A method for separating and enriching glycosphingolipids and its concentration, analysis and identification

Abstract

The application discloses a glycosphingolipid separation and enrichment method and concentration, analysis and identification thereof, and the separation and enrichment method comprises the following steps: adding magnetic titanium dioxide nanomaterial and a sample to be separated into a loading buffer, incubating to obtain incubated materials; the sample to be separated contains glycosphingolipids and phospholipids, and the loading buffer comprises acetonitrile and ammonia water; the incubated materials are washed by using a washing buffer; the washing buffer comprises methanol, ammonia water and an ammonium salt; the washed materials are added into an elution buffer, incubated, and an eluent of glycosphingolipids is obtained. + The method utilizes the coordination interaction between glycosphingolipids and magnetic titanium dioxide nanomaterial, uses an alkaline aprotic solvent loading buffer and an alkaline protic solvent washing buffer, and introduces NH4 + , realizes specific separation and enrichment of glycosphingolipids, and can be used for Paternò-Büchi reaction and various liquid chromatography-mass spectrometry platforms to identify fine structures of glycosphingolipids.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of sample separation, in particular, the present application relates to a method for separating and enriching glycosphingolipids and its concentration, analysis and identification. BACKGROUND

[0002] Glycosphingolipids are an important class of lipid signaling molecules, which show important research value in neurodegenerative diseases, lysosomal metabolic diseases and diabetes. The synthesis of glycosphingolipids is regulated by multiple enzymes, so the content and structural changes of glycosphingolipids can reflect the changes of enzyme levels in the process of disease occurrence or cell life activities. Mass spectrometry as a major tool for lipid structure identification has been applied in various lipid subclasses. In addition, the identification of fine structures of lipids such as carbon-carbon double bonds has been further achieved by the development of various chemical derivatization methods and different ion fragmentation methods.

[0003] At present, the mass spectrometry structure identification of glycosphingolipids still faces difficulties. The head group of glycosphingolipids composed of oligosaccharide chains has the diversity of sugar ring number, type, sequence and modification. Glycosphingolipids are low in abundance (<nM) in various biological samples, and the content of most glycosphingolipids is lower than the detection range of mass spectrometry. As a neutral lipid, glycosphingolipids are subject to signal inhibition by phosphatidylcholine lipids with fixed charge. The mass spectrometry identification of fine structure often requires higher abundance of lipids, so the identification of fine structure such as double bond position of glycosphingolipids is rarely reported.

[0004] In view of the current research status of glycosphingolipids, a glycosphingolipid separation and enrichment method with high recovery rate and good specificity of glycosphingolipids still needs to be developed. SUMMARY

[0005] The present application is based on the discovery and recognition of the inventors on the following facts and problems: In order to improve the mass spectrometric signal of glycosphingolipids, two common research directions are to improve the mass spectrometric response ability of glycosphingolipids by chemical derivatization or to develop mass spectrometric front-end separation and enrichment methods. At present, methylation has been used for the derivatization of glycosphingolipids, which effectively suppresses the loss of water peak of glycosphingolipids, and makes the signal normalized, and the methylation conditions need to be adjusted for different numbers of sugar ring head groups, thus bringing certain operation difficulty. In the separation of the front end of mass spectrometry, in the commonly used chromatographic column separation, the reverse phase chromatography cannot exclude the matrix effect of phospholipids, and the normal phase chromatography cannot avoid the overlapping of isotopic peaks caused by the difference in the number of unsaturation and the same isotope peaks caused by the modification of different numbers of hydroxyl groups. Recently, titanium dioxide has been used for the separation of sphingolipids. The cis-dihydroxyl (glycolipid sugar ring) and alpha-hydroxyl amine structure (sphingosine skeleton) in sphingolipids can form stable coordination with the metal center, while the coordination of phospholipids carrying phosphate functional groups with metal is destroyed, thereby realizing the separation of sphingolipids. However, this method has poor specificity for the separation of glycosphingolipids, and the recovery rate is low. The separation and analysis of glycosphingolipids in complex samples still face research difficulties. The specific separation and enrichment of glycosphingolipids before chromatographic separation has great application potential. In order to solve the problems of low recovery rate and poor specificity of glycosphingolipids, targeted sample pretreatment methods need to be developed.

[0006] The present application aims to at least solve one of the technical problems in the related art to some extent. To this end, the embodiments of the present application propose a glycosphingolipid separation and enrichment method and its concentration, analysis and identification, which utilizes the coordination interaction between glycosphingolipids and magnetic titanium dioxide nanomaterials, uses an alkaline aprotic solvent sample loading buffer and an alkaline protic solvent washing buffer, and introduces NH4 + in the washing buffer to realize the specific separation and enrichment of glycosphingolipids. The method can be used for Paternò-Büchi (PB) reaction and various liquid chromatography-mass spectrometry platforms, and can identify the fine structure of glycosphingolipids.

[0007] The embodiments of the present application provide a glycosphingolipid separation and enrichment method, which comprises the following steps:

[0008] (1) adding magnetic titanium dioxide nanomaterials and a sample to be separated into a sample loading buffer, incubating, and obtaining incubated magnetic titanium dioxide nanomaterials; wherein the sample to be separated comprises glycosphingolipids and phospholipids; and the sample loading buffer comprises acetonitrile and ammonia water;

[0009] (2) washing the incubated magnetic titanium dioxide nanomaterials in step (1) with a washing buffer; and the washing buffer comprises methanol, ammonia water and an ammonium salt;

[0010] (3) adding the washed magnetic titanium dioxide nanomaterials in step (2) into an elution buffer, incubating, and obtaining an eluate of glycosphingolipids.

[0011] The advantages and technical effects of the glycosphingolipid separation and enrichment method of the embodiments of the present application are as follows: 1. In the embodiments of the present application, the sample to be separated contains glycosphingolipids and interfering lipids phospholipids, and the glycosphingolipids in the sample to be separated are separated and enriched by using the magnetic titanium dioxide nanomaterial. The modified magnetic titanium dioxide nanomaterial has good magnetic response, can realize effective coordination interaction with the cis-dihydroxyl in the target substance sphingomyelin, can capture glycosphingolipids in the alkaline environment of the loading buffer, and can separate and carry glycosphingolipids from the complex sample to be separated with the help of an external magnetic field. The nanomaterial has a high specific surface area and can provide a large number of active sites to realize high loading capacity of glycosphingolipids. With the aid of an external magnetic field, the magnetic titanium dioxide nanomaterial can be quickly separated from the liquid phase, greatly simplifying the operation process and avoiding sample loss caused by pretreatment.

