Lipid oxidative metabolism characteristic analysis method and system based on REIMS and LC-MS / MS

Abstract

The invention discloses a lipid oxidative metabolism characteristic analysis method and system based on REIMS and LC-MS / MS, and belongs to the technical field of lipid oxidative metabolism detection. In order to solve the detection problems of various oxidized lipids, extremely low content in a biological sample and large metabolic fluctuation, the method provided by the invention optimizes a biological sample pretreatment process, sets accurate REIMS and LC-MS / MS technical parameters, adopts two mass spectrometry technologies for combined detection and adopts an internal standard method for quantification, so that the content of the oxidized lipids in the biological sample is greatly reduced, and the content of the oxidized lipids in the biological sample is greatly reduced. And accurate qualitation and quantification of trace lipid oxidation products in a biological sample are realized. The method has the advantages that the trace lipid oxidation products in the biological sample can be accurately qualitative and quantitative, and reliable data support is provided for mining novel biomarkers.

Description

Technical Field

[0001] This invention relates to the field of lipid oxidation metabolism detection technology, and in particular to a method and system for analyzing lipid oxidation metabolism characteristics based on REIMS and LC-MS / MS. Background Technology

[0002] Lipid oxidation is a ubiquitous metabolic process in living organisms. Oxidized lipids, as products of lipid oxidation, participate in various physiological and pathological processes, such as inflammatory responses, cardiovascular diseases, and tumor development. Therefore, oxidized lipids are considered potential biomarkers, and their accurate detection is of great significance for early disease diagnosis, disease monitoring, and drug development.

[0003] However, the detection of oxidized lipids faces many challenges:

[0004] First, there are many types of oxidized lipids. The oxidation products of unsaturated fatty acids alone include hundreds of types such as hydroxy fatty acids, epoxy fatty acids, ketone fatty acids, prostaglandins, and leukotrienes.

[0005] Secondly, the content of oxidized lipids in biological samples is extremely low, mostly in trace amounts (ng / mL or even pg / mL), and is easily affected by matrix interference.

[0006] Third, lipid oxidative metabolism fluctuates greatly, and the content varies significantly under different physiological and pathological conditions, which places extremely high demands on the sensitivity and accuracy of detection methods.

[0007] Existing methods for detecting oxidized lipids mainly include gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS / MS).

[0008] GC-MS requires derivatization of oxidized lipids, which is cumbersome and time-consuming. Furthermore, the derivatization process may alter the structure of oxidized lipids, affecting the accuracy of detection.

[0009] Although traditional LC-MS / MS does not require derivatization, it is limited by the sensitivity and resolution of the mass spectrometer, making it difficult to accurately quantify trace amounts of oxidized lipids in biological samples.

[0010] Rapid evaporation ionization mass spectrometry (REIMS) is a novel in-situ mass spectrometry technique with the advantages of rapid ionization and no need for complex pretreatment. However, when used alone, its qualitative accuracy is insufficient and it is easily affected by matrix interference, leading to false positive results.

[0011] Therefore, developing an analytical method that combines high sensitivity, high accuracy, and high stability to accurately characterize and quantify trace oxidized lipids in biological samples has become a pressing technical problem to be solved in this field. Summary of the Invention

[0012] The purpose of this invention is to provide a method and system for analyzing lipid oxidation metabolism characteristics based on REIMS and LC-MS / MS. This invention has the advantage of enabling accurate qualitative and quantitative analysis of trace lipid oxidation products in biological samples, providing reliable data support for the discovery of novel biomarkers.

[0013] The technical solution of the present invention:

[0014] A method for analyzing lipid oxidation metabolism characteristics based on REIMS and LC-MS / MS includes the following steps:

[0015] A. Pretreatment of biological samples: Mix biological samples with extraction reagent at a volume ratio of 1:2-1:8, centrifuge at 4℃ and 10000-15000r / min for 10-20min, take the supernatant and filter it through a 0.22μm organic phase filter membrane to obtain the sample to be tested;

[0016] B. Rapid Evaporation Ionization Mass Spectrometry (REIMS) Detection: The sample to be detected is introduced into a REIMS mass spectrometer equipped with an iKnife ion source, the iKnife and ionization parameters are set, and the raw REIMS mass spectrometry data is obtained.

