Method for analyzing lipid in biological sample

Abstract

The invention provides a method for analyzing lipid in a biological sample. The method comprises the processes of extraction of lipid in a biological sample, separation and purification, a high-resolution mass spectrometry detection method and the like, and specifically comprises the following steps: after the lipid in the biological sample is extracted, thin-layer chromatography rapid analysis is carried out, and the type of the contained lipid is determined; determining a silica gel column separation method and steps by combining an analysis result of thin-layer chromatography; and further separating and purifying the lipid by using a silica gel chromatographic column, eluting and separating by using liquid chromatography, and performing high-resolution mass spectrometry detection and analysis. The method further comprises the step of analyzing and processing the detected lipid by using software, and finally determining lipid type information. According to the method, the purity of the lipid and the separation degree of isomers of the lipid can be remarkably improved, a low-abundance lipid signal is improved, the lipid co-outflow behavior is reduced, a high-definition secondary mass spectrum is obtained, the accuracy and coverage rate of qualitative and quantitative analysis of the lipid are effectively improved, and the method is easy to operate, high in repeatability and high in universality.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry and lipidomics research, and particularly relates to a method for analyzing lipids in a biological sample. Background Art

[0002] Lipid compounds are very important substances in living organisms. As the main component of biological membranes, lipid compounds have molecular diversity and multiple biological functions, including providing a suitable environment for protein interactions, storing energy, and serving as important messenger molecules. In recent years, lipid compounds have attracted much attention in the study of various diseases and various biological problems, such as cell apoptosis, signal transduction, disease infection, immune function, growth and development, food nutrition, etc. At the same time, lipid metabolism is also closely related to diabetes, liver cancer, kidney disease, breast cancer and many other diseases.

[0003] Lipids are of many types, with complex and diverse structures, and there are a large number of isomers, including acyl isomers, double bond isomers, etc. At present, the identification of lipids mainly relies on high-quality secondary mass spectra for structural prediction. Therefore, obtaining high-definition and high-quality secondary mass spectra is the key to accurate lipid identification. Ultra-high performance liquid chromatography (HPLC)-high-resolution mass spectrometry (HPLC-HRMS) achieves effective separation of lipids through chromatography, and combines the high selectivity, high sensitivity, and qualitative ability of mass spectrometry to achieve the complementary advantages of chromatography and mass spectrometry. It is the most widely used and key application platform for lipidomics research. In the data-dependent acquisition (DDA) mode, the mass spectrometer performs a full scan and then performs a secondary scan on the parent ion (intensity-dependent) list selected in the full scan mass spectrum. It is currently the most widely used scanning mode for lipid identification. Relying on data intensity for ion selection analysis is not friendly to the detection of low-abundance and low-response lipids. At the same time, the DDA scanning mode usually also sets an isolation window of 0.5 to 3 m / z, so that its fragment spectrum is also derived from the convolution fragment peaks of multiple parent ions. The characteristics of lipids determine that if only one chromatographic column is used for separation, there will inevitably be a large amount of co-elution between and within lipid classes, which will affect the purity of the data points collected by mass spectrometry. Although mass spectrometry data can usually be deconvolved by mathematical algorithms through data processing software to re-parse the components that are not separated by chromatography and restore their true mass spectrometry information. However, deconvolution is based on algorithms and errors will still occur. Therefore, full coverage and high-accuracy qualitative and quantitative analysis of lipids in biological samples remains a challenge. Summary of the invention

[0004] The purpose of this application is to provide a universal deep lipid detection and analysis method and operation process in biological samples to solve the current problems of low-definition secondary mass spectrometry quality caused by lipid co-elution, and low response signals of low-abundance lipids that are difficult to detect, which affects the comprehensive characterization of lipids in biological samples.

[0005] In order to achieve this purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for analyzing lipids in a biological sample, and the method for analyzing lipids in a biological sample comprises the following steps:

[0007] (1) extracting lipids from a biological sample to obtain a first sample;

[0008] (2) determining the lipid categories of the first sample by thin layer chromatography, wherein the lipid categories include non-polar lipids, medium-polar lipids, and polar lipids;

[0009] (3) Based on the lipid type and content in (2), the first sample is separated and purified using a silica gel chromatography column to obtain a second sample:

[0010] (4) The second sample is subjected to gradient elution by liquid chromatography and then subjected to mass spectrometry analysis.

[0011] In some embodiments, the silica gel chromatographic column described in step (3) is an NH2-based SPE column, and the eluent of the silica gel chromatographic column includes one or more of a n-hexane phase, an isopropanol:dichloromethane (3:7 (v / v)) phase, and a methanol phase; wherein the n-hexane phase is used to elute non-polar lipids; the isopropanol:dichloromethane (3:7 (v / v)) phase is used to elute medium-polar lipids; and the methanol phase is used to elute polar lipids.

[0012] In some embodiments, the elution volume of each eluent is 3 to 6 column volumes.

[0013] In some embodiments, the silica gel chromatography column is activated with 6 column volumes of n-hexane.

[0014] In some embodiments, in step (3), the first sample is concentrated to dryness, re-dissolved in n-hexane and then loaded.

[0015] In some embodiments, step (3) comprises concentrating the first sample to dryness, re-dissolving it with n-hexane and then loading it; eluting weak polar lipids with n-hexane, eluting medium polar lipids with isopropanol: dichloromethane (3:7 (v / v)), and eluting strong polar lipids with methanol, and the elution volume is 6-12 times the column volume.

[0016] In some embodiments, the eluate obtained by separating and purifying the first sample using a silica gel chromatography column in step (3) is concentrated to dryness, the methanol phase eluted sample is re-dissolved with pure methanol, the n-hexane phase eluted sample or the isopropanol: dichloromethane (3:7 (v / v)) phase eluted sample is re-dissolved with dichloromethane: methanol (1:1 (v / v)), and a filter membrane is used; as a preferred embodiment, the filter membrane is a 0.22 micron filter membrane.

[0017] In some embodiments, the second sample includes one or more of a n-hexane phase elution sample, an isopropanol:dichloromethane (3:7 (v / v)) phase elution sample, and a methanol phase elution sample; in step (4), the chromatographic column used in the liquid chromatography includes one or more of a C30 chromatographic column, a C18 chromatographic column, and a HILIC chromatographic column, wherein the C30 chromatographic column is used to separate and purify the n-hexane phase elution sample, the C18 chromatographic column is used to separate and purify the isopropanol:dichloromethane (3:7 (v / v)) phase elution sample, and the HILIC chromatographic column is used to separate and purify the methanol phase elution sample.