[0012] 2. In the embodiments of the present application, the separation and enrichment method involves the use of three kinds of buffer solutions. The loading buffer is an alkaline aprotic solvent, including acetonitrile and ammonia water, which allows glycosphingolipids to form stable coordination interaction with titanium dioxide and makes a large amount of phospholipids not be adsorbed by titanium dioxide through the interference of the alkaline environment; the washing buffer is an alkaline protic solvent ammonium salt solution, which is used to wash the surface of the adsorbed lipid titanium dioxide to remove a large amount of non-specifically adsorbed phospholipids and improve the selectivity for the target substance sphingomyelin. The washing buffer uses an alkaline protic solvent and adds ammonium salt, specifically including methanol, ammonia water and ammonium salt. The ammonium salt provides additional large amounts of NH4 + ions to compete with the interfering lipid phospholipids remaining on the surface of titanium dioxide for the active sites of titanium dioxide, thereby further removing phospholipids and eliminating the adsorption of high-abundance interferents on the material. In addition, the addition of the protic solvent not only helps the dissolution of the ammonium salt, but also can remove the adsorbed phospholipids on the surface of the material through coordination with titanium dioxide; finally, the glycosphingolipids are eluted by the elution buffer to realize the specific separation of the complete glycosphingolipid molecules.

[0013] 3. In the embodiments of the present application, based on the use of the magnetic titanium dioxide nanomaterial and the washing buffer of the present application, high-specificity separation and enrichment of glycosphingolipids can be realized in a standard lipid mixture solution in which the concentration of interfering lipids is 100 times that of the target glycosphingolipids or in a complex biological sample pig brain extract. The method of the present application can be used for Paternò-Büchi reaction and various liquid chromatography platforms, and realizes high-throughput, sensitive and fine mass spectrum structure analysis of the fine structure of glycosphingolipids in complex samples, for example, identification of the double bond position of glycosphingolipids, improves the identification ability of mass spectrometry for low-abundance glycosphingolipids, and discovers a large number of new structures of low-abundance glycosphingolipids, laying a foundation for the research of low-abundance glycosphingolipids.

[0014] In some embodiments, in the step (1), the volume fraction of acetonitrile in the loading buffer is 92-96%, and the volume fraction of ammonia water is 4-8%.

[0015] And / or, the incubation time is 30-90 min.

[0016] In some embodiments, in the step (2), the volume fraction of methanol in the washing buffer is 94-96%, the volume fraction of ammonia water is 4-6%, and the concentration of ammonium salt is 10-20 mM; preferably, the ammonium salt comprises at least one of ammonium bicarbonate, ammonium formate or ammonium acetate.

[0017] And / or, the magnetic titania nanomaterial after the incubation in the step (1) is washed with the washing buffer for 1-3 times.

[0018] In some embodiments, in the step (3), the elution buffer comprises formic acid and methanol; preferably, the volume fraction of formic acid in the elution buffer is 2-8%, and the volume fraction of methanol is 92-98%.

[0019] And / or, the incubation time is 30-90 min.

[0020] In some embodiments, in the step (1), the magnetic titania nanomaterial comprises a titania-coated ferroferric oxide magnetic nanomaterial.

[0021] In some embodiments, the method for preparing the magnetic titania nanomaterial comprises the following steps:

[0022] (a) dissolving ferric chloride hexahydrate in ethylene glycol, then adding anhydrous sodium acetate, mixing, and reacting at 100-400℃ for 8-20 hours to obtain ferroferric oxide;

[0023] (b) dispersing the ferroferric oxide in a solvent, adding a base and a titania precursor, and reacting at 20-80℃ for 12-48 hours;

[0024] (c) calcining the product obtained in the step (b) at 200-800℃ for 1-10 hours to obtain the magnetic titania nanomaterial.

[0025] In some embodiments, in the steps (1)-(3), both the materials and the solutions are separated by a magnet.

[0026] In some embodiments, in the step (1), the sample to be separated is a biological sample.

[0027] The embodiment of the present application provides a method for concentrating glycosphingolipids. The eluent of the glycosphingolipids enriched by the method of the embodiment of the present application is blown dry by nitrogen, and is redissolved in methanol to obtain concentrated glycosphingolipids. In the embodiment of the present application, the separated glycosphingolipids are redissolved in methanol to realize concentration of the purified glycosphingolipids, improve the sample injection amount of the chromatography, and improve the identification ability of the mass spectrometry on the low-abundance glycosphingolipids. The detection limit of the glycosphingolipids is improved from 0.5 nM to 20 pM. The glycosphingolipids purified by the method are suitable for various chromatographic separation methods.

[0028] The embodiment of the present application provides a method for analyzing a sample containing glycosphingolipids. The eluent of the glycosphingolipids enriched by the method of the embodiment of the present application is subjected to mass spectrometry analysis and / or liquid chromatography separation. In the embodiment of the present application, based on the use of the magnetic titanium dioxide nanomaterial and the washing buffer of the present application, high-specificity separation and enrichment of glycosphingolipids can be realized in a standard lipid mixed solution or a biological complex sample pig brain extract with the concentration of interfering lipids being 100 times that of the target glycosphingolipids. The purified glycosphingolipids are suitable for various chromatographic separation methods and can also be used for various liquid mass platforms. The fine structure of the glycosphingolipids is analyzed and identified, for example, the identification of the double bond position of the glycosphingolipids. The identification ability of the mass spectrometry on the low-abundance glycosphingolipids is improved. A large number of low-abundance glycosphingolipid structures are found, which lays a foundation for the research on low-abundance glycosphingolipids.

[0029] The embodiment of the present application provides a method for identifying the double bond position of glycosphingolipids. The eluent of the glycosphingolipids enriched by the method of the embodiment of the present application is subjected to photo-derivatization Paternò-Büchi reaction, and the double bond position of the glycosphingolipids is identified by connecting secondary mass spectrometry. In the embodiment of the present application, the double bond position of the unsaturated glycosphingolipids is subjected to photo-derivatization, and the low-energy collision-induced dissociation of the mass spectrometry can identify the double bond position of the glycosphingolipids. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 It is a process and mechanism for specifically separating glycosphingolipids by the magnetic titanium dioxide nanomaterial.

[0031] Figure 2 It is a normal phase (HILIC) separation chromatogram of Example 1, wherein (a) is a standard lipid mixture; (b) is residual lipids in the sample buffer after the glycosphingolipids are captured by the magnetic titanium dioxide nanomaterial; (c) is lipids in the washing buffer after the magnetic titanium dioxide nanomaterial capturing lipids is washed; (d) is the elution buffer obtained by eluting the glycosphingolipids from the magnetic titanium dioxide nanomaterial after the lipids are captured and washed.

[0032] Figure 3is a normal phase separation chromatogram of Example 2, wherein (a) standard lipid mixture; (b) sphingoglycolipid eluate after separation and enrichment.

[0033] Figure 4 is a secondary mass spectrum of Example 3, wherein (a) HexCer d18:1 / 18:1 (0.5 nM); (b) 20 pM HexCer d18:1 / 18:1 after separation and enrichment.