[0017] C. Detection by liquid chromatography-mass spectrometry (LC-MS / MS): The sample to be tested in step A is introduced into the liquid chromatography-mass spectrometry system, separated by a reversed-phase column, and the chromatographic and mass spectrometric parameters are set to obtain raw LC-MS / MS mass spectrometry data.

[0018] D. Qualitative and quantitative analysis: The raw REIMS mass spectrometry data and LC-MS / MS raw mass spectrometry data were analyzed together. Accurate qualitative analysis of oxidized lipids was achieved by comparing with standard mass spectra. Quantitative calculation was performed using the internal standard method to obtain the qualitative results and quantitative concentration values ​​of trace lipid oxidation products in biological samples.

[0019] In the aforementioned lipid oxidation metabolism characteristic analysis method based on REIMS and LC-MS / MS, in step A, the extraction agent is a methanol-acetonitrile mixture, and the volume ratio of methanol to acetonitrile is 1:1-3:2.

[0020] In the aforementioned lipid oxidation metabolism characterization analysis method based on REIMS and LC-MS / MS, the biological sample pretreatment in step A further includes biological sample pretreatment, the details of which are as follows:

[0021] The biological samples include at least one or more of the following: serum samples, plasma samples, tissue homogenate samples, and cell lysate samples;

[0022] Serum / plasma samples: Take 50-100 μL of serum / plasma, add 200-400 μL of extraction reagent, vortex for 3-5 min and then centrifuge;

[0023] Tissue homogenate samples: Prepare a homogenate by mixing tissue samples with physiological saline at a mass-to-volume ratio of 1:5-1:10. Take 100-200 μL of homogenate, add 400-600 μL of extraction reagent, vortex for 5-8 min, and then centrifuge.

[0024] Cell lysis buffer sample: Take 100-150 μL of cell lysis buffer, add 300-450 μL of extraction reagent, vortex for 4-6 min and then centrifuge.

[0025] In the aforementioned lipid oxidation metabolism characterization method based on REIMS and LC-MS / MS, the iKnife and ionization parameters in step B are specifically as follows:

[0026] The iKnife cutting head temperature is 400-500℃, the cutting speed is 1-3mm / s, the distance between the ion source and the cutting head is 3-5mm, the ionization voltage is 3.5-5.0kV, the atomizing gas pressure is 0.3-0.6MPa, the drying gas temperature is 120-180℃, the drying gas flow rate is 5-10L / min, the ion transmission tube temperature is 200-280℃, and the scanning range is 100-1200 m / z.

[0027] In the aforementioned method for analyzing lipid oxidation metabolism characteristics based on REIMS and LC-MS / MS, in step C, the reversed-phase chromatographic column is a C18 column with dimensions of 2.1 mm × 100 mm and a particle size of 1.7 μm; the chromatographic parameters are as follows:

[0028] Mobile phase A: 0.1% (v / v) formic acid aqueous solution;

[0029] Mobile phase B: 0.1% (v / v) formic acid acetonitrile solution;

[0030] Flow rate: 0.2-0.3 mL / min;

[0031] Column temperature: 35-40℃;

[0032] Injection volume: 5-10 μL;

[0033] Gradient elution program: 0-2 min, 30%-40% B; 2-8 min, 40%-70% B; 8-12 min, 70%-90% B; 12-15 min, 90% B; 15-16 min, 90%-30% B; 16-20 min, 30% B.

[0034] In the aforementioned lipid oxidation metabolism characterization method based on REIMS and LC-MS / MS, the mass spectrometry parameters of LC-MS / MS in step C are as follows:

[0035] Ion source: Electrospray ionization (ESI), positive ion mode;

[0036] Ion source temperature: 500-550℃;

[0037] Spray voltage: 5500-6000V;

[0038] Air curtain pressure: 20-30 psi;

[0039] Nebulizer gas pressure: 50-60 psi;

[0040] Drying gas pressure: 55-65 psi;

[0041] Scanning mode: Multiple response monitoring (MRM);

[0042] Collision gas: Nitrogen;

[0043] Collision energy: 15-45 eV;

[0044] Declustering voltage: 30-80V.

[0045] In the aforementioned lipid oxidation metabolism characterization analysis method based on REIMS and LC-MS / MS, the internal standard used in step D is a deuterated lipid oxidation standard, which includes deuterated 13-hydroxyoctadecadienoic acid (13-HODE-d4), deuterated 9-hydroxyoctadecadienoic acid (9-HODE-d4), and deuterated prostaglandin E2 (PGE2-d4), with an internal standard concentration of 50 ng / mL.