[0018] In some embodiments, the mobile phase system of the C30 chromatographic column or the C18 chromatographic column is:

[0019] A1: acetonitrile: water (6:4 (v / v));

[0020] B1: isopropanol:acetonitrile (9:1 (v / v)), 5-10 mM ammonium formate and 0.1%-0.2% (v / v) formic acid were added to A1 and B1;

[0021] The mobile phase system of the HILIC column is:

[0022] A2: acetonitrile: water (1:3 (v / v));

[0023] B2: acetonitrile: water (95:5 (v / v)), 5-10 mM ammonium formate and 0.1%-0.2% (v / v) formic acid were added to A2 and B2.

[0024] In the present invention, the elution gradient of the chromatography can be adjusted according to the lipids to be analyzed to obtain a good chromatographic separation effect.

[0025] In some embodiments, the liquid chromatography elution procedure in step (4) is:

[0026] Elution program of C30 column: at 0 min, mobile phase B1 50% v / v; at 4 min, mobile phase B1 70% v / v; at 13 min, mobile phase B1 75% v / v; at 24 min, mobile phase B1 85% v / v; at 33 min, mobile phase B1 90% v / v; at 36 min, mobile phase B1 92% v / v; at 37 min, mobile phase B1 95% v / v; at 46 min, mobile phase B1 95% v / v; at 46.1 min, mobile phase B1 50% v / v; at 50 min, mobile phase B1 50% v / v;

[0027] C18 column elution program: at 0 min, mobile phase B1 40% v / v; at 3 min, mobile phase B1 50% v / v; at 6 min, mobile phase B1 62% v / v; at 14 min, mobile phase B1 72% v / v; at 15 min, mobile phase B1 85% v / v; at 16 min, mobile phase B1 95% v / v; at 19 min, mobile phase B1 95% v / v; at 19.1 min, mobile phase B1 40% v / v; at 22 min, mobile phase B1 40% v / v;

[0028] HILIC column elution program: at 0 min, mobile phase B2 99% v / v; at 6 min, mobile phase B2 94% v / v; at 11 min, mobile phase B2 92% v / v; at 16 min, mobile phase B2 88% v / v; at 15 min, mobile phase B2 85% v / v; at 16 min, mobile phase B2 95% v / v; at 19 min, mobile phase B2 95% v / v; at 19.1 min, mobile phase B2 40% v / v; at 22 min, mobile phase B2 40% v / v.

[0029] In some embodiments, the length of the chromatographic column used in the liquid chromatography is 100-150 mm; the injection volume is 2-10 μL, the column temperature is 30-50° C., and the flow rate is 0.2-0.4 mL / min.

[0030] In some embodiments, in step (4), the mass spectrum is acquired in CID mode to obtain the accurate ion mass-to-charge ratio, retention time and high-definition secondary mass spectrum of the lipid primary mass spectrum.

[0031] In some embodiments, in step (4), the mass spectrometer is collected in the following manners: using positive ion mode to detect and analyze the effluent after separation and purification by a C30 chromatographic column; using both positive and negative modes to detect and analyze the effluent after separation and purification by a C18 chromatographic column; using both positive and negative modes to detect and analyze the effluent after separation and purification by a HILIC chromatographic column.

[0032] In some embodiments, the mass spectrometer instrument comprises Thermo Scientific Q Exactive, Waters or Agilent.

[0033] In some embodiments, the primary scanning range of the mass spectrometer is 50-1200 m / z, and the secondary acquisition mass range is 50-1200 m / z.

[0034] In some embodiments, the mass spectrometry parameters of the Thermo Scientific Q Exactive are: heated electrospray ion source, a. the voltage in positive and negative ion modes is 3.0-4.0 kV; b. the ion transfer tube temperature is 300-320°C; c. the sheath gas pressure is 30-40 psi, and the auxiliary gas heating temperature is 300°C; d. the scanning mode is Full MS, NCE25-50eV.

[0035] In some embodiments, the mass spectrometry parameters of the Waters are: scan time of 0.5-1 s; high energy channel collision energy of 20-50 eV; ESI source, capillary voltage, 2.0-3.0 kv; ion source temperature, 90-110 °C; cone voltage, 35-45 V.

[0036] In some embodiments, the Agilent mass spectrometry parameters are: collision energy of 20-30 eV, gas temperature of 250°C, sheath gas temperature of 300°C; sheath gas flow rate of 12 L / min; capillary voltage positive ion mode 2500-3500 V; cone voltage 60-65 V; MS and MS / MS minimum acquisition rate 2-4 spectra / s.

[0037] In some embodiments, the mass spectrometry parameters of the Thermo Scientific Q Exactive are: heated electrospray ion source, collected in positive and negative modes respectively; a. the voltage in positive and negative ion modes is 4.00 KV; b. the ion transfer tube temperature is 320°C; c. the sheath gas pressure is 40psi and 30psi respectively, the auxiliary gas pressure is 10 arb, and the auxiliary gas heating temperature is 300°C; d. the scanning mode is Full MS, the instrument resolution is set to 70000, -dd / MS2, the resolution is 17500, NCE 30eV, and the scanning range is 50-1200m / z.

[0038] In some embodiments, the mass spectrometry parameters of the Waters are: mass range of 50~1200 m / z, scan time of 0.8 s; high energy channel collision energy of 20-50 eV; ESI source, positive (+) ion mode; capillary voltage, 2.5 kv(+); negative (-) ion mode; capillary voltage, 2.0 kv(-); ion source temperature, 100 ℃; cone voltage, 40 V.

[0039] In some embodiments, the mass spectrometry parameters of Agilent are: electrospray ion source, data-dependent mode data acquisition; primary mass spectrometry acquisition mass range m / z 100-1600, secondary mass spectrometry acquisition mass range m / z 50-1200; positive ion mode collision energy is 20 eV; negative ion mode collision energy is 25 eV; gas temperature is 250°C, sheath gas temperature is 300°C; sheath gas flow rate is 12 L / min; capillary voltage is 3000 V in positive ion mode and 3000 V in negative ion mode; fragmentation voltage is 150 V; cone voltage is 65 V; MS and MS / MS minimum acquisition rate is 3 spectra / s.

[0040] In some embodiments, the column temperature of the chromatography column is 35-50 °C.

[0041] In some embodiments, the injection volume of each HPLC column is 2-10 μL.

[0042] In some embodiments, the biological sample includes a sample comprising a lipid or a lipid analog.

[0043] In some embodiments, the biological sample includes any one of traditional Chinese medicine, biological tissue, food, in vitro detection sample of human or animal body, and artificial synthetic biological tissue.

[0044] In a specific embodiment of the present invention, the biological sample is milk or zebrafish brain tissue.