[0034] Figure 5 is a secondary mass spectrum of Example 4 and extracted ion chromatogram of sphingosine fragments, wherein (a) secondary fragmentation spectrum of HexCer d18:1 / 15:0; (b) secondary fragmentation spectrum of Hex2Cer d18:1 / 17:0; (c) sphingosine fragment ion [d18:1-2H2O+H] of HexCer d18:1 / 15:0 (10 nM) after separation by magnetic titanium dioxide nanomaterials + m / z 264.5 before and after separation by magnetic titanium dioxide nanomaterials; (d) sphingosine fragment ion [d18:1-2H2O+H] of Hex2Cer d18:1 / 17:0 (10 nM) after separation by magnetic titanium dioxide nanomaterials + m / z 264.5 before and after separation by magnetic titanium dioxide nanomaterials.

[0035] Figure 6 is a reverse phase separation chromatogram of Example 5, in which the two most abundant phosphatidylcholines and the two most abundant sphingoglycolipids are marked, and the interfering lipids such as phospholipids are white filled and the sphingoglycolipids are black filled, wherein (a) total lipids in pig brain (0.2 μg); (b) sphingoglycolipids in pig brain (3 μg) after separation and enrichment.

[0036] Figure 7 is an extracted ion chromatogram after reverse phase chromatographic separation in Example 6, wherein (a) extracted ion chromatogram of HexCer d18:1 / 18:1 (0.5 μM) after reverse phase chromatographic separation [HexCer d18:1 / 18:1+H] + m / z 726.5; (b) solution of HexCer d18:1 / 18:1 (0.5 μM) after reaction with diacetylpyridine at 254 nm light for 15 s after reverse phase chromatographic separation, against substrate [HexCer d18:1 / 18:1+H] + m / z 726.5 and PB product PB HexCer d18:1 / 18:1+H] +Extraction ion chromatogram of m / z 847.5; (c) Secondary mass spectrum detection of the PB product flowing out at 4.3 min, with a collision energy of 45 eV; (d) Secondary mass spectrum detection of the PB product flowing out at 4.5 min, with a collision energy of 45 eV.

[0037] Figure 8 is the detection result of lipids in the elution buffer, wherein (a) is the detection result of lipids in the elution buffer in the comparative example 1 using no NH4 + washing buffer treatment; (b) is the detection result of lipids in the elution buffer in the example 1 using the washing buffer added with NH4 + washing buffer treatment. DETAILED DESCRIPTION

[0038] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings. The embodiments described below by reference to the drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0039] As shown in Figure 1 , the glycosphingolipid separation and enrichment method of the embodiments of the present application comprises the following steps:

[0040] (1) adding the magnetic titanium dioxide nanomaterial and the sample to be separated into a loading buffer, incubating, and obtaining the incubated magnetic titanium dioxide nanomaterial; wherein the sample to be separated contains glycosphingolipids and phospholipids; the loading buffer comprises acetonitrile and ammonia water;

[0041] Optionally, adding the magnetic titanium dioxide nanomaterial and the sample to be separated into the loading buffer is specifically adding the magnetic titanium dioxide nanomaterial into the loading buffer, and then adding the sample to be separated;

[0042] (2) washing the incubated magnetic titanium dioxide nanomaterial in the step (1) with a washing buffer; the washing buffer comprises methanol, ammonia water and ammonium salt;

[0043] (3) adding the washed magnetic titanium dioxide nanomaterial in the step (2) into an elution buffer, incubating, and obtaining an eluent of glycosphingolipids.

[0044] The glycolipid separation and enrichment method of the embodiment of the present application utilizes the magnetic titanium dioxide nanomaterial to separate and enrich the glycolipid in the sample to be separated containing glycolipid and interfering lipid phospholipid. The magnetic nanomaterial modified with titanium dioxide has good magnetic response and can realize effective coordination interaction with the cis-dihydroxyl in the target substance sphingomyelin, capture the glycolipid in the alkaline environment of the sample buffer, and separate the glycolipid from the complex sample system with the help of the external magnetic field. The nanomaterial has a high specific surface area and can provide a large number of active sites to realize high loading capacity of the glycolipid. With the aid of the external magnetic field, the magnetic titanium dioxide nanomaterial can be quickly separated from the liquid phase, greatly simplifying the operation process and avoiding sample loss caused by pretreatment.

[0045] In the embodiment of the present application, the separation and enrichment method involves the use of three kinds of buffer solutions. The sample buffer is an alkaline aprotic solvent including acetonitrile and ammonia water, which allows the glycolipid to form a stable coordination interaction with titanium dioxide and makes a large amount of phospholipid not be adsorbed by titanium dioxide through the interference of the alkaline environment; the washing buffer is an alkaline protic solvent ammonium salt solution, which is used to wash the titanium dioxide surface adsorbed with lipids and remove a large amount of non-specifically adsorbed phospholipid, thereby improving the selectivity for the target substance sphingomyelin. The washing buffer uses an alkaline protic solvent and adds ammonium salt, specifically including methanol, ammonia water and ammonium salt. The ammonium salt provides a large amount of NH4 + ions, which compete with the interfering lipid phospholipid remaining on the titanium dioxide surface for the active sites of the titanium dioxide, thereby further removing the phospholipid and eliminating the adsorption of the high-abundance interferent on the material. In addition, the addition of the protic solvent not only helps the dissolution of the ammonium salt, but also can remove the adsorbed phospholipid on the surface of the material through coordination with titanium dioxide. Finally, the glycolipid is eluted by the elution buffer to realize the specific separation of the complete glycolipid molecule.

[0046] In the embodiment of the present application, based on the use of the magnetic titanium dioxide nanomaterial and the washing buffer of the present application, high-specificity separation and enrichment of glycolipid can be realized in a standard lipid mixture solution or a biological complex sample pig brain extract in which the concentration of the interfering lipid is 100 times that of the target glycolipid. The method of the present application can be used for Paternò-Büchi (PB) reaction and various liquid chromatography-mass spectrometry platforms, and realizes high-throughput, sensitive and fine mass spectrum structure analysis of the fine structure of the glycolipid in the complex sample, for example, identification of the double bond position of the glycolipid, improves the identification ability of the mass spectrum for low-abundance glycolipid, and discovers a large number of low-abundance glycolipid new structures, which lays a foundation for the research of low-abundance glycolipid.

[0047] In some embodiments, in step (1), the volume fraction of acetonitrile in the sample buffer is 92-96%, specifically, for example, 92%, 94%, 96%, and the volume fraction of ammonia water is 4%-8%, specifically, for example, 4%, 6%, 8%.

[0048] and / or, the time of the incubation is 30-90 min, in particular, for example, 30 min, 60 min, 90 min;

[0049] and / or, the temperature of the incubation is room temperature (25℃);

[0050] and / or, the ratio of the magnetic titanium dioxide nanomaterial and the loading buffer is 5-20 mg:400 μL, in particular, for example, 5 mg:400 μL, 10 mg:400 μL, 20 mg:400 μL.