[0046] In the aforementioned lipid oxidation metabolism characterization analysis methods based on REIMS and LC-MS / MS, the method for constructing the quantitative standard curve in step D is specifically as follows:

[0047] A series of lipid oxidization standard solutions with concentrations ranging from 0.1 to 1000 ng / mL were prepared. After adding an equal amount of internal standard, LC-MS / MS was performed. The standard curve equation was obtained by using weighted least squares linear regression with the standard concentration as the x-axis and the peak area ratio of the standard to the internal standard as the y-axis.

[0048] A lipid oxidation metabolism characterization analysis system based on REIMS and LC-MS / MS includes a sample preprocessing module, a REIMS detection module, an LC-MS / MS detection module, and a data processing module;

[0049] The sample pretreatment module is used to extract, centrifuge, and filter biological samples to obtain samples to be tested.

[0050] The REIMS detection module uses a rapid evaporation ionization mass spectrometer to perform preliminary ionization detection on the sample to be tested and obtain raw REIMS mass spectrometry data.

[0051] The LC-MS / MS detection module uses an AB SCIEX 6500Qtrap liquid chromatography-tandem mass spectrometer, equipped with a C18 reversed-phase column, for efficient separation and high-sensitivity mass spectrometry detection of the sample to be tested, and to obtain raw LC-MS / MS mass spectrometry data;

[0052] The data processing module is used to perform joint analysis of REIMS raw mass spectrometry data and LC-MS / MS raw mass spectrometry data to achieve qualitative and quantitative calculations of oxidized lipids and output detection results.

[0053] In the aforementioned lipid oxidation metabolism characterization system based on REIMS and LC-MS / MS, the REIMS detection module includes an iKnife front-end ion source.

[0054] Compared with the prior art, the beneficial effects of this application are as follows:

[0055] 1. This invention employs a combined detection technique of REIMS and LC-MS / MS, which combines the advantages of rapid ionization of REIMS and high sensitivity and high resolution of LC-MS / MS, thus solving the problem of insufficient sensitivity or inaccurate qualitative analysis when detecting oxidized lipids using a single mass spectrometry technique.

[0056] 2. The biological sample pretreatment process and mass spectrometry detection parameters were optimized, which significantly improved the detection sensitivity. The detection limit can reach 0.01-0.05 ng / mL, and the quantitation limit can reach 0.03-0.15 ng / mL, which can realize the accurate detection of trace oxidized lipids in biological samples.

[0057] 3. The internal standard method is used for quantification, which effectively eliminates the influence of matrix interference and instrument fluctuations on the detection results. The relative standard deviation (RSD) is ≤8.5%, and the spiked recovery rate is 85.2%-112.6%, which has high accuracy and high stability.

[0058] 4. The detection process is simple and fast, requiring no derivatization, and can simultaneously detect multiple oxidized lipids. It is suitable for different types of biological samples, providing a reliable technical means for the discovery of biomarkers related to lipid oxidation metabolism, and has broad application prospects. Attached Figure Description

[0059] Figure 1 This is a flowchart illustrating the analytical method of the present invention. Detailed Implementation

[0060] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0061] Example 1. Analysis of lipid oxidation metabolism characteristics based on human serum

[0062] (I) Experimental Materials and Instruments

[0063] 1. Experimental materials

[0064] Oxidized lipid standards: 13-hydroxyoctadecadienoic acid (13-HODE), 9-hydroxyoctadecadienoic acid (9-HODE), prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and epoxy eicosatotrienoic acid (EETs), with a purity of ≥98%, purchased from Sigma-Aldrich.

[0065] Internal standards: deuterated 13-HODE (13-HODE-d4), deuterated 9-HODE (9-HODE-d4), and deuterated PGE2 (PGE2-d4), with a purity of ≥98%, purchased from Cayman Chemical.

[0066] Reagents: Methanol, acetonitrile (chromatographic grade, Merck), formic acid (mass spectrometry grade, Thermo Fisher Scientific), physiological saline (analytical grade, Sinopharm Group);

[0067] Biological samples: serum from healthy individuals, homogenate of mouse liver tissue, and lysate of human hepatocellular carcinoma cells (HepG2), all derived from clinical sample banks or laboratory cultures.