[0045] In the method of the present invention, the Folch method is preferably used to extract lipids from biological samples, but other lipid extraction methods are also acceptable.

[0046] In some embodiments, the method for extracting lipids from a biological sample in step (1) is as follows:

[0047] Add an extractant to the biological sample, ultrasonically disrupt or let it stand, then add water and centrifuge to collect the organic phase; repeat the extraction of the remaining part, combine the organic phases, remove the solvent, redissolve, filter, and obtain the first sample; the extractant includes methanol and dichloromethane.

[0048] In some embodiments, ultrasonic crushing is performed using an ultrasonic crusher. In some embodiments, the parameters of the ultrasonic crusher are: 10-20 s per crushing, 20-40 s interval per crushing, power 500-650 W, and total crushing time 10-30 min.

[0049] In some embodiments, the method for removing the solvent is nitrogen blowing.

[0050] In some embodiments, methanol and dichloromethane are used for reconstitution.

[0051] In some embodiments, the volume ratio of methanol to dichloromethane in the extractant is 1:1-1:2.

[0052] In some embodiments, the biological sample includes a solid sample and a liquid sample. As a preferred embodiment, the solid sample is ≥20 mg, and the liquid sample is ≥100 μL.

[0053] In some embodiments, step (2) comprises:

[0054] Spot the first sample and the standard using a silica gel thin layer chromatography plate;

[0055] The secondary developing method is used for development, wherein the first developing agent includes n-hexane and ether, and the second developing agent includes dichloromethane, isopropanol, methanol and formic acid. After development, the color is developed in a color developing tank after the reagents are completely volatilized. Preferably, the color developing agent of the secondary developing method is iodine.

[0056] In some embodiments, the spotting method is: using a silica gel thin layer chromatography plate to spot the first sample and the standard on a silica gel plate pre-dried at 60°C for 5 min: the spotting diameter is ≤3 mm, 10 mm from the bottom edge of the plate, the standard spotting volume is 10 μL, and the first sample spotting volume is 3 μL.

[0057] In some embodiments, the first developing solvent is a mixed solution of n-hexane and diethyl ether, and the second developing solvent is a mixed solution of dichloromethane, isopropanol, methanol and formic acid.

[0058] In some embodiments, the volume ratio of n-hexane to diethyl ether in the first developing solvent is 1:1.

[0059] In some embodiments, the volume ratio of dichloromethane, isopropanol, methanol and formic acid in the second developing solvent is 5:3:2:0.5.

[0060] In some embodiments, the secondary development method includes developing in a first developing agent to 45% of the height of the silica gel plate, taking out the silica gel plate from the development tank, and developing in a second developing agent to 90% of the height of the silica gel plate after the organic solvent is completely evaporated.

[0061] According to the thin layer chromatography result in step (2), the present invention can quickly determine the lipid types and contents in the sample by comparing the relative migration value of the lipid standard on the thin layer plate and the color depth of the color spots, thereby optimizing the lipid separation and purification method and process.

[0062] The present invention can classify lipids into non-polar lipids such as triglycerides, medium-polar lipids such as sphingolipids, diglycerides, sterol lipids, and polar lipids such as phospholipids according to the moving distance of lipids in the thin layer plate.

[0063] The present invention separates sample lipids into three parts: non-polar lipids, medium-polar lipids and polar lipids, and further optimizes lipid separation by screening liquid chromatography columns and optimizing the elution gradient of the chromatography columns.

[0064] In some embodiments, the method for analyzing lipids in a biological sample further comprises step (5): analyzing mass spectrometry data.

[0065] In some embodiments, the step (5) includes: using software to deconvolute the mass spectrometry data, matching the lipid database for processing and analysis, preliminarily selecting possible lipid categories, types, and fatty acid chain compositions, and then manually annotating and screening to determine the structure of the isomers and exclude false positives to determine the lipid molecular structure.

[0066] In some embodiments, step (5) includes: processing data using MSDIAL, selecting identified lipids according to the highest score, selecting lipids with fill%>50 in QC samples (quality control samples), RSD<30%, and analyzing after deleting duplicates.

[0067] Beneficial effects:

[0068] In view of the currently commonly used lipid analysis methods, this application optimizes the pre-treatment to elute lipids according to different polarities to achieve effective separation between lipid classes, and completes the separation within lipid classes by optimizing chromatographic separation, minimizing the co-elution of lipids, obtaining high-quality secondary mass spectra, and achieving high coverage and accurate qualitative and quantitative analysis of lipids in biological samples. At the same time, lipids are separated within lipid classes according to lipid unsaturation and carbon chain length in the chromatographic column, and separated between lipid classes according to lipid polarity. This retention rule can be used to exclude lipids from false positive ions in the results of lipid identification within and between lipid classes to improve the reliability of lipid identification analysis.

[0069] The present invention proposes that after extracting lipids from biological samples, the lipid types and contents in the samples are first quickly determined by thin layer chromatography; the lipids are then eluted in order of weak polarity, medium polarity and polarity by silica gel chromatography, and the lipids are separated and purified, and then separated by gradient elution of C30, C18 and HILIC columns before entering high-resolution mass spectrometry for detection and analysis. This method can improve the purity of lipids by customizing the silica gel chromatography method, and improve the separation between and within lipid classes by elution separation with liquid chromatography, reduce lipid co-elution behavior, and not only improve the clarity of the secondary mass spectrogram, but also improve the response signal of low-abundance lipids.

[0070] The method provided by the present invention can quickly determine the type and content of lipids contained in the biological sample through thin layer chromatography analysis after biological sample extraction, and can effectively determine the next step of silica gel column separation, thereby reducing solvents and experimental operation procedures;

[0071] Silica gel chromatography columns are used to separate, purify and enrich lipids according to polarity, followed by liquid chromatography elution and separation, and then high-resolution mass spectrometry detection and analysis, which can effectively reduce lipid co-elution, increase the content of low-abundance lipids, enhance mass spectrometry signal response, and improve the coverage of lipid detection;

[0072] The method of the present invention has stable retention behavior of lipids in the chromatographic column and little interference within and between lipid classes, which is conducive to judging false positives in lipid identification through retention behavior, improves identification accuracy, reduces identification difficulty, and saves time and economic costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 It is the TIC diagram of the sample C30 chromatographic column and the lipid retention position diagram in Example 1 of the present application;

[0074] Figure 2 It is the TIC diagram of the sample C18 chromatographic column and the lipid retention position diagram in Example 1 of the present application;

[0075] Figure 3 It is the TIC diagram of the sample HILIC chromatographic column and the lipid retention position diagram in Example 1 of the present application;