[0051] In the embodiments of the present application, by optimizing the volume fraction of acetonitrile and ammonia water, the adsorption and separation and enrichment of glycosphingolipids are further improved. When the content of ammonia water is too high, the amide bond in sphingolipids or is hydrolyzed in a strong alkali environment; when the content of ammonia water is too low, it is not enough to destroy the coordination of titanium dioxide and phosphoric acid functional groups.

[0052] In some embodiments, in the step (2), the volume fraction of methanol in the washing buffer is 94%-96%, in particular, for example, 94%, 95%, 96%, the volume fraction of ammonia water is 4%-6%, in particular, for example, 4%, 5%, 6%, and the concentration of ammonium salt is 10-20 mM, in particular, for example, 10 mM, 20 mM; preferably, the ammonium salt includes at least one of ammonium bicarbonate, ammonium formate or ammonium acetate;

[0053] and / or, the incubated magnetic titanium dioxide nanomaterial in the step (1) is washed with the washing buffer for 1-3 times, in particular, for example, 1 time, 2 times, 3 times;

[0054] and / or, the ratio of the incubated magnetic titanium dioxide nanomaterial and the washing buffer is 5-20 mg:400 μL, in particular, for example, 5 mg:400 μL, 10 mg:400 μL, 20 mg:400 μL. In the embodiments of the present application, by optimizing the content of methanol, ammonia water and ammonium bicarbonate, further elution of residual phospholipids is facilitated. When the content of ammonia water is too high, the amide bond in sphingolipids or is hydrolyzed in a strong alkali environment; when the content of ammonia water is too low, it is not enough to destroy the coordination of titanium dioxide and phosphoric acid functional groups. The content of ammonium salt is limited by the solubility of methanol solvent, so 20 mM is a relatively saturated state; when the content of ammonium salt is too low, it cannot provide sufficient NH4 + for stabilizing the negative electric phosphoric acid functional group and the titanium dioxide surface.

[0055] In some embodiments, the mass fraction of ammonia water is 28 wt%.

[0056] In some embodiments, in step (3), the elution buffer comprises formic acid and methanol; preferably, the volume fraction of formic acid in the elution buffer is 2% to 8%, and specifically, for example, 2%, 4%, 6%, 8%, the volume fraction of methanol is 92% to 98%, and specifically, for example, 92%, 94%, 96%, 98%;

[0057] And / or, the incubation time is 30-90 min, and specifically, for example, 30 min, 60 min, 90 min;

[0058] And / or, the incubation temperature is room temperature;

[0059] And / or, the ratio of the washed magnetic titanium dioxide nanomaterial to the elution buffer is 5-20 mg:400 μL, and specifically, for example, 5 mg:400 μL, 10 mg:400 μL, 20 mg:400 μL. In the embodiments of the present application, the elution buffer of the glycosphingolipid preferably comprises formic acid and methanol, the elution buffer is an acidic proton solvent, the cis-dihydroxyl group of the glycosphingolipid is protonated in the acidic environment, the coordination with titanium dioxide is destroyed, and thus the elution of the glycosphingolipid is realized. By optimizing the content of formic acid and methanol, the rapid elution of the glycosphingolipid is further realized.

[0060] In some embodiments, in step (1), the magnetic titanium dioxide nanomaterial comprises a titanium dioxide-coated ferroferric oxide magnetic nanomaterial, and in specific embodiments, the magnetic titanium dioxide nanomaterial is a titanium dioxide-coated ferroferric oxide magnetic nanomaterial. In the embodiments of the present application, the magnetic titanium dioxide nanomaterial comprises a titanium dioxide-coated ferroferric oxide magnetic nanomaterial, which is beneficial to separating the solid-phase nanomaterial from the solution system under the guidance of an external magnetic field, effectively improving the operation efficiency in subsequent multi-step washing and elution operations, and reducing the loss of target substances.

[0061] In some embodiments, the preparation method of the magnetic titanium dioxide nanomaterial comprises the following steps:

[0062] (a) dissolving ferric chloride hexahydrate in ethylene glycol, then adding anhydrous sodium acetate, mixing, and reacting at 100-400°C for 8-20 hours to obtain ferroferric oxide;

[0063] Preferably, the reaction is carried out in a reaction kettle; after the reaction, the reaction product is cooled to room temperature, washed with deionized water and anhydrous ethanol, and then vacuum dried at 40-75°C; the mass ratio of the ferric chloride hexahydrate to the anhydrous sodium acetate is 3:8;

[0064] (b) dispersing the ferroferric oxide in a solvent, adding a base and a titanium dioxide precursor, and reacting at 20-80°C for 12-48 hours;

[0065] Preferably, the base comprises ammonia water; the titanium dioxide precursor comprises tetrabutyl titanate; the solvent comprises anhydrous ethanol; the reaction is carried out with stirring; and the reaction product is washed with deionized water and anhydrous ethanol after the reaction.

[0066] (c) calcining the product obtained in step (b) at 200-800 DEG C for 1-10 hours to obtain the magnetic titanium dioxide nanomaterial.

[0067] In some embodiments, in steps (1)-(3), both the material and the solution are separated by a magnet. In the embodiments of the present application, the modified magnetic titanium dioxide nanomaterial has good magnetic response, and under the assistance of an external magnetic field, the magnetic titanium dioxide nanomaterial can be quickly separated from the liquid phase, greatly simplifying the operation process and avoiding sample loss caused by pretreatment.

[0068] In some embodiments, in step (1), the sample to be separated is a biological sample, which is optionally a total lipid extract of the biological sample, and specifically, for example, a porcine brain polar lipid extract. In the embodiments of the present application, the method for separating and enriching glycosphingolipids can be used for the separation of biological complex samples, and the method is applied to the analysis of porcine brain extract. By separating and enriching glycosphingolipids in the porcine brain through the method, 365 glycosphingolipid molecules are identified in the porcine brain lipid extract by reverse phase chromatography and secondary mass spectrometry. PB-MS 2 Flow analysis, 197 glycosphingolipid molecules are identified at the structural level of the double bond position, the separation and enrichment of glycosphingolipids in complex biological samples are achieved, and a large number of low-abundance glycosphingolipid new structures are discovered, which lays a foundation for the research of low-abundance glycosphingolipids.

[0069] In some embodiments, the eluate of the separated and enriched glycosphingolipids is blown dry with nitrogen and redissolved in methanol to obtain concentrated glycosphingolipids.