[0068] 2. Experimental apparatus

[0069] Rapid evaporation ionization mass spectrometer (REIMS, model: Xevo REIMS, Waters Corporation);

[0070] Liquid chromatography-tandem mass spectrometry (LC-MS / MS, model AB SCIEX 6500Qtrap, AB SCIEX).

[0071] High-speed refrigerated centrifuge (model: Centrifuge 5810R, Eppendorf).

[0072] Vortex oscillator (Model: Vortex-Genie 2, Scientific Industries);

[0073] Ultrapure water system (Model: Milli-Q IQ 7000, Merck Millipore);

[0074] Pipettes (0.5-10μL, 10-100μL, 100-1000μL, Eppendorf).

[0075] (II) Experimental Methods

[0076] Methods for analyzing lipid oxidation metabolism characteristics based on REIMS and LC-MS / MS, the steps are as follows: Figure 1 As shown, the details are as follows:

[0077] 1. Biological sample pretreatment

[0078] Serum sample processing: Take 80 μL of healthy human serum and place it in a 1.5 mL centrifuge tube. Add 320 μL of extraction reagent (methanol:acetonitrile = 2:1, v / v), vortex for 4 min, centrifuge at 4℃ and 12000 r / min for 15 min, take the supernatant and filter it through a 0.22 μm organic phase filter membrane. Collect the filtrate as the sample to be tested, and add 50 ng / mL of internal standard mixture at the same time.

[0079] Tissue homogenate sample processing: Take 0.1g of mouse liver tissue, add 1mL of physiological saline, prepare homogenate using a tissue homogenizer, take 150μL of homogenate and place it in a 1.5mL centrifuge tube, add 450μL of extraction reagent (methanol:acetonitrile = 2:1, v / v), vortex for 6min, centrifuge at 4℃ and 13000r / min for 18min, take the supernatant and filter it through a 0.22μm organic phase filter membrane, collect the filtrate as the sample to be tested, and add 50ng / mL of internal standard mixture;

[0080] Cell lysate sample processing: Take 120 μL of HepG2 cell lysate and place it in a 1.5 mL centrifuge tube. Add 360 μL of extraction reagent (methanol:acetonitrile = 2:1, v / v), vortex for 5 min, and centrifuge at 4℃ and 14000 r / min for 12 min. Take the supernatant and filter it through a 0.22 μm organic phase filter membrane. Collect the filtrate as the sample to be tested and add 50 ng / mL of internal standard mixture.

[0081] 2. Construction of Standard Curve

[0082] Accurately weigh appropriate amounts of each oxidized lipid standard, dissolve and dilute with methanol to prepare a standard stock solution with a concentration of 1000 ng / mL, and store at -20℃. When using, dilute the standard stock solution with extraction solvent to prepare a series of standard working solutions with concentrations of 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL.

[0083] 50 ng / mL of internal standard solution was added to each concentration of standard working solution, and the analysis was performed according to the subsequent LC-MS / MS detection conditions. The standard concentration (x) was plotted on the x-axis, and the peak area ratio of the standard to the internal standard (y) was plotted on the y-axis. Weighted least squares method (weighting coefficient 1 / x) was used. 2 Linear regression was performed to obtain the standard curve equations and correlation coefficients (R²) for each oxidized lipid. 2 ).

[0084] 3. REIMS detection

[0085] The sample to be tested was imported into a REIMS mass spectrometer equipped with an iKnife ion source. The detection parameters were set as follows: iKnife tip temperature 450℃, tip cutting speed 2mm / s, distance between ion source and tip 4mm, ionization voltage 4.2kV, nebulizing gas pressure 0.45MPa, drying gas temperature 150℃, drying gas flow rate 7.5L / min, ion transmission tube temperature 240℃, and scanning range m / z 100-1200. Each sample was scanned 3 times, and the average value was taken as the raw REIMS mass spectrometry data.

[0086] 4. LC-MS / MS detection

[0087] (1) Chromatographic conditions: A C18 reversed-phase column (2.1 mm × 100 mm, 1.7 μm) was used. The mobile phase A was 0.1% formic acid aqueous solution, the mobile phase B was 0.1% formic acid acetonitrile solution, the flow rate was 0.25 mL / min, the column temperature was 37 °C, and the injection volume was 8 μL.