[0076] Figure 4 It is the lipid thin layer chromatogram of the samples in Examples 1 and 2 of the present application;

[0077] Figure 5 This is a general comparison of lipids (major categories) identified by the present application and the reference group in Example 1 of the present application;

[0078] Figure 6 is a comparison chart of the number of lipids (subclasses) identified by the present application and the reference method in Example 1 of the present application;

[0079] Figure 7 is a mapping relationship diagram of m / z and RT of the present application and the reference group TG in Example 1 of the present application;

[0080] Figure 8 is a mapping relationship diagram of m / z and RT of the present application and the reference group DG in Example 1 of the present application;

[0081] Fig. 9 It is the DG chromatogram mass spectrum of the method of the present application in Example 1 of the present application;

[0082] Fig.10 It is the DG chromatogram mass spectrum of the reference group method in Example 1 of the present application;

[0083] Fig.11 This is a general comparison of lipids (major categories) identified by the present application and the reference group in Example 2 of the present application;

[0084] Fig.12is a comparison chart of the number of lipids (subclasses) identified by the present application and the reference method in Example 2 of the present application;

[0085] Fig.13 is a mapping relationship diagram of M / Z to RT of the present application and reference group PC in Example 2 of the present application;

[0086] Fig.14 This is the PC 34:0 mass spectrum acquisition and analysis diagram in Example 2 of this application;

[0087] Fig.15 This is the mass spectrum acquisition diagram of the reference group PC 34:0 in Example 2 of the present application;

[0088] Fig.16 Solvent selection for optimal elution efficiency of lipids in NH2-based SPE columns;

[0089] Fig.17 is the lipid elution recovery rate of NH2-based SPE column. DETAILED DESCRIPTION

[0090] Definitions of Abbreviations and Key Terms

[0091] Liquid chromatography-high resolution mass spectrometry (HPLC-HRMS)

[0092] Lipidomics: A method that enables high-throughput detection of lipids

[0093] Chromatographic column model: C30\C18\HILIC, suitable for separation of substances with different properties

[0094] High-definition secondary mass spectrometry: fragment information is consistent with parent ion attribution, with no impurity interfering ions

[0095] Collision Cross Section (CCS): An intrinsic property of the material that determines the flight time of the mobility cell

[0096] Isomers: They have the same molecular weight and generally have similar structures, so it is difficult to separate them simply. Because of their similar retention time and molecular weight, they will enter the mass spectrometer at the same time, resulting in unclean data collected by the mass spectrometer.

[0097] Convolution peak: A chromatographic peak formed by the mixing of multiple ions.

[0098] In order to enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. The described embodiments are only embodiments of a part of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present application.

[0099] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0100] Unless otherwise stated, all experimental methods, detection methods and preparation methods not disclosed in the present invention adopt conventional techniques in the technical field.

[0101] It should be noted that the term "comprising" in the specification and claims of the present application and the above-mentioned drawings is intended to cover non-exclusive inclusions. For example, a process comprising a series of steps is not necessarily limited to those steps explicitly listed, but may include other steps that are not explicitly listed or inherent to these processes.

[0102] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0103] In the embodiments of the present application, after the biological sample tissue homogenate is extracted, thin layer chromatography is performed to determine the type and content of lipids contained; the lipids are further separated and purified using a silica gel column, and the silica gel column elution method and steps are determined in combination with the analysis results of thin layer chromatography; the separated and purified lipids are separated into a liquid chromatography and a high-resolution mass spectrometer, and the detected lipids are identified and analyzed using software to determine the lipid type information. The method provided in the present application optimizes the pretreatment of lipids in biological samples, improves the separation of lipids, especially isomers, reduces the co-elution phenomenon of lipids, obtains high-quality secondary mass spectrometry, improves the accuracy of qualitative and quantitative analysis of lipids, and is simple to operate, highly repeatable, and highly versatile.

[0104] Example 1 Comparison of lipid analysis results in zebrafish brain tissue

[0105] Extraction method: This application and the reference group both adopted the modified Folch lipid extraction method. The specific operation process is as follows: 20 mg of zebrafish brain tissue was accurately weighed into a centrifuge tube, 480 µL CH2Cl2:MeOH (2:1 v / v) was added, and ultrasonic extraction was performed at 4 °C for 10 min (Q800R3 (Qsonica, USA)), 150 µL water was added, and centrifuged for 10 min. The organic phase was recovered and 250 µL CH2Cl2:MEOH (2:1 v / v) was added. This was repeated once, the organic phases were combined, nitrogen blown, and re-dissolved with 100 µL CH2CL2:MeOH (2:1 v / v).

[0106] The zebrafish brain lipids in the reference group were extracted by the above lipid extraction method, filtered through a 0.2 μm PTFE filter (CytivaPall), separated by advanced liquid chromatography, and then entered high-resolution mass spectrometry acquisition and analysis. 18 Column (Waters 2.1×150 mm, 1.7 µm) was used for separation, and the column temperature was maintained at 50 °C. Mobile phase A (acetonitrile: water (6:4, v / v)) and mobile phase B (isopropanol: acetonitrile (9:1, v / v)) were used, both containing 10 mM ammonium formate and 0.1% formic acid. The flow rate was 0.28 mL / min, and the elution gradient was as follows: 0, 60% A; 4, 55% A; 4.1, 50% A; 12, 44% A; 12.1, 30% A; 26, 1% A; followed by re-equilibration to 60% A for 6 min.

[0107] This application is processed according to the chromatographic conditions of the present invention 9. After the sample is analyzed by thin layer chromatography, the sample is eluted into three types of strong, medium and weak polarity using an NH2 column, and then eluted and analyzed using C30\C18\HILIC chromatographic columns respectively. The specific operation steps are as follows:

[0108] 1. The sample was spotted on a thin layer plate (Qingdao Ocean, GF254), ① developed with n-hexane / ether (1:1 v / v) to 45% height of the silica gel plate, and the silica gel plate was taken out from the developing tank and after the organic solvent evaporated completely, it was developed in the developing agent ② dichloromethane / isopropanol / methanol / formic acid (5:3:2:0.5, volume ratio) to 90% height of the silica gel plate, and iodine was used for color development. The results are as follows Figure 4 Thin layer chromatography showed that the lipids in the sample of Example 1 can be divided into three parts: weak polarity, medium polarity, and polar lipids.

[0109] The spotting method is as follows: use a silica gel thin layer chromatography plate to spot the sample and the standard on a silica gel plate that has been pre-dried at 60 °C for 5 minutes: the spotting diameter is ≤3 mm, 10 mm from the bottom edge of the plate, the standard spotting volume is 10 μL, and the sample spotting volume is 3 μL.

