substances, reducing the extraction efficiency. Therefore, the preferred dosage of the adsorbent TiO2 is 10 mg.

[0093] Examples 13 - 16

[0094] The analysis methods provided in Examples 13 - 16 are similar to that in Example 1, except that in step (1), the temperatures of the extraction tank 5 are set to 40 °C, 45 °C, 50 °C, and 55 °C respectively.

[0095] Please refer to Figure 16a , when the temperature of the extraction tank 5 is set to 35 °C, the response intensity of the epoxidation product is the highest. With the increase in temperature, the responses of most target substances slightly decrease. It shows that this system may be more suitable for the analysis of biological samples that are not heat - resistant.

[0096] Examples 17 - 19

[0097] The analysis methods provided in Examples 17 - 19 are similar to that in Example 1, except that in step (1), the pressures of the second back - pressure regulator 9 are set to 10 MPa, 20 MPa, and 25 MPa respectively.

[0098] Please refer to Figure 16b , when the pressure of the second back - pressure regulator 9 is 15 MPa, the response intensity of the epoxidation product is the highest.

[0099] Examples 20 - 23

[0100] The analysis methods provided in Examples 20 - 23 are similar to that in Example 1, except that the derivatization extraction times are 5 min, 15 min, 20 min, and 25 min.

[0101] Please refer to Figure 16c , when the derivatization extraction time is 10 min, the response intensity of the epoxidation product is the highest. Maybe due to too long a reaction time, the epoxidation product slightly decomposes in the pressurized extraction tank.

[0102] Examples 24 - 27

[0103] The analysis methods provided in Examples 24 - 27 are similar to that in Example 1, except that in step (1), the proportions of the modifier are 0%, 2%, 4%, and 6% respectively.

[0104] Please refer to Figure 16d , when the delivery proportion of the modifier is 8%, the response intensity of the epoxidation product is the highest.

[0105] In summary, the present invention has the following advantages compared with the prior art:

[0106] (1) The on-line supercritical fluid derivatization extraction-variable phase focusing-chromatography mass spectrometry on-line analysis system of the present invention integrates derivatization-extraction-purification-separation-detection. Derivatization and extraction can be carried out in the same container, and extracts for direct analysis by supercritical fluid chromatography mass spectrometry can be provided, reducing sample handling and preparation steps. It solves the problems of cumbersome sample handling steps, long time consumption, and sample loss, reduces the human operation errors generated in sample pretreatment, and improves the reproducibility of analysis.

[0107] (2) For this system, only the sample and the derivatization reagent need to be dropped into the extraction tank 5, and according to the pre-set analysis program, the derivatization reaction and selective extraction of lipids in complex samples can be completed. This method is simple and easy to operate, without complex sample pretreatment and without modifying the instrument. In addition, by combining the variable phase focusing technology, the response of low-abundance lipids can be improved, which is beneficial to the qualitative and quantitative analysis of low-abundance lipids.

[0108] (3) The method of the present invention realizes the derivatization reaction of the carbon-carbon double bond in unsaturated lipids with the epoxidation reagent m-CPBA to generate an epoxidation product. After the epoxidation product of unsaturated lipids undergoes CID fragmentation in the mass spectrometer, each epoxy group generates two characteristic diagnostic ions, and the mass-to-charge ratios of the two detectable characteristic fragment ions differ by 16 Da, so as to accurately determine the position of C=C.

[0109] (4) By combining the variable phase focusing technology, only by adjusting the pressure and temperature, the focusing of the analyte at the chromatographic column head can be realized, and it has the effects of improving the peak shape and sensitivity, which is beneficial to quantitative analysis.

[0110] (5) By introducing a selective adsorbent, the pollution of the mass spectrometer by excessive derivatization reagent and the influence on the response are reduced; impurities in the complex sample matrix are removed, which is beneficial to reducing the matrix effect and enriching the target analyte.

[0111] The above embodiments only represent several implementation manners of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A method for analyzing unsaturated lipids, characterized in that: The following steps are involved: S1. Under the action of a selective adsorbent, an unsaturated lipid is subjected to an epoxidation reaction with a derivatization reagent in supercritical carbon dioxide to obtain an extract; the selective adsorbent is TiO2, and the derivatization reagent is m-CPBA; S2, by controlling the temperature and / or pressure to change the supercritical carbon dioxide in the extract into a gaseous state, so that the epoxidation product of the unsaturated lipid is enriched; S3. Analyze the epoxidation product by chromatography-mass spectrometry to obtain diagnostic ions of the epoxidation product, and determine the double bond positions in the unsaturated lipid according to the diagnostic ions.