[0088] The gradient elution program is as follows: 0-2 min, 35% B; 2-8 min, 35%-70% B; 8-12 min, 70%-90% B; 12-15 min, 90% B; 15-16 min, 90%-35% B; 16-20 min, 35% B.

[0089] (2) Mass spectrometry conditions: The ion source was an electrospray ion source (ESI), positive ion mode, ion source temperature 520℃, spray voltage 5800V, curtain gas pressure 25psi, nebulizer gas pressure 55psi, drying gas pressure 60psi, scanning mode was multiple reaction monitoring (MRM), and collision gas was nitrogen.

[0090] The MRM parameters of each oxidized lipid are shown in Table 1.

[0091] 5. Qualitative and quantitative analysis

[0092] Qualitative analysis: The REIMS and LC-MS / MS mass spectra of the sample to be tested are compared with the mass spectra of the standard. The oxidized lipids are accurately identified based on the retention time and mass-to-charge ratio (m / z) of the characteristic ion peaks. If the sample to be tested shows the same retention time and characteristic ion peak as the standard, it is determined that the sample contains the corresponding oxidized lipids.

[0093] Quantitative analysis: Based on the constructed standard curve equation, the peak area ratio of oxidized lipids to internal standard in the sample to be tested is substituted to calculate the concentration value of each oxidized lipid in the sample to be tested.

[0094] 6. Methodological Validation

[0095] The method of this invention was validated, including indicators such as limit of detection (LOD), limit of quantitation (LOQ), precision, accuracy, and stability.

[0096] Limit of detection and limit of quantitation: The limit of detection is the concentration corresponding to a signal-to-noise ratio (S / N) of 3, and the limit of quantitation is the concentration corresponding to an S / N of 10.

[0097] Precision: Standard working solutions at three concentrations (1 ng / mL, 50 ng / mL, and 500 ng / mL) were used, and each concentration was measured in parallel six times. The intra-day precision (RSD) was calculated. r ) and daytime precision (RSD) R (Continuous testing for 3 days)

[0098] Accuracy: A spiked recovery experiment was conducted. Biological samples of known concentrations were taken, and standards of low, medium, and high concentrations were added respectively. The spiked recovery rate was then calculated.

[0099] Stability: The same sample was placed at room temperature for 0h, 2h, 4h, 6h, 8h, and 12h before testing. The RSD of the peak area was calculated to assess the short-term stability of the sample. The sample was stored at -20℃ and tested after 0 days, 3 days, 7 days, 14 days, and 30 days. The RSD of the peak area was calculated to assess the long-term stability of the sample.

[0100] Table 1. MRM parameters of various oxidized lipids

[0101]

[0102] (III) Experimental Results

[0103] 1. Standard Curve Results

[0104] Standard curve equations and correlation coefficients (R) for various oxidized lipids 2 The results of the limit of detection (LOD) and limit of quantitation (LOQ) are shown in Table 2.

[0105] Table 2 Results of various lipid oxidation methods

[0106]

[0107] Table 2 shows that the various oxidized lipids exhibited good linearity within the concentration range of 0.1-1000 ng / mL, with R... 2 All values ​​were ≥0.995, LOD was 0.01-0.05 ng / mL, and LOQ was 0.03-0.15 ng / mL, indicating that the method of the present invention has a good linear range and high sensitivity.

[0108] 2. Precision results

[0109] The results of the precision experiment are shown in Table 3.

[0110] Table 3 Precision Experiment Results

[0111]

[0112] As shown in Table 3, the intraday RSD for low, medium, and high concentrations is 2.3%-7.2%, and the interday RSD is 3.5%-8.5%, all ≤8.5%, indicating that the method of the present invention has good precision.

[0113] 3. Accuracy Results

[0114] The results of the spiked recovery experiment are shown in Table 4.

[0115] Table 4 Results of Spiked Recovery Experiment

[0116]

[0117] As shown in Table 4, the recoveries of spiked samples at low, medium, and high concentrations ranged from 85.2% to 112.6%, which meet the accuracy requirements for trace analysis, indicating that the method of the present invention has good accuracy.

[0118] 4. Stability Results

[0119] Stability test results showed that the peak area RSD of the sample was 2.1%-5.8% within 12 hours of being placed at room temperature; and the peak area RSD was 3.2%-7.3% within 30 days of storage in a -20℃ refrigerator, both ≤8%, indicating that the sample has good stability during detection and storage and will not affect the accuracy of the detection results.