[0110] 2. Use NH2-based SPE cartridge (Maiborui, LBSNH25006), activate it with 6 column volumes of n-hexane, and re-dissolve lipids with 1 mL of n-hexane for loading. Elute weak polar lipids with n-hexane, medium polar lipids with isopropanol: dichloromethane (3:7 (v / v)), and polar lipids with methanol in sequence, with an elution volume of 6 column volumes.

[0111] 3. After further concentration of the eluate, the methanol eluate phase was reconstituted with 200 µL of pure methanol, and the other two phases were reconstituted with 200 µL of dichloromethane:methanol (1:1 (v / v)), and passed through a 0.20 μm filter membrane for analysis.

[0112] 4. Liquid chromatography analysis: the n-hexane phase was analyzed using a C30 column, the isopropanol: dichloromethane (3:7 (v / v)) phase was analyzed using a C18 column, and the mobile phases were A, acetonitrile: water (6:4 (v / v)); B, isopropanol: acetonitrile (9:1 (v / v)), both containing 0.1% formic acid and 10 mM ammonium formate. The methanol phase was analyzed using a HILIC column, with mobile phases A, acetonitrile: water (1:3 (v / v)), B, acetonitrile: water (95:5 (v / v)); A and B both contained 0.2% v / v formic acid and 10 mM ammonium formate.

[0113] 5. In this embodiment, the chromatographic conditions are:

[0114] C30 chromatographic column Thermo Fisher 2.6μm, 150 x2.1 mm, elution program: at 0 min, mobile phase B 50% v / v; at 4 min, mobile phase B 70% v / v; at 13 min, mobile phase B 75% v / v; at 24 min, mobile phase B 85% v / v; at 33 min, mobile phase B 90% v / v; at 36 min, mobile phase B 92% v / v; at 37 min, mobile phase B 95% v / v; at 46 min, mobile phase B 95% v / v; at 46.1 min, mobile phase B 50% v / v; at 50 min, mobile phase B 50% v / v; injection volume was 2μL, column temperature was 50℃, and flow rate was 0.3 mL / min.

[0115] The C18 chromatographic column was a Waters X Bridge 3.5μm, 150 x2.1 mm column. The elution program was as follows: at 0 min, mobile phase B 40% v / v; at 3 min, mobile phase B 50% v / v; at 6 min, mobile phase B 62% v / v; at 14 min, mobile phase B 72% v / v; at 15 min, mobile phase B 85% v / v; at 16 min, mobile phase B 95% v / v; at 19 min, mobile phase B 95% v / v; at 19.1 min, mobile phase B 40% v / v; at 22 min, mobile phase B 40% v / v; the injection volume was 2μL, the column temperature was 50°C, and the flow rate was 0.3mL / min.

[0116] The Hilic column is waters HILIC 1.7μm, 150 x2.1 mm, elution program: at 0 min, mobile phase B 99% v / v; at 6 min, mobile phase B 94% v / v; at 11 min, mobile phase B 92% v / v; at 16 min, mobile phase B 88% v / v; at 15 min, mobile phase B 85% v / v; at 16 min, mobile phase B 95% v / v; at 19 min, mobile phase B 95% v / v; at 19.1 min, mobile phase B 40% v / v; at 22 min, mobile phase B 40% v / v; the injection volume was 2μL, the column temperature was 40℃, and the flow rate was 0.3mL / min.

[0117] Instrumental analysis: The reference group and this application were analyzed using Thermo Scientific Q Exactive, and the mass spectrometry parameters were preferably selected as follows, with positive and negative modes for acquisition; a. the voltage in both positive and negative ion modes was 4.00 kV; b. the ion transfer tube temperature was 320 °C; c. the sheath gas pressures were 40 psi and 30 psi, respectively, the auxiliary gas pressures were 10 arb, and the auxiliary gas heating temperatures were 300 °C; d. the scanning mode was Full MS, the instrument resolution was set to 70000, -dd / MS2, the resolution was 17500, NCE 30eV, and the scanning range was 50-1200 m / z.

[0118] Data processing: MSDIAL was used to process the data with default parameters. The identified lipids were selected according to the highest score, and the lipids with fill%>50 in the QC samples and RSD<30% were selected, and the duplicates were removed before analysis.

[0119] Result analysis:

[0120] Figure 1 The TIC diagram of the sample C30 chromatographic column and the lipid retention position diagram in Example 1 of the present application are shown; Figure 2The TIC diagram of the sample C18 chromatographic column and the lipid retention position diagram in Example 1 of the present application are shown; Figure 3 The TIC diagram of the sample HILIC chromatographic column and the lipid retention position diagram in Example 1 of the present application are shown. Figure 4 Sample lipid thin layer chromatograms are shown.

[0121] Figure 5 The results showed that in the zebrafish brain tissue of the reference group, a total of 5 major lipids were detected, including 45 lipid subclasses and 1103 lipid molecules. They were mainly: fatty acids (FA) with 7 subclasses and 65 lipids; glycerolipids (GL) with 8 subclasses and 370 lipids; glycerophospholipids (GP) with 15 subclasses and 387 lipid types; sphingolipids (SP) with 12 subclasses and 275 lipid types; and sterols with 3 subclasses and 6 lipids. After zebrafish brain tissue was processed by the method of the present application, 5 major lipid classes, 52 lipid subclasses, and 1542 lipid types were identified, mainly: fatty acids with a total of 9 subclasses and 86 lipids; glycerolipids (GL) with a total of 11 subclasses and 579 lipids; glycerophospholipids (GP) with a total of 15 subclasses and 495 lipids; sphingolipids (SP) with a total of 13 subclasses and 361 lipid types; sterols with 4 subclasses and 21 lipids.

[0122] Compared with the reference group, the method of the present application not only comprehensively improves the number of lipid identifications but also improves the accuracy of lipid identification. Figure 6 The results show that the number of lipids identified in this application increased by 41.97% compared with the reference group, among which glycerides increased by 56.48%, glycerophospholipids increased by 27.9%, sphingolipids increased by 31.27%, fatty acids increased by 32.3%, and sterols increased by 200%.

[0123] Figure 6 The identification number of glycerolipids TG, DG, and PC was significantly improved in this application, among which the identification rate of TG was 43.06%; DG was increased by 93.75%; and PC was increased by 75%. In order to further verify the accuracy of lipid identification, the present invention mapped the m / z pairs of the two groups of treatments to RT. Figure 7 This is a mapping relationship diagram of the M / Z to RT of TG in the application group and the reference group. The results show that the carbon number (m / z) of the TG lipid molecules identified in the present application is well positively correlated with the retention time (RT). At the same time, as the number of double bonds increases, the retention time decreases, showing a good negative correlation. The results are neat and orderly, which is consistent with the retention behavior of lipid molecules on the chromatography, proving that the lipid identification results are highly reliable. The mapping relationship of TG identified in the reference group is relatively complex, indicating that the identified lipid molecules have interference results such as false positives, and the reliability is relatively low. The same results are presented in the mapping relationship diagram of DG's m / z and RT, as shown in Figure 8 shown.