[0070] The embodiments of the present application provide a method for concentrating glycosphingolipids, the eluate of the glycosphingolipids enriched by the method of the embodiments of the present application is blown dry with nitrogen and redissolved in methanol to obtain concentrated glycosphingolipids. In the embodiments of the present application, the separated glycosphingolipids are redissolved in methanol to realize the concentration of the purified glycosphingolipids, improve the sample injection amount of the chromatography, and improve the identification ability of the mass spectrometry for low-abundance glycosphingolipids, and the detection limit of the glycosphingolipids is improved from 0.5 nM to 20 pM. The glycosphingolipids purified by the method are suitable for various chromatographic separation methods.

[0071] The application provides a method for analyzing a sample containing glycosphingolipids. The sample containing glycosphingolipids is enriched by the method to obtain an eluent of glycosphingolipids, and the eluent of glycosphingolipids is subjected to mass spectrometry analysis and / or liquid chromatography separation, specifically, for example, liquid chromatography-mass spectrometry analysis. In the application, based on the use of magnetic titanium dioxide nanomaterials and the washing buffer, high-specificity separation and enrichment of glycosphingolipids can be achieved in a standard lipid mixture solution or a biological complex sample pig brain extract with the concentration of interfering lipids being 100 times that of the target glycosphingolipids. The purified glycosphingolipids are suitable for various chromatography separation methods and can also be used in various liquid chromatography-mass spectrometry platforms. The fine structure of glycosphingolipids, for example, the identification of the double bond position of glycosphingolipids, is analyzed and identified. The identification ability of mass spectrometry for low-abundance glycosphingolipids is improved, and a large number of low-abundance glycosphingolipid new structures are found, which lays a foundation for the research of low-abundance glycosphingolipids.

[0072] In some embodiments, the concentrated glycosphingolipids are subjected to mass spectrometry analysis and / or liquid chromatography separation.

[0073] The application provides a method for identifying the double bond position of glycosphingolipids. The eluent of glycosphingolipids is enriched by the separation and enrichment method, the eluent of glycosphingolipids is subjected to photo-derivatization Paternò-Büchi reaction, and secondary mass spectrometry is connected to identify the double bond position of glycosphingolipids. In the application, the photo-derivatization of the double bond position of unsaturated glycosphingolipids is combined with low-energy collision-induced dissociation of mass spectrometry, so that the double bond position of glycosphingolipids can be identified.

[0074] In some embodiments, the concentrated glycosphingolipids are subjected to photo-derivatization Paternò-Büchi reaction, and secondary mass spectrometry is connected to identify the double bond position of glycosphingolipids.

[0075] In some embodiments, diacetylpyridine is used as a PB reagent to perform photo-derivatization of the double bond position of unsaturated glycosphingolipids.

[0076] The application is described below with reference to specific embodiments, and it should be noted that these embodiments are merely descriptive and do not limit the application in any way.

[0077] The liquid chromatography is ExionLC AC system (Sciex), and the liquid chromatography is equipped with a degassing device, two pumps, an automatic sampler and a column oven.

[0078] The lipids in Examples 1-2 were separated by a normal phase chromatographic column, model: 150 mm x 2.1 mm, silica spheres, 2.7 pm (Sigma-Aldrich). The column temperature was 30 °C. Mobile phase A: 10 mM ammonium acetate in water, mobile phase B: acetonitrile solution. The mobile phase gradient was: 0-1 min: 90% B; 1-5 min: 90% to 85% B; 5-8 min: 85% to 80% B: 8-13.5 min: 80% B. The flow rate of the mobile phase was 0.2 mL / min.

[0079] The lipids in Examples 3-6 were separated by a reverse phase chromatographic column, model: 100 mm x 2.1 mm, silica spheres, 2.5 pm (Sigma-Aldrich). The column temperature was 40 °C. Mobile phase A: 10 mM ammonium formate in water containing 0.1% formic acid, mobile phase B: solution containing 10% isopropanol and 90% acetonitrile. The mobile phase gradient was: 0-1 min: 90% B; 1-10 min: 90% to 93% B; 10-12 min: 93% B; 12-32 min: 93% to 99% B; 32-35 min: 99% B. The flow rate of the mobile phase was 0.5 mL / min.

[0080] The mass spectrometer was X500R of Sciex. The parameters for the first mass spectrometry scan in the positive ion mode were set as follows: gas curtain gas 30 psi; collision gas high; voltage 4500 V; temperature 400 °C; nebulizer gas 30 psi; auxiliary heater gas 30 psi; de-clustering voltage 80 eV. The collision energy for the second mass spectrometry scan in the positive ion mode was 40-45 eV, and the other parameters were consistent with the above. In the following examples, unless otherwise specified, the mass spectrometry detection mode was the above mass spectrometry detection mode.

[0081] Example 1

[0082] Separation and enrichment of glycosphingolipids in standard lipid mixture:

[0083] 1. Mass spectrometry analysis of standard lipid mixture

[0084] Glycosphingolipid standard lipid (1 mM) and interfering substances sphingomyelin (5 mM), phosphatidylcholine (5 mM), phosphatidylethanolamine (5 mM) were added to the loading buffer and directly sent to liquid chromatography-mass spectrometry analysis.

[0085] 2. Mass spectrometry analysis of standard lipid mixture after separation and enrichment

[0086] The preparation method of the magnetic titanium dioxide nanomaterial comprises the following steps:

[0087] Dissolve 2.7 g of ferric chloride hexahydrate in 75 mL of ethylene glycol, after the solution is clear and transparent, add 7.2 g of anhydrous sodium acetate, after ultrasonic stirring, transfer to a reaction kettle, heat at 200°C for 16 hours, after the reaction is completed, cool the reaction kettle to room temperature, wash the obtained product with deionized water and anhydrous ethanol, and dry in a vacuum at 45°C; uniformly disperse the obtained product in a solvent anhydrous ethanol, add 0.72 mL of ammonia water and 1.6 mL of tetrabutyl titanate, stir the obtained mixed solution at 45°C for 24 hours, and wash with deionized water and anhydrous ethanol; calcine the obtained product in air at 400°C for 2 hours, to obtain a titanium dioxide coated ferroferric oxide magnetic nanomaterial.

[0088] The separation and enrichment of the glycosphingolipid analysis sample includes:

[0089] (1) Take 10 mg of magnetic titanium dioxide material and add 400 μL of sample loading buffer, add 1 μM target glycosphingolipid standard lipid (monosaccharide head group glycosphingolipid, HexCer) and interferents 5 μM sphingomyelin (SM), 5 μM phosphatidylcholine (PC), and 5 μM phosphatidylethanolamine (PE) to the sample loading buffer, incubate at room temperature for 60 minutes, and collect the sample loading buffer; the sample loading buffer is a buffer solution containing 94% by volume acetonitrile and 6% by volume ammonia water;

[0090] (2) Wash the material twice with 400 μL of washing buffer, the washing buffer contains 96% by volume methanol, 4% by volume ammonia water, and 20 mM ammonium bicarbonate, and collect the washing buffer;

[0091] (3) Use 5% formic acid by volume and 95% methanol as elution buffer (400 μL), incubate at room temperature for 90 minutes, collect the eluent, and send it to liquid chromatography-mass spectrometry analysis.