[0120] 5. Actual biological sample test results

[0121] The method of this invention was used to detect oxidized lipids in serum of healthy individuals, homogenate of mouse liver tissue, and lysate of HepG2 cells. The results are shown in Table 5.

[0122] Table 5 Results of lipid oxidation detection

[0123]

[0124] As shown in Table 5, the method of the present invention can successfully detect a variety of oxidized lipids in different biological samples, and the detection results are stable and reliable, proving that the method of the present invention is applicable to the lipid oxidation metabolism characteristic analysis of a variety of biological samples.

[0125] Example 2. Analysis of lipid oxidation metabolism characteristics in serum from a mouse inflammation model.

[0126] 1. Experimental Materials and Methods

[0127] Biological samples: serum from a mouse inflammation model (an inflammation model was constructed by intraperitoneal injection of lipopolysaccharide) and serum from normal mice, with 3 parallel samples from each;

[0128] Other experimental materials and instruments are the same as in Example 1.

[0129] The sample pretreatment, REIMS detection, and LC-MS / MS detection parameters were all consistent with those in Example 1.

[0130] 2. Experimental Results

[0131] The test results are shown in Table 6.

[0132] Table 6 Test Results

[0133]

[0134] As shown in Table 6, compared with normal mouse serum, the concentrations of 13-HODE, 9-HODE, PGE2, and LTB4 in the serum of inflammatory model mice were significantly increased (P<0.05), with the LTB4 concentration increasing from 0.48±0.03 ng / mL to 2.85±0.12 ng / mL, an increase of 493.8%.

[0135] These results indicate that the method of the present invention can effectively distinguish the differences in lipid oxidation metabolism between pathological and normal states, and can provide reliable data for the mining of biomarkers for inflammation-related diseases, further verifying the clinical application value of the method of the present invention.

Claims

1. A method for analyzing lipid oxidation metabolism characteristics based on REIMS and LC-MS / MS, characterized in that, Includes the following steps: A. Pretreatment of biological samples: Mix biological samples with extraction reagent at a volume ratio of 1:2-1:8, centrifuge at 4℃ and 10000-15000r / min for 10-20min, take the supernatant and filter it through a 0.22μm organic phase filter membrane to obtain the sample to be tested; B. Rapid Evaporation Ionization Mass Spectrometry (REIMS) Detection: The sample to be detected is introduced into a REIMS mass spectrometer equipped with an iKnife ion source, the iKnife and ionization parameters are set, and the raw REIMS mass spectrometry data is obtained. C. Detection by liquid chromatography-mass spectrometry (LC-MS / MS): The sample to be tested in step A is introduced into the liquid chromatography-mass spectrometry system, separated by a reversed-phase column, and the chromatographic and mass spectrometric parameters are set to obtain raw LC-MS / MS mass spectrometry data. D. Qualitative and quantitative analysis: The raw REIMS mass spectrometry data and LC-MS / MS raw mass spectrometry data were analyzed together. Accurate qualitative analysis of oxidized lipids was achieved by comparing with standard mass spectra. Quantitative calculation was performed using the internal standard method to obtain the qualitative results and quantitative concentration values ​​of trace lipid oxidation products in biological samples.

2. The method for analyzing lipid oxidation metabolism characteristics based on REIMS and LC-MS / MS according to claim 1, characterized in that: In step A, the extractant is a methanol-acetonitrile mixture, with a volume ratio of methanol to acetonitrile of 1:1 to 3:

2.

3. The method for analyzing lipid oxidation metabolism characteristics based on REIMS and LC-MS / MS according to claim 1, characterized in that, Step A, the biological sample pretreatment, also includes biological sample pretreatment, the details of which are as follows: The biological samples include at least one or more of the following: serum samples, plasma samples, tissue homogenate samples, and cell lysate samples; Serum / plasma samples: Take 50-100 μL of serum / plasma, add 200-400 μL of extraction reagent, vortex for 3-5 min and then centrifuge; Tissue homogenate samples: Prepare a homogenate by mixing tissue samples with physiological saline at a mass-to-volume ratio of 1:5-1:

10. Take 100-200 μL of homogenate, add 400-600 μL of extraction reagent, vortex for 5-8 min, and then centrifuge. Cell lysis buffer sample: Take 100-150 μL of cell lysis buffer, add 300-450 μL of extraction reagent, vortex for 4-6 min and then centrifuge.