[0124] Figure 7 , Figure 8 The results showed that the same m / z could be detected at multiple retention times, and according to the identification results, it was known to be an isomer of TG lipid. Figure 8 The lipid molecule labeled in is DG 38:6, Fig. 9 This is the DG 38:6 mass spectrum acquisition and analysis diagram. Fig. 9 A in the figure is the chromatographic separation diagram of DG38:6, which shows that there are three isomers. Fig. 9 B in the middle is the enantiomeric mass spectrum, which has a clear mass spectrum and a high signal response, and is identified as: DG 38:6|DG 18:2_20:4, DG 38:6|DG 18:1_20:5, and DG 38:6|DG 18:1_20:5.

[0125] Fig.10 This is the mass spectrum acquisition diagram of the reference group DG 38:6. Fig.10 A in the middle is the chromatographic separation diagram, from which it can be seen that the response intensity decreases and the peak shape is asymmetric, and no separation of the isomers of DG 38:6 is observed. Fig.10 The mass spectrum of B showed that the identification result was DG38:6|DG 18:2_20:4.

[0126] Example 2 Comparison of lipid analysis results in milk

[0127] The extraction method includes the following steps: add 1 mL of milk sample to a 15 mL centrifuge tube, and then add 2 mL of methanol (containing 0.2% formic acid by volume), 4 mL of dichloromethane, vortex mix for 2 min after each addition and stand in an ice water bath for 10 min; then add 1 mL of ultrapure water to the centrifuge tube to induce phase separation, vortex mix for 2 min, and centrifuge at 10000 rpm / min for 5 min at 4°C. Transfer the lower organic phase to a new tube, re-extract the remaining part according to the previous steps, combine the lower organic phase, blow dry under mild N2 gas, and re-dissolve with 1 mL of dichloromethane: methanol (2:1, v / v), and store the obtained total lipids at -80°C until use.

[0128] The reference group was directly sampled through Hippocampus 0.2 μm PTFE for analysis; the reference group was sampled using Xbridge BEH C 18Column (Waters 2.1×150 mm, 3.5µm) was used for separation and maintained at 50°C. Mobile phase A (acetonitrile: water (6:4, v / v)) and mobile phase B (isopropanol: acetonitrile (9:1, v / v)) were used, both containing 10 mM ammonium formate and 0.1% formic acid. The flow rate was 0.28 mL / min for a total of 32 min, and the elution gradient was as follows: 0, 60% A; 4, 55% A; 4.1, 50% A; 12, 44% A; 12.1, 30% A; 26, 1% A; followed by 6 min re-equilibration to 60% A.

[0129] The specific operations of this application are as follows:

[0130] 1. After lipid extraction, the sample was spotted on a thin layer plate (Qingdao Ocean, GF254), ① developed with n-hexane / ether (1:1 v / v) to 45% height of the silica gel plate, and the silica gel plate was taken out from the developing tank and after the organic solvent evaporated completely, it was developed in the developing agent ② dichloromethane / isopropanol / methanol / formic acid (5:3:2:0.5, volume ratio) to 90% height of the silica gel plate, and iodine was used for color development. The results are as follows Figure 4 It shows that the lipids in the sample of Example 2 can be divided into two parts: weak polar lipids and polar lipids.

[0131] The spotting method is as follows: use a silica gel thin layer chromatography plate to spot the sample and the standard on a silica gel plate pre-dried at 60 °C for 5 minutes, the spot diameter is ≤3 mm, 10 mm from the bottom edge of the plate, the standard spotting volume is 10 μL, and the sample spotting volume is 3 μL.

[0132] 2. Use NH2-based column for separation, activate with 6 column volumes of n-hexane, and re-dissolve lipids with 1 mL of n-hexane for loading. Elute weak polar lipids with n-hexane and polar lipids with methanol in sequence, with an elution volume of 8 column volumes.

[0133] 3. After further concentration of the eluate, the methanol elution phase was re-dissolved with pure methanol, and the n-hexane phase was re-dissolved with dichloromethane:methanol (2:1 (v / v)), and passed through a 0.20 μm filter membrane for analysis.

[0134] 4. Liquid chromatography analysis: The n-hexane phase was analyzed using a C30 column, and the mobile phase was A, acetonitrile: water (6:4 (v / v)); B, isopropanol: acetonitrile (9:1 (v / v)), A and B both contained 0.1% formic acid and 10 mM ammonium formate; the methanol phase was analyzed using a HILIC column, and the mobile phase was A, acetonitrile: water (1:3 (v / v)), B, acetonitrile: water (95:5 (v / v)). A and B both contained 0.2% formic acid and 10 mM ammonium formate; the injection volume was 2 μL, the column temperature was 50 ℃, and the flow rate was 0.3 mL / min.

[0135] 5. In this embodiment, the chromatographic conditions are:

[0136] C30 chromatographic column Thermo Fisher 2.6μm, 150 x2.1 mm, elution program: at 0 min, mobile phase B 50%v / v; at 4 min, mobile phase B 70%v / v; at 13 min, mobile phase B 75%v / v; at 24 min, mobile phase B 85%v / v; at 33 min, mobile phase B 90%v / v; at 36 min, mobile phase B 92%v / v; at 37 min, mobile phase B 95%v / v; at 46 min, mobile phase B 95%v / v; at 46.1 min, mobile phase B 50%v / v; at 50 min, mobile phase B 50%v / v; injection volume was 2μL, column temperature was 50℃, and flow rate was 0.3 mL / min.

[0137] The Hilic column is waters HILIC 1.7μm, 150x2.1 mm, elution program: at 0 min, mobile phase B 99% v / v; at 6 min, mobile phase B 94% v / v; at 11 min, mobile phase B 92% v / v; at 16 min, mobile phase B 88% v / v; at 15 min, mobile phase B 85% v / v; at 16 min, mobile phase B 95% v / v; at 19 min, mobile phase B 95% v / v; at 19.1 min, mobile phase B 40% v / v; at 22 min, mobile phase B 40% v / v; the injection volume was 2μL, the column temperature was 40℃, and the flow rate was 0.3mL / min.