[0092] Mass spectrometry analysis: send the sample loading buffer, washing buffer, and elution buffer to normal phase chromatography separation of lipid species, and connect mass spectrometry for primary mass spectrometry detection in positive ion mode, with an injection amount of 2 μL.

[0093] Figure 2This is a normal-phase (HILIC) separation chromatogram. The lipid standard mixture contains 1 μM glycosphingolipid HexCer d18:1 / 18:1, 5 μM phosphatidylethanolamine PE 16:0-18:1, 5 μM phosphatidylcholine PC 16:0-18:1, and 5 μM sphingomyelin SM d18:1 / 12:0. Among them, (a) is the extracted ion chromatogram of the standard lipid mixture in the loading buffer, HexCer d18:1 / 18:1 m / z 726.5, PE 16:0_18:1 m / z 718.5, PC 16:0_18:1 m / z 760.5, SM d18:1 / 12:0 m / z 647.5; (b) is the lipids remaining in the loading buffer after the magnetic titanium dioxide nanomaterials captured glycosphingolipids; (c) is the lipids in the washing buffer after washing the magnetic titanium dioxide nanomaterials that captured lipids; (d) is the elution buffer obtained by eluting glycosphingolipids from the magnetic titanium dioxide nanomaterials after lipid capture and washing.

[0094] Depend on Figure 2 It was observed that a large amount of phospholipids remained in the loading buffer, while no glycosphingolipids were observed, indicating that the glycosphingolipids were completely adsorbed by the magnetic titanium dioxide nanomaterials. In the washing buffer, a large amount of phospholipids and a small amount of glycosphingolipids were observed, indicating that the washing buffer effectively washed away the residual phospholipids from the material while preserving the coordination interaction between the glycosphingolipids and the magnetic titanium dioxide nanomaterials. In the elution buffer, a large amount of glycosphingolipids was observed, indicating that the interfering lipids were largely removed.

[0095] Example 2

[0096] Separation and enrichment of glycosphingolipids from standard lipid mixtures containing high abundance of interfering lipids:

[0097] 1. Mass spectrometry analysis of standard lipid mixtures containing high abundance interfering lipids

[0098] The standard lipid glycosphingolipid (100 nM) and interfering substances sphingomyelin (10 μM), phosphatidylcholine (10 μM), and phosphatidylethanolamine (10 μM) were added to the loading buffer and directly sent to the liquid chromatography-mass spectrometry (LC-MS) analysis.

[0099] 2. Mass spectrometry analysis of standard lipid mixtures after separation and enrichment

[0100] The preparation method of the magnetic titanium dioxide nanomaterials is exactly the same as that in Example 1.

[0101] Separation and enrichment of glycosphingolipids in samples include:

[0102] (1) Weigh 10 mg of magnetic titanium dioxide material and add it to 400 μL of loading buffer. Add the target glycosphingolipid standard lipid (monosaccharide head glycosphingolipid, HexCer, 100 nM) and the interfering substances sphingomyelin (SM, 10 μM), phosphatidylcholine (PC, 10 μM), and phosphatidylethanolamine (PE, 10 μM) to the loading buffer. Incubate at room temperature for 60 minutes and collect the loading buffer. The loading buffer is a buffer solution containing 94% acetonitrile and 6% ammonia by volume.

[0103] (2) Wash the material twice with 400 μL of washing buffer containing 96% methanol, 4% ammonia and 20 mM ammonium bicarbonate by volume.

[0104] (3) Use 5% formic acid and 95% methanol as elution buffer (400 μL) and incubate at room temperature for 90 minutes. Collect the eluent and send it to liquid chromatography-mass spectrometry.

[0105] Mass spectrometry analysis: The elution buffer was sent to normal phase chromatography to separate lipid species, and then connected to a mass spectrometer for primary mass spectrometry detection in positive ion mode. The injection volume was 2 μL.

[0106] Figure 3 The HILIC separation chromatogram shows that (a) is a standard lipid mixture containing 0.1 μM glycosphingolipid HexCer d18:1 / 18:1, 10 μM phosphatidylethanolamine PE 16:0-18:1, 10 μM phosphatidylcholine PC 16:0-18:1, and 10 μM sphingomyelin SM d18:1 / 12:0; and (b) is the elution buffer after the glycosphingolipids in the above standard lipid mixture have been separated and enriched by magnetic titanium dioxide nanomaterials.

[0107] Depend on Figure 3 It can be seen that magnetic titanium dioxide nanomaterials can still effectively separate the target glycosphingolipid and remove a large amount of interfering lipids when the concentration of interfering substances reaches 100 times that of the target substance.

[0108] Example 3

[0109] Separation and enrichment of low-abundance glycosphingolipids in a standard lipid mixture:

[0110] 1. Mass spectrometry analysis of standard lipid mixtures

[0111] The glycosphingolipid standard lipid at a concentration of 0.5 nM was directly sent to liquid chromatography-mass spectrometry (LC-MS).

[0112] 2. Mass spectrometry analysis of standard lipid mixtures after separation and enrichment

[0113] The preparation method of the magnetic titanium dioxide nanomaterials is exactly the same as that in Example 1.

[0114] Separation and enrichment of glycosphingolipids in samples include:

[0115] (1) Weigh 10 mg of magnetic titanium dioxide material and add it to 400 μL of loading buffer. Add the target glycosphingolipid standard lipid (monosaccharide head glycosphingolipid, HexCer, 20 pM) and the interfering substances sphingomyelin (SM, 5 nM), phosphatidylcholine (PC, 5 nM), and phosphatidylethanolamine (PE, 5 nM) to the loading buffer and incubate at room temperature for 60 minutes. The loading buffer is a buffer solution containing 94% acetonitrile and 6% ammonia by volume.

[0116] (2) Wash the material twice with 400 μL of washing buffer containing 96% methanol, 4% ammonia and 20 mM ammonium bicarbonate by volume.

[0117] (3) Use 5% formic acid and 95% methanol as elution buffer (400 μL) and incubate at room temperature for 90 minutes. Collect the eluent and dry it with nitrogen gas. Redissolve it in 20 μL of methanol and send it to liquid chromatography-mass spectrometry.

[0118] Mass spectrometry analysis: Glycosphingolipids separated by magnetic titanium dioxide nanomaterials were sent to reversed-phase chromatography for further separation and then connected to a mass spectrometer for secondary mass spectrometry detection in positive ion mode. The injection volume was 2 μL.

[0119] Figure 4 (a) is the second-order mass spectrum of HexCer d18:1 / 18:1 (0.5 nM) (collision energy at 40 eV); Figure 4 (b) is the secondary mass spectrum of 20pM HexCer d18:1 / 18:1 separated and enriched by magnetic titanium dioxide nanomaterials (collision energy around 40eV).