4. The method for analyzing lipid oxidation metabolism characteristics based on REIMS and LC-MS / MS according to claim 1, characterized in that, The specific iKnife and ionization parameters in step B are as follows: The iKnife cutting head temperature is 400-500℃, the cutting speed is 1-3mm / s, the distance between the ion source and the cutting head is 3-5mm, the ionization voltage is 3.5-5.0kV, the atomizing gas pressure is 0.3-0.6MPa, the drying gas temperature is 120-180℃, the drying gas flow rate is 5-10L / min, the ion transmission tube temperature is 200-280℃, and the scanning range is 100-1200 m / z.

5. The method for analyzing lipid oxidation metabolism characteristics based on REIMS and LC-MS / MS according to claim 1, characterized in that, In step C, the reversed-phase chromatographic column is a C18 column with dimensions of 2.1 mm × 100 mm and a particle size of 1.7 μm; the chromatographic parameters are as follows: Mobile phase A: 0.1% (v / v) formic acid aqueous solution; Mobile phase B: 0.1% (v / v) formic acid acetonitrile solution; Flow rate: 0.2-0.3 mL / min; Column temperature: 35-40℃; Injection volume: 5-10 μL; Gradient elution program: 0-2 min, 30%-40% B; 2-8 min, 40%-70% B; 8-12 min, 70%-90% B; 12-15 min, 90% B; 15-16 min, 90%-30% B; 16-20 min, 30% B.

6. The method for analyzing lipid oxidation metabolism characteristics based on REIMS and LC-MS / MS according to claim 1, characterized in that, The mass spectrometry parameters of the LC-MS / MS mentioned in step C are as follows: Ion source: Electrospray ionization (ESI), positive ion mode; Ion source temperature: 500-550℃; Spray voltage: 5500-6000V; Air curtain pressure: 20-30 psi; Nebulizer gas pressure: 50-60 psi; Drying gas pressure: 55-65 psi; Scanning mode: Multiple response monitoring (MRM); Collision gas: Nitrogen; Collision energy: 15-45 eV; Declustering voltage: 30-80V.

7. The method for analyzing lipid oxidation metabolism characteristics based on REIMS and LC-MS / MS according to claim 1, characterized in that: The internal standard used in step D is a deuterated lipid oxide standard, which includes deuterated 13-hydroxyoctadecadienoic acid, deuterated 9-hydroxyoctadecadienoic acid, and deuterated prostaglandin E2, with a concentration of 50 ng / mL.

8. The method for analyzing lipid oxidation metabolism characteristics based on REIMS and LC-MS / MS according to claim 1, characterized in that, The method for constructing the standard curve for quantitative calculation described in step D is as follows: A series of lipid oxidization standard solutions with concentrations ranging from 0.1 to 1000 ng / mL were prepared. After adding an equal amount of internal standard, LC-MS / MS was performed. The standard curve equation was obtained by using weighted least squares linear regression with the standard concentration as the x-axis and the peak area ratio of the standard to the internal standard as the y-axis.

9. A lipid oxidation metabolism characterization analysis system based on REIMS and LC-MS / MS according to any one of claims 1-8, characterized in that: It includes a sample preprocessing module, a REIMS detection module, an LC-MS / MS detection module, and a data processing module; The sample pretreatment module is used to extract, centrifuge, and filter biological samples to obtain samples to be tested. The REIMS detection module uses a rapid evaporation ionization mass spectrometer to perform preliminary ionization detection on the sample to be tested and obtain raw REIMS mass spectrometry data. The LC-MS / MS detection module uses an AB SCIEX 6500Qtrap liquid chromatography-tandem mass spectrometer, equipped with a C18 reversed-phase column, for efficient separation and high-sensitivity mass spectrometry detection of the sample to be tested, and to obtain raw LC-MS / MS mass spectrometry data; The data processing module is used to perform joint analysis of REIMS raw mass spectrometry data and LC-MS / MS raw mass spectrometry data to achieve qualitative and quantitative calculations of oxidized lipids and output detection results.

10. A lipid oxidation metabolism characterization analysis system based on REIMS and LC-MS / MS according to claim 9, characterized in that: The REIMS detection module includes an iKnife front-end ion source.

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