[0138] Agilent 6545 instrument analysis: electrospray ion source, data-dependent mode acquisition; primary mass spectrometry acquisition mass range m / z 100-1600, secondary mass spectrometry acquisition mass range m / z 50-1200; positive ion mode collision energy 35 eV; negative ion mode collision energy 30 eV; gas temperature 250 °C, sheath gas temperature 300 °C; sheath gas flow rate 12 L / min; capillary voltage 3000 V in positive ion mode, 3000 V in negative ion mode; fragmentor voltage 150 V; cone voltage 65 V; MS and MS / MS minimum acquisition rate 3 spectra / s.

[0139] Data processing: MSDIAL was used to process the data with default parameters. The identified lipids were selected according to the highest score, and the lipids with fill%>50 in the QC samples and RSD<30% were selected, and the duplicates were removed before analysis.

[0140] Result analysis:

[0141] A total of 31 lipid subclasses and 735 lipid types were detected in milk tissues by the software processing of the reference group data, mainly: fatty acids with a total of 5 subclasses and 26 lipid types; glycerolipids (GL) with a total of 6 subclasses and 347 lipid types; glycerophospholipids (GP) with a total of 11 subclasses and 314 lipid types; sphingolipids (SP) with a total of 7 subclasses and 46 lipid types, sterols with 2 subclasses and 2 lipid types;

[0142] After the method of the present application was used, 38 lipid subclasses and 1062 lipid species were identified after sample analysis, mainly: fatty acids (FA) with 5 subclasses and 31 lipid species; glycerolipids (GL) with 7 subclasses and 442 lipid species; glycerophospholipids (GP) with 16 subclasses and 495 lipid species; sphingolipids (SP) with 8 subclasses and 73 lipid species, and 5 sterol molecules in 3 sterol lipid subclasses;

[0143] Compared with the reference group, the method of the present application comprehensively improves the number of lipid identifications and also improves the accuracy of lipid identification. Fig.11 The results show that the number of lipids identified in this application increased by 44.48% compared with the reference group, among which glycerides increased by 31.98%, glycerophospholipids increased by 57.64%, sphingolipids increased by 62.79%, fatty acids increased by 19.23%, and sterols increased by 150%.

[0144] Fig.12 The identification number of glycerolipids PC, PE, and PC was significantly improved in this application. The results of the thin layer showed that the phospholipid content in milk was lower than that of glycerolipids. Direct analysis in the reference group would be interfered by the high abundance of glycerolipids. After treatment by this application, the identification number of PC increased by 69.23%; DG increased by 93.33%; SM increased by 81.39%. In order to further verify the accuracy of lipid identification, the present invention mapped the m / z pairs of the two groups of treatments to RT. Fig.13 The mapping relationship diagram of M / Z to RT of PC in the application group and the reference group shows that the carbon number (m / z) of the PC lipid molecules identified in the present application is well positively correlated with the retention time (RT). At the same time, as the number of double bonds increases, the retention time decreases, showing a good negative correlation. The results are neat and orderly, which is consistent with the retention behavior of lipid molecules on the chromatography, proving that the lipid identification results are highly reliable. The mapping relationship of the PC identified in the reference group shows that the lipid detection rate is reduced and the retention behavior of lipids is unstable. The results show that the signal collected by the mass spectrometry of the reference group is relatively false positive compared with the processing of the present application, which affects the accuracy and coverage of lipid identification.

[0145] Fig.14 This is the PC 34:0 mass spectrum acquisition and analysis chart. Fig.14A in the middle is the chromatogram of PC 34:1, which shows that the peak shape is sharp and symmetrical, and the signal intensity is high. Fig.14 B in the figure is the mass spectrum of PC 34:0, which has a clear mass spectrum and a high signal response and is identified as PC 34:0|PC 16:0_18:0. Fig.15 This is the mass spectrum acquisition diagram of the reference group PC 34:0. Fig.15 A in the middle is the chromatographic separation diagram, which has a low response intensity and an asymmetric peak shape. Fig.15 The mass spectrum of B only has the parent ion m / z 762.5971 and the PC head group m / z 184.0751, and the key information of the acyl chain is missing in the middle. The lipid can only be identified as a subclass, PC 34:0.

[0146] According to the comparison of the experimental results of the above embodiments, the lipid analysis method of the present application uses thin layer chromatography to quickly determine the lipid type and content in the sample after lipid extraction; silica gel chromatography separates and purifies lipids according to weak polarity, medium polarity and polarity, and then elutes through C30, C18, and HILIC chromatographic columns with customized gradients, which improves the separation between and within lipid classes, reduces lipid co-elution behavior, and not only improves the clarity of the secondary mass spectrogram, but also improves the response signal of low-abundance lipids. Compared with the reference group analysis method, the customized method of the present application can increase the concentration of lipid molecules and increase the separation effect of isomers, thereby improving the coverage, reliability and identification efficiency of lipid detection.

[0147] Comparative Example

[0148] According to thin layer chromatography, lipids can be divided into three categories: strong, medium and weak polar lipids. NH2-based SPE columns are used as a means of further separation and purification. Select TG(TG 16:0_16:0_16:0,TG 15:0_15:0_15:0,TG 12:0_12:0_12:0,TG10:0_10:0_10:0), DG(DG 20:5_20:5,DG 20:3_20:3,DG 18:3_18:3,DG 15:0_18:1(d7)), SP(HexCer 18:1;O2 / 12:0,HexCer 18:1;O2 / 18:1,Cer 18:1;O2 / 12:0,Cer 18:1;O2 / 18:1,Cer 18:0;O2 / 18:1, SM24:1;O2, SM 36:2;O2,SM 36:1;O2), PC (PC 16:0_18:1, PC 15:0_18:1(d7), PC 14:0_14:0), PE (PE 16:0_18:1, PE 15:0_18:1(d7), PE 15:0_15:0), MG 18:0, LPC 18:1(d7), C18(plasm)-18:1(d9) PC standards were loaded with hexane (HEX) and then washed with HEX, HEX:DCM 7:3, HEX:DCM 5:5, HEX:DCM 3:7, DCM, DCM:IPA 7:3, DCM:IPA 5:5, DCM:IPA3:7, IPA, IPA:MeOH 7:3, IPA:MeOH 5:5, IPA:MeOH 3:7, MeOH, MeOH+FA1% (volume ratio), elution volume is 3 times column volume (3 times column volume is 3mL, Fig.16 The HEX in the solution was eluted twice with 3 column volumes (6 mL) and then analyzed by mass spectrometry.