[0120] Depend on Figure 4 It was found that the mass spectrometry detection limit for direct detection of glycosphingolipid standard lipids was 0.5 nM. Glycosphingolipids at a concentration of 20 pM, after separation and concentration (the concentration of glycosphingolipids in 400 μL of loading buffer was 20 pM, which was then separated and concentrated to 20 μL before injection), could still be detected by mass spectrometry, indicating that the separation and enrichment method can be used for the separation and enrichment of low-abundance glycosphingolipids and can effectively reduce the detection limit of glycosphingolipid chain composition and structure.

[0121] Example 4

[0122] Separation and enrichment of different glycosphingolipid types in a standard lipid mixture:

[0123] 1. Mass spectrometry analysis of standard lipid mixtures

[0124] 10 nM glycosphingolipid (monosaccharide head) standard lipids and 10 nM glycosphingolipid (disaccharide head) standard lipids were sent to liquid chromatography-mass spectrometry (LC-MS) analysis.

[0125] 2. Mass spectrometry analysis of standard lipid mixtures after separation and enrichment

[0126] The preparation method of the magnetic titanium dioxide nanomaterials is exactly the same as that in Example 1.

[0127] Separation and enrichment of glycosphingolipids in samples include:

[0128] (1) Weigh 10 mg of magnetic titanium dioxide material and add it to 400 μL of loading buffer. Add 10 nM glycosphingolipid (monosaccharide head) standard lipid (HexCer), 10 nM glycosphingolipid (disaccharide head) standard lipid (Hex2Cer) and interfering substances sphingomyelin (SM, 50 nM), phosphatidylcholine (PC, 50 nM), and phosphatidylethanolamine (PE, 50 nM) to the loading buffer and incubate at room temperature for 60 minutes. The loading buffer is a buffer solution containing 94% acetonitrile and 6% ammonia by volume.

[0129] (2) Wash the material twice with 400 μL of washing buffer containing 96% methanol, 4% ammonia and 20 mM ammonium bicarbonate by volume.

[0130] (3) Use 5% formic acid and 95% methanol as elution buffer (400 μL) and incubate at room temperature for 90 minutes. Collect the elution solution.

[0131] Mass spectrometry analysis: The glycosphingolipid eluent separated by magnetic titanium dioxide nanomaterials was sent to reversed-phase chromatography for separation and then connected to a mass spectrometer for secondary mass spectrometry detection in positive ion mode. The injection volume was 2 μL.

[0132] Figure 5 (a) is the second-order spectrum of HexCer d18:1 / 15:0 (10 nM); Figure 5 (b) is the second-order spectrum of Hex2Cer d18:1 / 17:0 (10 nM); Figure 5 (c) Secondary spectra of HexCer d18:1 / 15:0 (10 nM) before and after separation by magnetic titanium dioxide nanomaterials, showing sphingosine fragment ions [d18:1-2H2O+H]. + Extraction ion chromatography at m / z 264.5 (collision energy at 40 eV); Figure 5 (d) Sphingosine fragment ions [d18:1-2H2O+H] of Hex2Cer d18:1 / 17:0 (10 nM) before and after separation using magnetic titanium dioxide nanomaterials. + Extraction ion chromatography at m / z 264.5.

[0133] By Figure 5 It can be seen that, by comparing the signal intensity of the glycosphingolipid standard and the glycosphingolipid separated from the lipid mixture at the same concentration, it can be found that the method of the present application achieves a higher recovery rate of glycosphingolipid, and the glycosphingolipid with monosaccharide head group and disaccharide head group has similar recovery capacity, indicating that the separation and enrichment method has no preference for the separation and enrichment of the glycosphingolipid with the two sugar head groups.

[0134] Example 5

[0135] Separation of glycosphingolipid in pig brain:

[0136] The preparation method of the magnetic titanium dioxide nanomaterial is exactly the same as in Example 1.

[0137] Separation and enrichment of glycosphingolipid in pig brain lipid extract, comprising:

[0138] (1) 10 mg of magnetic titanium dioxide material was weighed and added to 400 μL of sample buffer, 5 μL of pig brain polar lipid extract (20 mg / mL, purchased from Avanti) was added to the sample buffer, and incubated at room temperature for 90 minutes; the sample buffer is a buffer solution containing 94% acetonitrile by volume and 6% ammonia water by volume;

[0139] (2) The material was washed with 400 μL of washing buffer for 3 times, and the washing buffer contains 96% methanol by volume, 4% ammonia water by volume and 20 mM ammonium bicarbonate;

[0140] (3) 5% formic acid by volume, 95% methanol as elution buffer (400 μL), incubated at room temperature for 90 minutes, the eluent was collected and blown dry, and resuspended in 150 μL of methanol.

[0141] Mass spectrometry analysis: The pig brain polar lipid extract (0.1 mg / mL) was sent into the reverse phase chromatography separation mass spectrometry detection, and the separated and enriched pig brain glycosphingolipid was sent into the reverse phase chromatography separation, and the injection amount was 2 μL. The detection adopts Sequential Windowed Acquisition of all Theoretical fragment ions (SWATH) mode, the window width is 1 Da, the detection window number is 81, the collision energy is 30-55 eV, and the collection time of each window for primary mass spectrum and secondary mass spectrum is 0.1 s.

[0142] Figure 6 (a) is the reverse phase separation chromatogram of the total lipid (0.2 μg) in pig brain, the interfering lipids such as phospholipids are white filled, and the glycosphingolipids are black filled, and two kinds of most abundant phosphatidylcholines and two kinds of most abundant glycosphingolipids are marked in the figure; Figure 6(b) is the reverse phase separation chromatogram of the glycosphingolipids in the pig brain (3 μg) after separation and enrichment by magnetic titanium dioxide nanomaterials, and two kinds of phosphatidylcholine with the highest abundance and two kinds of glycosphingolipids with the highest abundance are marked in the figure.

[0143] The lipid species are identified by secondary mass spectrometry, and the lipids are identified and divided into glycosphingolipids and phospholipids corresponding to the parent ions in the primary mass spectrometry. Figure 6 It can be seen from the direct detection results of the pig brain polar lipid extract that the high-abundance phosphatidylcholine occupies the main peak of the chromatogram, and after separation and enrichment by magnetic titanium dioxide nanomaterials, various glycosphingolipids are identified, and the two kinds of phosphatidylcholine with the highest abundance, PC 34:1 and PC 36:1, are largely removed by pretreatment.

[0144] Example 6

[0145] Diacetyl pyridine is used as a charge PB reagent for the derivatization of unsaturated glycosphingolipids and the identification of double bond positions by mass spectrometry detection.