2. An online supercritical fluid derivatization extraction-phase-changing focusing device, characterized in that: include: a carbon dioxide pump, a modifier pump, a mixer, an extraction unit, a three-way connector, a separation unit, a first back pressure regulator, and a second back pressure regulator; The mixer has a first inlet, a second inlet and an outlet, which are respectively connected to the outlet of the carbon dioxide pump, the outlet of the modifier pump and the inlet of the extraction unit; the three interfaces of the three-way connector are respectively connected to the outlet of the extraction unit, the inlet of the separation unit, and the inlet of the second back pressure regulator; the outlet of the separation unit is connected to the inlet of the first back pressure regulator; The extraction unit comprises an extraction tank for loading a sample and a derivatization reagent; the extraction tank is filled with a selective adsorbent, and the extraction tank has a top inlet and a bottom inlet; the extraction unit comprises a static mode and a dynamic mode, and the static mode is that the outlet of the mixer is connected to the top inlet of the extraction tank and the three-way connector respectively; The dynamic mode is that the outlet of the mixer is connected to the bottom inlet, and the top inlet is connected to the three-way connector.

3. The online supercritical fluid derivatization extraction-phase-changing focusing device according to claim 2, characterized in that: The extraction unit also includes an extraction valve, which is provided with a liquid inlet, a first liquid outlet and at least one second liquid outlet, wherein the liquid inlet is connected to the outlet of the mixer, and each of the second liquid outlets is respectively connected to the bottom inlet of an extraction tank; the extraction valve can switch the valve position so that the liquid inlet is connected to the first liquid outlet or to any second liquid outlet.

4. The online supercritical fluid derivatization extraction-phase-changing focusing device according to claim 3, characterized in that: The extraction unit also includes a drain valve, a T-shaped tube and an injection needle. The drain valve is provided with a first interface, a second interface, a third interface and a fourth interface. The fourth interface is connected to the inlet of the three-way connector, and the third interface is connected to the first liquid outlet; the T-shaped tube is connected to the first interface, the second interface and the injection needle respectively, and the injection needle can be connected to the top inlet of any extraction tank; the drain valve can switch the valve position to an extraction position and a drain position. The extraction position is that the fourth interface is connected to the first interface, and the second interface is connected to the third interface; the drain position is that the fourth interface is connected to the third interface.

5. The on-line supercritical fluid derivatization extraction-phase-changing focusing device according to claim 4, characterized in that: The static mode is that the drain valve is in the extraction position, and the liquid inlet is connected to the first liquid outlet; the dynamic mode is that the drain valve is in the extraction position, the liquid inlet is connected to any second liquid outlet, and any second liquid outlet is connected to the T-shaped tube through the extraction tank; the extraction unit also includes an analysis mode, and the analysis mode is that the drain valve is in the drain position, and the liquid inlet is connected to the first liquid outlet; wherein, in the dynamic mode and the analysis mode, the pressure of the second back pressure regulator is greater than the pressure of the first back pressure regulator.

6. The online supercritical fluid derivatization extraction-phase-changing focusing device according to claim 5 is characterized in that: The static mode includes two working modes, namely, a derivative extraction mode: the pressure of the first back pressure regulator is greater than the pressure of the second back pressure regulator; and a phase change mode: the pressure of the first back pressure regulator is less than the pressure of the second back pressure regulator, and the pressure inside the device is less than the supercritical point pressure of carbon dioxide.

7. The online supercritical fluid derivatization extraction-phase-changing focusing device according to claim 5 is characterized in that: The separation unit is a chromatographic column.

8. The online supercritical fluid derivatization extraction-phase-changing focusing device according to claim 5 is characterized in that: In the dynamic mode and the analysis mode, the pressure of the first back pressure regulator is set to a fixed value between 10-30 MPa, and the pressure of the second back pressure regulator is set to 40 MPa.

9. The online supercritical fluid derivatization extraction-phase-changing focusing device according to claim 5 is characterized in that: The temperature of the extraction tank is a fixed value between 31-60°C.

10. An unsaturated lipid analysis system, characterized in that: It comprises a mass spectrometer detector and the online supercritical fluid derivatization extraction-phase-changing focusing device according to any one of claims 2 to 9, wherein the inlet of the mass spectrometer detector is connected to the outlet of the first back pressure regulator.

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