[0149] like Fig.16 As shown in the figure, the hexane (HEX) phase can effectively elute TG and has weak elution ability for other lipids, so it can be used as an elution solvent. Dichloromethane: isopropanol 7:3 (DCM:IPA 7:3) can be used as an elution solvent for medium-polar lipids, and has a strong elution ability for SP, DG and other lipids. The remaining strong polar lipids such as PC, PE, LPC, SM, etc. can be eluted by methanol.

[0150] After selecting the lipid strong, medium and weak polar elution solvents, the elution solvent and efficiency were optimized by adding Avanti lipid internal standards LM6004, LM6002, LM6000 and 330709w. The elution recovery results showed that TG lipid LM6000 was mainly eluted in the HEX phase, SP lipid LM6002 (except SM) and DG lipid LM6004 were mainly eluted in DCM:IPA 7:3, and the internal standard 330709wGP lipid and SM lipid had the best elution effect in the methanol phase (e.g. Fig.17 The elution volume was 6 column volumes.

[0151] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for analyzing lipids in a biological sample, characterized in that: The method for analyzing lipids in a biological sample comprises the following steps: (1) extracting lipids from a biological sample to obtain a first sample; (2) determining the lipid categories of the first sample by thin layer chromatography, wherein the lipid categories include non-polar lipids, medium-polar lipids, and polar lipids; (3) Based on the lipid type and content in (2), the first sample is separated and purified using a silica gel chromatography column to obtain a second sample: (4) The second sample is subjected to gradient elution by liquid chromatography and then subjected to mass spectrometry analysis.

2. The method for analyzing lipids in a biological sample according to claim 1, characterized in that: The silica gel chromatographic column described in step (3) is an NH2-based SPE column, and the eluent of the silica gel chromatographic column includes one or more of a n-hexane phase, an isopropanol: dichloromethane (3:7 (v / v)) phase, and a methanol phase; wherein the n-hexane phase is used to elute non-polar lipids; the isopropanol: dichloromethane (3:7 (v / v)) phase is used to elute medium-polar lipids; and the methanol phase is used to elute polar lipids.

3. The method for analyzing lipids in a biological sample according to claim 1, characterized in that: The second sample comprises one or more of a n-hexane phase elution sample, an isopropanol: dichloromethane (3:7 (v / v)) phase elution sample, and a methanol phase elution sample; In step (4), the chromatographic column used in the liquid chromatography includes one or more of a C30 chromatographic column, a C18 chromatographic column and a HILIC chromatographic column, wherein the C30 chromatographic column is used to separate and purify samples eluted with n-hexane phase, the C18 chromatographic column is used to separate and purify samples eluted with isopropanol: dichloromethane (3:7 (v / v)), and the HILIC chromatographic column is used to separate and purify samples eluted with methanol phase.

4. The method for analyzing lipids in a biological sample according to claim 3, characterized in that: The mobile phase system of the C30 chromatographic column or C18 chromatographic column is: A1: acetonitrile: water (6:4 (v / v)); B1: isopropanol:acetonitrile (9:1 (v / v)), 5-10 mM ammonium formate and 0.1%-0.2% formic acid were added to A1 and B1; The mobile phase system of the HILIC chromatographic column is: A2: acetonitrile: water (1:3 (v / v)); B2: acetonitrile: water (95:5 (v / v)), 5-10 mM ammonium formate and 0.1%-0.2% formic acid were added to A2 and B2.

5. The method for analyzing lipids in a biological sample according to claim 1, characterized in that: The liquid chromatography elution procedure in step (4) is: Elution program of C30 column: at 0 min, mobile phase B1 50% v / v; at 4 min, mobile phase B1 70% v / v; at 13 min, mobile phase B1 75% v / v; at 24 min, mobile phase B1 85% v / v; at 33 min, mobile phase B1 90% v / v; at 36 min, mobile phase B1 92% v / v; at 37 min, mobile phase B1 95% v / v; at 46 min, mobile phase B1 95% v / v; at 46.1 min, mobile phase B1 50% v / v; at 50 min, mobile phase B1 50% v / v; C18 column elution program: at 0 min, mobile phase B1 40% v / v; at 3 min, mobile phase B1 50% v / v; at 6 min, mobile phase B1 62% v / v; at 14 min, mobile phase B1 72% v / v; at 15 min, mobile phase B1 85% v / v; at 16 min, mobile phase B1 95% v / v; at 19 min, mobile phase B1 95% v / v; at 19.1 min, mobile phase B1 40% v / v; at 22 min, mobile phase B1 40% v / v; HILIC column elution program: at 0 min, mobile phase B2 99% v / v; at 6 min, mobile phase B2 94% v / v; at 11 min, mobile phase B2 92% v / v; at 16 min, mobile phase B2 88% v / v; at 15 min, mobile phase B2 85% v / v; at 16 min, mobile phase B2 95% v / v; at 19 min, mobile phase B2 95% v / v; at 19.1 min, mobile phase B2 40% v / v; at 22 min, mobile phase B2 40% v / v.

6. The method for analyzing lipids in a biological sample according to claim 5, characterized in that: In step (4), the mass spectrum is collected in CID mode to obtain the accurate ion mass-to-charge ratio, retention time and high-definition secondary mass spectrum of the lipid primary mass spectrum; in step (4), the mass spectrum is collected in the following ways: using positive ion mode to detect and analyze the effluent after separation and purification by C30 chromatographic column; using positive and negative modes to detect and analyze the effluent after separation and purification by C18 chromatographic column; using positive and negative modes to detect and analyze the effluent after separation and purification by HILIC chromatographic column.

7. The method for analyzing lipids in a biological sample according to claim 5, characterized in that: The elution conditions of the chromatographic column are: the flow rate of the mobile phase is 0.2-0.4 mL / min, and the column temperature is 35-50°C.

8. The method for analyzing lipids in a biological sample according to any one of claims 1 to 7, characterized in that: The biological sample includes a sample containing lipids or lipid analogs.

9. The method for analyzing lipids in a biological sample according to any one of claims 1 to 7, characterized in that: The method for extracting lipids from the biological sample in step (1) is as follows: Add an extractant to the biological sample, ultrasonically disrupt or let it stand, then add water and centrifuge, collect the organic phase, then repeat the extraction of the remaining part, combine the organic phases, remove the solvent, redissolve, filter, and obtain the first sample; the extractant includes methanol and dichloromethane.

10. The method for analyzing lipids in a biological sample according to any one of claims 1 to 7, characterized in that: Step (2) includes: Spot the first sample and the standard using a silica gel thin layer chromatography plate; The samples were developed by the secondary development method. The developing agents were ① n-hexane / ethyl ether, ② dichloromethane / isopropanol / methanol / water / formic acid. After development, the samples were taken out and the color was developed in a color developing tank after the reagents were completely evaporated.

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