[0146] Add glycosphingolipid HexCer d18:1 / 18:1 (9Z) (0.5 μM) to the photoreaction solution, and the volume ratio of acetonitrile to water in the photoreaction solution is 5:1. Add diacetyl pyridine PB reagent (10 mM). Load the reaction solution into the offline microflow reaction device after deoxygenation under nitrogen flow for 3 minutes. A low-pressure ultraviolet mercury lamp at 254 nm is placed in parallel with a fused quartz capillary (outer diameter: 363 μm, inner diameter: 100 μm), with a distance of about 0.5 cm and an irradiation length of about 5 cm. The fused quartz capillary is used as a microflow reactor, which is connected to a syringe through a two-way valve. The syringe is used to adjust the flow rate of the sample under the push of a syringe pump, thereby adjusting the reaction time. The reaction time is controlled at 15 s, and the reacted liquid is collected.

[0147] Mass spectrometry analysis: The unsaturated glycosphingolipid without reaction and the solution collected after reaction are respectively sent into reverse phase chromatography separation mass spectrometry detection, and the injection amount is 2 μL.

[0148] Figure 7 (a) is the extracted ion chromatogram of HexCer d18:1 / 18:1 (0.5 μM) after reverse phase chromatography separation [HexCer d18:1 / 18:1 + H] + m / z 726.5; Figure 7 (b) is the solution of HexCer d18:1 / 18:1 (0.5 μM) and diacetyl pyridine after 15 s of reaction under 254 nm light, which is separated by reverse phase chromatography. The substrate [HexCer d18:1 / 18:1 + H] + m / z 726.5 and PB product [ PB HexCer d18:1 / 18:1 + H]+ Extraction ion chromatogram at m / z 847.5; Figure 7 (c) Secondary mass spectrometry was used to detect the PB product that elutes at 4.3 min, with a collision energy of 45 eV; Figure 7 (d) Secondary mass spectrometry was used to detect the PB product that flowed out after 4.5 min, with a collision energy of 45 eV.

[0149] Depend on Figure 7 As can be seen, extractive ion chromatography analysis of the PB products revealed that the main PB product eluted within 4-5 minutes. This main product was separated into two peaks in reversed-phase chromatography, attributed to the addition isomer of the PB reagent. Secondary mass spectrometry analysis of the two PB products showed the formation of two types of PB fragment ions, which can be used to identify the two ends of the double bond positions.

[0150] Comparative Example 1

[0151] The method for separating and enriching glycosphingolipids in the standard lipid mixture in Example 1 is the same, except that ammonium bicarbonate is not added to the washing buffer in step (2).

[0152] Depend on Figure 8 It can be seen that in Comparative Example 1, a large amount of phospholipids can be observed in the elution buffer obtained in step (3), indicating that the washing buffer in step (2) did not effectively remove the phospholipids adsorbed on the magnetic titanium dioxide nanomaterials.

[0153] In Example 1, the washing buffer containing ammonium ions effectively removed the phospholipids adsorbed on the magnetic titanium dioxide nanomaterials. After washing twice with the washing buffer containing ammonium ions, the residual phospholipids on the material were basically removed.

[0154] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0155] Although the above embodiments have been shown and described, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Any changes, modifications, substitutions and variations made to the above embodiments by those skilled in the art are within the protection scope of the present invention.

Claims

1. A method for separating and enriching glycosphingolipids, characterized by, The method comprises the following steps: (1) adding the magnetic titanium dioxide nanomaterial and a sample to be separated into a sample loading buffer, incubating, and obtaining the incubated magnetic titanium dioxide nanomaterial; wherein the sample to be separated comprises glycosphingolipids and phospholipids; and the sample loading buffer comprises acetonitrile and ammonia water; (2) washing the incubated magnetic titanium dioxide nanomaterial in step (1) with a washing buffer; the washing buffer comprises methanol, ammonia water and an ammonium salt; the volume fraction of methanol in the washing buffer is 94% to 96%, the volume fraction of ammonia water is 4% to 6%, and the concentration of the ammonium salt is 10 to 20 mM; (3) adding the washed magnetic titanium dioxide nanomaterial in step (2) into an elution buffer, incubating, and obtaining an eluent of glycosphingolipids; the elution buffer comprises formic acid and methanol.

2. The glycolipid separation and enrichment method according to claim 1, characterized by, In step (1), the volume fraction of acetonitrile in the sample loading buffer is 92% to 96%, and the volume fraction of ammonia water is 4% to 8%; And / or, the incubation time is 30 to 90 minutes.

3. The glycolipid separation and enrichment method according to claim 1, characterized by, In step (2), the ammonium salt comprises at least one of ammonium bicarbonate, ammonium formate or ammonium acetate; And / or, the incubated magnetic titanium dioxide nanomaterial in step (1) is washed with the washing buffer for 1 to 3 times.

4. The glycolipid separation and enrichment method according to claim 1, characterized by, In step (3), the volume fraction of formic acid in the elution buffer is 2% to 8%, and the volume fraction of methanol is 92% to 98%; And / or, the incubation time is 30 to 90 minutes.

5. The glycolipid separation and enrichment method according to claim 1, characterized by, In step (1), the magnetic titanium dioxide nanomaterial comprises a titanium dioxide-coated ferroferric oxide magnetic nanomaterial.

6. The glycolipid separation and enrichment method according to claim 5, characterized by, In step (1), the preparation method of the magnetic titanium dioxide nanomaterial comprises the following steps: (a) dissolving ferric chloride hexahydrate in ethylene glycol, then adding anhydrous sodium acetate, mixing, and reacting at 100 to 400°C for 8 to 20 hours to obtain ferroferric oxide; (b) dispersing the ferroferric oxide in a solvent, adding a base and a titanium dioxide precursor, and reacting at 20 to 80°C for 12 to 48 hours; (c) calcining the product obtained in step (b) at 200 to 800°C for 1 to 10 hours to obtain the magnetic titanium dioxide nanomaterial.

7. The glycolipid separation and enrichment method according to claim 1, characterized by, In steps (1) to (3), the materials and solutions are separated by a magnet.

8. The glycolipid separation and enrichment method according to claim 1, characterized by, In step (1), the sample to be separated is a biological sample.

9. A method for concentrating glycosphingolipids, characterized by, The eluent of glycosphingolipids obtained by the method of any one of claims 1 to 8 is blown dry with nitrogen, and redissolved in methanol to obtain concentrated glycosphingolipids.

10. A method of analysis of a sample containing glycosphingolipids, characterized in that, The eluent of glycosphingolipids obtained by the method of any one of claims 1 to 8 is subjected to mass spectrometric analysis and / or liquid chromatographic separation.

11. A method for identifying the position of a double bond of a glycosphingolipid, characterized by, The eluent of glycosphingolipids obtained by the method of any one of claims 1 to 8 is subjected to a photo-derivative Paternò-Büchi reaction, and a secondary mass spectrometer is used to identify the double bond position of the glycosphingolipids.

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